48 results on '"Giacomo, Oliveira"'
Search Results
2. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma
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Tomasz Kula, Gavin MacBeath, Genevieve M. Boland, Wandi Zhang, Donna Neuberg, Phuong M. Le, Giacomo Oliveira, Catherine J. Wu, Steven A. Carr, Qikai Xu, Patrick A. Ott, Moshe Sade-Feldman, Sachet A. Shukla, Nir Hacohen, Susan Klaeger, Juliet Forman, Dennie T. Frederick, Karl R. Clauser, Edward F. Fritsch, Nicoletta Cieri, Shuqiang Li, Derin B. Keskin, Kenneth J. Livak, Kari Stromhaug, Teddy Huang, and Sune Justesen
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Multidisciplinary ,Immune system ,medicine.anatomical_structure ,Antigen ,T cell ,T-cell receptor ,Cancer research ,medicine ,Cytotoxic T cell ,Human leukocyte antigen ,Biology ,CD8 ,Immune checkpoint - Abstract
Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1–3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide–human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire. The authors use single-cell profiling and T cell receptor specificity screening to show how tumour antigen recognition shapes the phenotypes of CD8+ T cells and antitumour immune responses.
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- 2021
3. CD4+ Memory Stem T Cells Recognizing Citrullinated Epitopes Are Expanded in Patients With Rheumatoid Arthritis and Sensitive to Tumor Necrosis Factor Blockade
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Chiara Bonini, M. Tassara, Angelo A. Manfredi, Beatrice Claudia Cianciotti, Elena Baldissera, Zulma Magnani, Eliana Ruggiero, Fabio Ciceri, Corrado Campochiaro, Mattia Baldini, Nicoletta Cieri, Giacomo Oliveira, Matteo Doglio, Cianciotti, B. C., Ruggiero, E., Campochiaro, C., Oliveira, G., Magnani, Z. I., Baldini, M., Doglio, M., Tassara, M., Manfredi, A. A., Baldissera, E., Ciceri, F., Cieri, N., and Bonini, C.
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Adult ,CD4-Positive T-Lymphocytes ,Male ,0301 basic medicine ,T cell ,Immunology ,Cell ,Epitopes, T-Lymphocyte ,Arthritis ,Biology ,Epitope ,Flow cytometry ,Arthritis, Rheumatoid ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Rheumatology ,medicine ,Humans ,Immunology and Allergy ,Aged ,medicine.diagnostic_test ,T-cell receptor ,Middle Aged ,Cell sorting ,medicine.disease ,030104 developmental biology ,medicine.anatomical_structure ,Antirheumatic Agents ,030220 oncology & carcinogenesis ,Female ,Tumor Necrosis Factor Inhibitors ,Tumor necrosis factor alpha ,Immunologic Memory - Abstract
Objective Memory stem T (Tscm) cells are long-lived, self-renewing T cells that play a relevant role in immunologic memory. This study was undertaken to investigate whether Tscm cells accumulate in rheumatoid arthritis (RA). Methods The polarization and differentiation profiles of circulating T cells were assessed by flow cytometry. Antigen-specific T cells were characterized by staining with major histocompatibility complex class II tetramers. The T cell receptor (TCR) repertoire was analyzed by high-throughput sequencing using an unbiased RNA-based approach in CD4+ T cell subpopulations sorted by fluorescence-activated cell sorting. Results We analyzed the dynamics of circulating Tscm cells (identified as CD45RA+CD62L+CD95+ T cells) by flow cytometry in 27 RA patients, 16 of whom were also studied during treatment with the anti-tumor necrosis factor (anti-TNF) agent etanercept. Age-matched healthy donors were used as controls. CD4+ Tscm cells were selectively and significantly expanded in RA patients in terms of frequency and absolute numbers, and significantly contracted upon anti-TNF treatment. Expanded CD4+ Tscm cells displayed a prevalent Th17 phenotype and a skewed TCR repertoire in RA patients, with the 10 most abundant clones representing up to 53.7% of the detected sequences. CD4+ lymphocytes specific for a citrullinated vimentin (Cit-vimentin) epitope were expanded in RA patients with active disease. Tscm cells accounted for a large fraction of Cit-vimentin-specific CD4+ cells. Conclusion Our results indicate that Tscm cells, including expanded clones specific for relevant autoantigens, accumulate in RA patients not exposed to biologic agents, and might be involved in the natural history of the disease. Further analysis of Tscm cell dynamics in autoimmune disorders may have implications for the design and efficacy assessment of innovative therapies.
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- 2020
4. Optimized liquid and gas phase fractionation increase HLA-peptidome coverage for primary cell and tissue samples
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Vipheaviny Chea, David A. Braun, Nir Hacohen, Anna Tarren, Jennifer G. Abelin, Phuong M. Le, Steven A. Carr, Karl R. Clauser, Annie Apffel, Derin B. Keskin, Hasmik Keshishian, Patrick A. Ott, Catherine J. Wu, Siranush Sarkizova, Giacomo Oliveira, Suzanna Rachimi, and Susan Klaeger
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chemistry.chemical_classification ,medicine.anatomical_structure ,Chromatography ,chemistry ,Cell ,medicine ,Separation method ,Peptide ,Fractionation ,Human leukocyte antigen ,Mass spectrometry ,Epitope ,Gas phase - Abstract
Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require many millions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 100 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens.
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- 2021
5. Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples
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Hasmik Keshishian, Phuong M. Le, Karl R. Clauser, Steven A. Carr, Suzanna Rachimi, Patrick A. Ott, Annie Apffel, Vipheaviny Chea, Susan Klaeger, Giacomo Oliveira, Catherine J. Wu, Jennifer G. Abelin, Anna Tarren, Derin B. Keskin, Siranush Sarkizova, Nir Hacohen, and David A. Braun
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Cell ,Peptide ,Chemical Fractionation ,FAIMS ,Biochemistry ,Epitope ,Analytical Chemistry ,CE, collision energy ,basic reversed-phase fractionation ,Tandem Mass Spectrometry ,Neoplasms ,CV, compensation voltage ,SM, Spectrum Mill ,FA, formic acid ,FAIMS, high field asymmetric waveform ion mobility spectrometry ,chemistry.chemical_classification ,Chemistry ,Technological Innovation and Resources ,immunopeptidomics ,IP, immunoprecipitation ,PIP, precursor isolation purity ,HCD, higher energy collisional dissociation ,HLA ,medicine.anatomical_structure ,RCC, renal cell carcinoma ,BCS, backbone cleavage score ,PSM, peptide spectrum match ,PBMC, peripheral blood mononuclear cell ,HLA-I, human leukocyte antigen class I ,MEL, melanoma ,Fractionation ,Human leukocyte antigen ,Cell Line ,ion mobility ,Special Issue: Immunopeptidomics ,Antigens, Neoplasm ,Ion Mobility Spectrometry ,medicine ,Humans ,bRP, basic reversed-phase ,Antigen-presenting cell ,Molecular Biology ,Chromatography ,HLA, human leukocyte antigen ,Selected reaction monitoring ,T-cell receptor ,Histocompatibility Antigens Class I ,ACN, acetonitrile ,IL, interleukin ,APC, antigen-presenting cell ,TCR, T-cell receptor ,GBM, glioblastoma ,Peptides ,Chromatography, Liquid ,MRM, multiple reaction monitoring - Abstract
MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC–MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens., Graphical Abstract, Highlights • Deep immunopeptidome coverage using liquid and gas phase separation. • Up to 50% more HLA-I peptides using microscaled basic reversed-phase fractionation. • Ion mobility separation (FAIMS) increases HLA-I peptide identifications by up to 58%. • Increased sensitivity provided by these methods enables detection of neoantigens., In Brief Here, we evaluated off-line microscaled basic reversed-phase fractionation as well as the use of ion mobility coupled to LC–MS/MS for analysis of peptides presented on HLA-I. The two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone starting with as few as 100 million cells. The increased sensitivity obtained using our methods can enable detection of low abundant but clinically relevant epitopes such as neoantigens.
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- 2021
6. Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma
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Patrick C. Lee, Shuqiang Li, Charles H. Yoon, Jing Sun, Jason Pyrdol, Zoe B. Ciantra, J. Bryan Iorgulescu, Jinyan Liu, Donna E. Leet, Anita Giobbie-Hurder, Donna Neuberg, Satyen H. Gohil, Teddy Huang, Edward F. Fritsch, Wandi Zhang, Adrienne M. Luoma, Mohamed Uduman, Siranush Sarkizova, Kai W. Wucherpfennig, Giacomo Oliveira, Dan H. Barouch, Liudmila Elagina, Nir Hacohen, Oriol Olive, Lars Rønn Olsen, Zhuting Hu, Elizabeth I. Buchbinder, Derin B. Keskin, Sachet A. Shukla, Robert A. Redd, Bradley L. Pentelute, Patrick A. Ott, Scott J. Rodig, Catherine J. Wu, Keerthi Shetty, Kenneth J. Livak, Pavan Bachireddy, Rosa Lundbye Allesøe, Rebecca L. Holden, Lauren Peter, and Juliet Forman
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0301 basic medicine ,integumentary system ,business.industry ,medicine.medical_treatment ,T cell ,General Medicine ,General Biochemistry, Genetics and Molecular Biology ,Neoantigen Peptide ,Article ,Vaccination ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Immune system ,medicine.anatomical_structure ,Cancer immunotherapy ,Antigen ,SDG 3 - Good Health and Well-being ,030220 oncology & carcinogenesis ,Immunology ,Medicine ,Cytotoxic T cell ,business ,Memory T cell - Abstract
Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma. Personalized neoantigen vaccination in patients with melanoma elicits durable and specific memory T cell clones that have cytotoxic gene signatures and can diversify to include nonvaccine neoantigen specificities.
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- 2021
7. Thousands of novel unannotated proteins expand the MHC I immunopeptidome in cancer
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Sachet A. Shukla, Wandi Zhang, Annie Apffel, Irwin Jungreis, Susan Klaeger, Aviv Regev, Bo Li, François Aguet, Zhe Ji, Tamara Ouspenskaia, Christina R. Hartigan, Nir Hacohen, Yuen Ting Chow, Catherine J. Wu, Gad Getz, Siranush Sarkizova, Travis Law, Phuong M. Le, Giacomo Oliveira, Steven A. Carr, Manolis Kellis, Derin B. Keskin, Karl R. Clauser, Hasmik Keshishian, Elena Christian, Binyamin A. Knisbacher, and Pavan Bachireddy
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Open reading frame ,biology ,Antigen ,Cancer immunotherapy ,medicine.medical_treatment ,MHC class I ,Proteome ,biology.protein ,medicine ,Ribosome profiling ,Computational biology ,Major histocompatibility complex ,Epitope - Abstract
Tumor epitopes – peptides that are presented on surface-bound MHC I proteins - provide targets for cancer immunotherapy and have been identified extensively in the annotated protein-coding regions of the genome. Motivated by the recent discovery of translated novel unannotated open reading frames (nuORFs) using ribosome profiling (Ribo-seq), we hypothesized that cancer-associated processes could generate nuORFs that can serve as a new source of tumor antigens that harbor somatic mutations or show tumor-specific expression. To identify cancer-specific nuORFs, we generated Ribo-seq profiles for 29 malignant and healthy samples, developed a sensitive analytic approach for hierarchical ORF prediction, and constructed a high-confidence database of translated nuORFs across tissues. Peptides from 3,555 unique translated nuORFs were presented on MHC I, based on analysis of an extensive dataset of MHC I-bound peptides detected by mass spectrometry, with >20-fold more nuORF peptides detected in the MHC I immunopeptidomes compared to whole proteomes. We further detected somatic mutations in nuORFs of cancer samples and identified nuORFs with tumor-specific translation in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs thus expand the pool of MHC I-presented, tumor-specific peptides, targetable by immunotherapies.
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- 2020
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8. Unannotated proteins expand the MHC-I-restricted immunopeptidome in cancer
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Karl R. Clauser, Gad Getz, Wandi Zhang, Elena Christian, Siranush Sarkizova, Susan Klaeger, Bo Li, Yuen Ting Chow, Annie Apffel, Sune Justesen, Binyamin A. Knisbacher, François Aguet, Pavan Bachireddy, Catherine J. Wu, Aviv Regev, Christina R. Hartigan, Nir Hacohen, Sarah Chen, Irwin Jungreis, Zhe Ji, Hasmik Keshishian, Sachet A. Shukla, Phuong M. Le, Steven A. Carr, Tamara Ouspenskaia, Giacomo Oliveira, Travis Law, Manolis Kellis, and Derin B. Keskin
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medicine.medical_treatment ,Biomedical Engineering ,Bioengineering ,Computational biology ,Major histocompatibility complex ,Applied Microbiology and Biotechnology ,Epitope ,Mass Spectrometry ,Immune system ,Antigen ,Antigens, Neoplasm ,MHC class I ,medicine ,Humans ,Ribosome profiling ,Melanoma ,biology ,Histocompatibility Antigens Class I ,Cancer ,Immunotherapy ,medicine.disease ,biology.protein ,Molecular Medicine ,Peptides ,Biotechnology - Abstract
Tumor-associated epitopes presented on MHC-I that can activate the immune system against cancer cells are typically identified from annotated protein-coding regions of the genome, but whether peptides originating from novel or unannotated open reading frames (nuORFs) can contribute to antitumor immune responses remains unclear. Here we show that peptides originating from nuORFs detected by ribosome profiling of malignant and healthy samples can be displayed on MHC-I of cancer cells, acting as additional sources of cancer antigens. We constructed a high-confidence database of translated nuORFs across tissues (nuORFdb) and used it to detect 3,555 translated nuORFs from MHC-I immunopeptidome mass spectrometry analysis, including peptides that result from somatic mutations in nuORFs of cancer samples as well as tumor-specific nuORFs translated in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs are an unexplored pool of MHC-I-presented, tumor-specific peptides with potential as immunotherapy targets. New tumor epitopes are discovered by ribosome profiling and immunopeptidome mass spectrometry.
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- 2020
9. 655 Landscape of helper and regulatory CD4+ T cells in melanoma
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Giacomo Oliveira, Moshe Sade-Feldman, Derin B. Keskin, Megan Wind-Rotolo, Edward F. Fritsch, Kenneth J. Livak, Scott J. Rodig, Genevive Boland, Dennie Tompers, David A. Braun, Bryan Iorgulescu, Patrick A. Ott, Shuqiang Li, Donna Neuberg, Steven A. Carr, Catherine J. Wu, Susan Klaeger, Kari Stromhaug, Nir Hacohen, and Nicoletta Cieri
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Pharmacology ,Cancer Research ,Oncology ,Melanoma ,Immunology ,Cancer research ,medicine ,Molecular Medicine ,Immunology and Allergy ,Biology ,medicine.disease - Abstract
BackgroundWithin the tumor microenvironment, distinct CD4+ T cell subsets can play different and even opposite roles either promoting or suppressing anti-tumor responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules. However, how cancers co-opt these processes to shape the intratumoral CD4+ landscape and achieve immune evasion remains incompletely understood.MethodsWe performed single-cell characterization of CD4+ tumor infiltrating lymphocytes (TILs) collected from four human melanoma with low or high HLA-class II expression and we utilized TCR reconstruction and antigen specificity screening to unambiguously discover the tumor reactivity of CD4+ TILs. By testing TCR-transduced T cells against autologous patient-derived melanoma cell lines or against autologous antigen presenting cells (APCs) loaded with tumor lysates, we assessed the capacity of CD4+ TCRs to directly or indirectly recognize tumor cells. We defined the antigen-specificity of antitumor CD4+ TCRs by assessing their reactivity towards personal neoantigens (NeoAg) or public melanoma associated antigens (MAAs). Finally, we correlated NeoAg burden and HLA-class II expression in a series of 116 melanoma specimens from 4 independent cohorts of patients.ResultsAnalysis of single-cell data showed that the cluster distribution of cells within each CD4+ TCR clonotype family was highly homogeneous and appeared to follow 3 distinct major phenotypes, corresponding to non-exhausted memory cells, exhausted cells and regulatory cells (TRegs). Strikingly, clonally expanded CD4+ TReg-TILs were highly abundant within the tumor microenvironment of HLA class IIpos melanomas. We found that TCRs from exhausted cytotoxic CD4+ T cells could be directly triggered by melanoma cells not only through recognition of HLA class II restricted antigens, but also through presentation of HLA class I restricted MAAs. TReg-TCRs could be indirectly elicited through presentation of tumor antigens via APCs. Notably, numerous tumor-reactive CD4+ TReg-TCRs were directly stimulated by HLA class IIpos melanoma and demonstrated specificity for melanoma NeoAgs. In HLA class IIpos melanomas, the clonal expansion of numerous tumor-reactive and NeoAg-specific TRegs-clones appeared to be favored by a dramatically high tumor NeoAg load. Analysis of 116 melanoma specimens confirmed the association of elevated HLA-class II expression with extremely high NeoAg burden.ConclusionsOur data elucidate the landscape of infiltrating CD4+ T cells in melanoma and point to presentation of HLA-class II restricted NeoAgs and direct engagement of immunosuppressive CD4+ TRegs as a novel mechanism of immune evasion favored in HLA class IIpos melanoma.
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- 2021
10. Immune signature drives leukemia escape and relapse after hematopoietic cell transplantation
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Leo Luznik, Luca Vago, Dietrich W. Beelen, Elisa Montaldo, Matteo Barcella, Robert Zeiser, Bernhard Gentner, Gabriele Bucci, Raynier Devillier, Renato Ostuni, Matteo Carrabba, Masahiro Onozawa, Valentina Gambacorta, Orietta Spinelli, Miguel Waterhouse, Katharina Fleischhauer, Elia Stupka, Ivana Gojo, Chiara Bonini, Cristina Toffalori, Lara Crucitti, Laura Zito, Raffaella Greco, Michela Riba, Matteo Maria Naldini, Dejan Lazarevic, Massimo Bernardi, Maddalena Noviello, Davide Cittaro, Takanori Teshima, Didier Blaise, Jacopo Peccatori, Cristina Barlassina, Francesco Manfredi, Giovanni Tonon, Giacomo Oliveira, Alessandro Rambaldi, Constantijn J.M. Halkes, Marieke Griffioen, Maher Hanoun, Nicoletta Cieri, Fabio Ciceri, Jürgen Finke, Toffalori, C., Zito, L., Gambacorta, V., Riba, M., Oliveira, G., Bucci, G., Barcella, M., Spinelli, O., Greco, R., Crucitti, L., Cieri, N., Noviello, M., Manfredi, F., Montaldo, E., Ostuni, R., Naldini, M. M., Gentner, B., Waterhouse, M., Zeiser, R., Finke, J., Hanoun, M., Beelen, D. W., Gojo, I., Luznik, L., Onozawa, M., Teshima, T., Devillier, R., Blaise, D., Halkes, C. J. M., Griffioen, M., Carrabba, M. G., Bernardi, M., Peccatori, J., Barlassina, C., Stupka, E., Lazarevic, D., Tonon, G., Rambaldi, A., Cittaro, D., Bonini, C., Fleischhauer, K., Ciceri, F., Vago, L., Toffalori, C, Zito, L, Gambacorta, V, Riba, M, Oliveira, G, Bucci, G, Barcella, M, Spinelli, O, Greco, R, Crucitti, L, Cieri, N, Noviello, M, Manfredi, F, Montaldo, E, Ostuni, R, Naldini, M, Gentner, B, Waterhouse, M, Zeiser, R, Finke, J, Hanoun, M, Beelen, D, Gojo, I, Luznik, L, Onozawa, M, Teshima, T, Devillier, R, Blaise, D, Halkes, C, Griffioen, M, Carrabba, M, Bernardi, M, Peccatori, J, Barlassina, C, Stupka, E, Lazarevic, D, Tonon, G, Rambaldi, A, Cittaro, D, Bonini, C, Fleischhauer, K, Ciceri, F, and Vago, L
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0301 basic medicine ,Myeloid ,medicine.medical_treatment ,Antigen presentation ,Medizin ,Reproducibility of Result ,Hematopoietic stem cell transplantation ,Lymphocyte Activation ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Recurrence ,hemic and lymphatic diseases ,medicine ,Humans ,Transplantation, Homologous ,RNA, Messenger ,Transplantation, Homologou ,business.industry ,Gene Expression Regulation, Leukemic ,Gene Expression Profiling ,Histocompatibility Antigens Class II ,Hematopoietic Stem Cell Transplantation ,Reproducibility of Results ,Myeloid leukemia ,General Medicine ,medicine.disease ,Transplantation ,Haematopoiesis ,Leukemia ,Leukemia, Myeloid, Acute ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Cancer research ,business ,CD80 ,Human - Abstract
Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-gamma or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.
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- 2019
11. Bone marrow central memory and memory stem T-cell exhaustion in AML patients relapsing after HSCT
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Masahiro Onozawa, Francesco Manfredi, Marieke Griffioen, Filippo Cortesi, Martin Bornhäuser, Luca Vago, Valentina Gambacorta, Chiara Bonini, Cristina Toffalori, Monica Casucci, Jhf Falkenburg, Raffaella Greco, Giacomo Oliveira, Tommaso Perini, Miguel Waterhouse, Maddalena Noviello, Fabio Ciceri, Jürgen Finke, Takanori Teshima, Constantijn J.M. Halkes, Robert Zeiser, Paolo Dellabona, Giulia Casorati, Pantaleo De Simone, Eliana Ruggiero, Heidi Altmann, Friedrich Stölzel, Attilio Bondanza, Nicoletta Cieri, Jacopo Peccatori, Noviello, M., Manfredi, F., Ruggiero, E., Perini, T., Oliveira, G., Cortesi, F., De Simone, P., Toffalori, C., Gambacorta, V., Greco, R., Peccatori, J., Casucci, M., Casorati, G., Dellabona, P., Onozawa, M., Teshima, T., Griffioen, M., Halkes, C. J. M., Falkenburg, J. H. F., Stolzel, F., Altmann, H., Bornhauser, M., Waterhouse, M., Zeiser, R., Finke, J., Cieri, N., Bondanza, A., Vago, L., Ciceri, F., Bonini, C., Noviello, M, Manfredi, F, Ruggiero, E, Perini, T, Oliveira, G, Cortesi, F, De Simone, P, Toffalori, C, Gambacorta, V, Greco, R, Peccatori, J, Casucci, M, Casorati, G, Dellabona, P, Onozawa, M, Teshima, T, Griffioen, M, Halkes, C, Falkenburg, J, Stolzel, F, Altmann, H, Bornhauser, M, Waterhouse, M, Zeiser, R, Finke, J, Cieri, N, Bondanza, A, Vago, L, Ciceri, F, and Bonini, C
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0301 basic medicine ,Male ,Myeloid ,T-Lymphocytes ,medicine.medical_treatment ,Programmed Cell Death 1 Receptor ,General Physics and Astronomy ,02 engineering and technology ,Hematopoietic stem cell transplantation ,Antineoplastic Agent ,Receptors, KIR ,Recurrence ,Retrospective Studie ,hemic and lymphatic diseases ,CTLA-4 Antigen ,lcsh:Science ,Hepatitis A Virus Cellular Receptor 2 ,Transplantation, Homologou ,Multidisciplinary ,Clonal anergy ,Gene Expression Regulation, Leukemic ,Remission Induction ,Hematopoietic Stem Cell Transplantation ,Myeloid leukemia ,Middle Aged ,021001 nanoscience & nanotechnology ,Lymphocyte Activation Gene 3 Protein ,3. Good health ,Leukemia ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Bone Marrow Cell ,Female ,0210 nano-technology ,Human ,Signal Transduction ,Adult ,T cell ,Science ,Antineoplastic Agents ,Bone Marrow Cells ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Antigens, CD ,Signaling Lymphocytic Activation Molecule Family ,Glucocorticoid-Induced TNFR-Related Protein ,medicine ,Humans ,Transplantation, Homologous ,Retrospective Studies ,Clonal Anergy ,business.industry ,General Chemistry ,medicine.disease ,Transplantation ,030104 developmental biology ,T-Lymphocyte ,Immunology ,lcsh:Q ,Bone marrow ,business ,Immunologic Memory - Abstract
The major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors (IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (TSCM) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1+Eomes+T-bet−) BM-TSCM predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease., Allogeneic hematopoietic cell transplantation is the standard treatment of acute myeloid leukemia, but many patients relapse. Here the authors show increased markers of exhaustion and cancer antigen specificity within bone marrow-residing memory T cells precede and potentially predict the relapse.
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- 2019
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12. Acquired mechanisms of immune escape in cancer following immunotherapy
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Giacomo Oliveira, J. Bryan Iorgulescu, David A. Braun, Derin B. Keskin, and Catherine J. Wu
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0301 basic medicine ,Antigenicity ,lcsh:QH426-470 ,medicine.medical_treatment ,Antigen presentation ,lcsh:Medicine ,chemical and pharmacologic phenomena ,03 medical and health sciences ,Neoplasms ,Genetics ,medicine ,Humans ,Molecular Biology ,Genetics (clinical) ,Antigen Presentation ,business.industry ,Immunogenicity ,lcsh:R ,Comment ,Immune escape ,Cancer ,Immunotherapy ,medicine.disease ,3. Good health ,Clinical trial ,lcsh:Genetics ,030104 developmental biology ,Tumor Escape ,Immunology ,Molecular Medicine ,business - Abstract
Immunotherapy has revolutionized the management of numerous cancers; however, a substantial proportion that initially respond subsequently acquire means of immune escape and relapse. Analysis of recent clinical trials permits us to preliminarily understand how immunotherapies exert evolutionary pressures: selecting cancer subclones deficient in antigenicity and/or immunogenicity, thereby facilitating immune escape.
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- 2018
13. CMV-Specific T Cells Restricted By Shared and Donor, but Not By Host HLA Molecules Reconstitute in the First 180 Days after Allogeneic HSCT and Protect from CMV Reactivation: Results of a Prospective Observational Study
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Fabio Ciceri, Massimo Bernardi, Elena Tassi, Maddalena Noviello, Liselotte Brix, Daniela Clerici, Jacopo Peccatori, Lorenzo Lazzari, Francesca Serio, Chiara Bonini, Fabio Giglio, Consuelo Corti, Roee Dvir, Maria Teresa Lupo Stanghellini, Raffaella Greco, Sara Racca, Pantaleo De Simone, Giacomo Oliveira, and Francesca Lorentino
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education.field_of_study ,biology ,business.industry ,medicine.medical_treatment ,CD3 ,T cell ,Immunology ,Population ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,Biochemistry ,Transplantation ,medicine.anatomical_structure ,Cord blood ,biology.protein ,Medicine ,business ,education ,CD8 - Abstract
Introduction: Cytomegalovirus (CMV) reactivation and disease are important risk factors after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and strongly affect morbidity and mortality after transplant. CMV-specific T cell reconstitution controls CMV reactivation and protects against serious adverse events but a protective level of CMV-specific T cell response or standardized method for its monitoring have not been yet determined. Methods: We designed a prospective, single-center observational study to assess if the kinetic and quality of CMV specific T-cell reconstitution impact the incidence and severity of CMV reactivations. We have enrolled 84 consecutive patients affected by hematological malignancies receiving allo-HSCT followed by Cyclophosphamide and Rapamycin between December 2017 and February 2019. Here we report preliminary data on the first 61 patients. Patients received allo-HSCT from family (siblings=10; HLA haploidentical=24), unrelated HLA-matched (n= 24) donors or cord blood (CB, n=3). The CMV serostatus of host (H) and donor (D) pairs was: H+/D+(n=40, 65%), H+/D-(n=20, 33%) and H-/D+ (n=1, 2%); H-/D-(7% of the overall transplanted population at our center) were excluded. CMV DNAemia was assessed weekly in whole blood (WB). Absolute numbers of polyclonal and CMV-specific T cells were quantified by flow cytometry using Troucount™Tubes (BD) and Dextramer®CMV-Kit (Immudex), respectively, in the graft and fresh WB at days -7, +30, +45, +60, +90, +120, +150, +180 and +360. Dextramer CMV kit includes reagents for the identification of CMV-specific lymphocytes restricted for several HLA class I molecules: A*01:01/*02:01/*03:01/*24:02 and B*07:02/*08:01/*35:01. These alleles allowed the longitudinal evaluation of 54 out of 61 (89%) patients. Results: At a median follow-up of 226 days post-HSCT, 31 (57%) patients experienced a CMV-related clinically relevant event (CRE, median +63 days), including 8 patients (15%) with CMV disease (median +59 days). Univariate analyses showed that the incidence of CMV clinically-relevant reactivation (CRE) was influenced by H/D CMV serostatus (0.90 in H+/D- versus 0.44 in H+/D+pairs, p=0.015) and by previous acute Graft-versus-Host Disease (aGvHD) requiring systemic immunosuppression (0.82 in aGvHD grade II-IV versus 0.52 in aGvHD grade 0-I, p=0.051). The disease status at transplant, the donor type (HLA-matched versus HLA-haploidentical/CB donors), donor's or host's age did not significantly affect the probability to develop CRE. For each time-point, we compared the absolute number of CMV-specific lymphocytes in patients experiencing or not a subsequent CRE. Our data demonstrate that higher levels of CMV-specific CD8+T cells in the donor apheresis and at +45 days after allo-HSCT are associated with reduced risk of subsequent CRE (median CMV-specific CD8+cells/kg in the apheresis=5x103in CRE-positive patients (CRE+) and 5x105in CRE-negative patients (CRE-), p=0.012; median CMV-specific CD8+at +45 days=0.14 cells/μL in CRE+and 1.21 cells/μL in CRE-, p=0.034). Furthermore, patients with any Dextramer positivity at +45 days displayed a lower incidence of CRE compared with subjects who were negative (CRE probability: 0.5 vs 1.0, p=0.003). Conversely, the absolute number of neither polyclonal CD3+CD8+T lymphocytes nor total CD3+T cells correlate with subsequent CRE. Taking advantage of the HLA mismatched-HSCT setting, we then dissected CMV-specific T-cell response according to HLA restriction elements (H/D=shared n=45, D-restricted n=14, H-restricted n=11). In H+/D+pairs, we observed a fast and similar kinetic of reconstitution of CMV-specific lymphocytes restricted by H/D and D HLAs. Conversely, in H+/D-pairs, we detected only CMV-specific CD8+lymphocytes restricted for H/D haplotypes. Host-restricted cells remained undetectable for the first 180 days after HSCT. Conclusion: Early after allo-HSCT and in the donor apheresis, the level of CMV-specific CD8+T cells measured by Dextramer staining differs in patients experiencing or not subsequent CRE. Furthermore, our findings indicate that CMV reactivations can prime H/D-restricted T cells presumably educated in the donor thymus; conversely, D- and H-restricted donor-derived lymphocytes have not yet undergone neither cross-priming nor thymic education respectively. Disclosures Brix: Immudex: Employment. Bonini:Kite/Gilead: Consultancy; Intellia Therapeutics: Consultancy; Intellia Therapeutics: Research Funding; Novartis: Consultancy; GSK: Consultancy; Allogene: Consultancy; Molmed: Consultancy; TxCell: Consultancy; -: Patents & Royalties: Adoptive T cell therapy field.
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- 2019
14. T Cell Determinants of Response and Resistance to PD-1 Blockade in Richter's Transformation
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Aina Zurita Martinez, Donna Neuberg, William G. Wierda, Lars Rønn Olsen, Gad Getz, Nitin Jain, Peter V. Kharchenko, Shuqiang Li, Kenneth Livak, Ana Lako, Scott J. Rodig, Catherine J. Wu, Camilla Koldbæk Lemvigh, Ignaty Leshchiner, Giacomo Oliveira, Erin M. Parry, Lillian Werner, Annabelle J. Anandappa, Pavan Bachireddy, Noelia Purroy, Satyen H. Gohil, and Teddy Huang
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Chemistry ,T cell ,Chronic lymphocytic leukemia ,Immunology ,T-cell receptor ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Richter's transformation ,medicine.anatomical_structure ,Cancer research ,medicine ,Pd 1 blockade ,Bone marrow specimen ,health care economics and organizations - Abstract
Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the transformation of CLL to an aggressive lymphoma, or Richter's transformation (RT), remains a clinical challenge, as it responds poorly to standard therapies and shortens survival. Recent studies demonstrate that RT, but not underlying CLL, responds to PD-1 checkpoint blockade (CPB) with an overall response rate of 43-65%. Given the central role of T cells in anti-tumor immunity, we hypothesized that differences in T cell populations underlie response and resistance to CPB in RT. We focused on a discovery cohort of 6 patients with RT (4 responders, 2 non-responders) and 2 patients with relapsed/refractory CLL enrolled on a study in which patients were initiated with anti-PD1 therapy (nivolumab 3 mg/kg every 2 weeks), with subsequent concurrent ibrutinib (420 mg daily)(NCT 02420912). We examined a total of 15 serial study marrow specimens collected at treatment initiation and 3 month response evaluation, as well as 2 healthy marrow donors. To systematically discover the T cell populations and states associated with CPB response in RT, we performed single-cell RNA-sequencing (scRNA-seq, 10x Genomics) of non-lymphoma (CD5-CD19-) cells isolated by flow cytometry from marrow samples. A total of 60,727 T and NK cells were captured with average detection of 1001 genes/cell. Using the novel joint clustering approach Conos, 11 transcriptionally distinct clusters of lymphocytes were identified. We first contrasted baseline RT/CLL with normal marrow and observed differences across T cell populations, which we confirmed through the examination of publicly available marrow scRNA-seq data from 28 healthy donors. Compared to normal marrow, RT/CLL marrow was enriched for cytotoxic populations, including both CD8 effector/effector memory (E/EM) (p=0.001, t-test) and cytotoxic CD4 (p=0.001) T cells as well as for cells expressing multiple exhaustion markers, including PDCD1, LAG3 and TIGIT (p=0.001). In contrast, normal marrow contained increased T cells with a naïve-like phenotype (p=0.06). When we focused on the pre-treatment samples from RT patients, RT responders had a larger CD8 E/EM population (p=0.04) and fewer T regulatory cells (p=0.006, t-test) than RT non-responders. Using DESeq2 to compare clusters from all samples, we evaluated if there were differences in gene expression between RT responders and non-responders. CD8 E/EM T cells of RT non-responders showed increased expression of TOX, a recently uncovered master regulator of cell exhaustion (padj =0.00016), while this cell subtype in RT responders upregulated a contrasting program of activating transcription factors as well as the co-stimulatory gene CD226 (padj =0.04). As for CD4 T cells, RT responders revealed an enriched cytotoxic gene program compared to RT non-responders (padjPRF1 5.9 x 10-10, GZMH 6.0 x 10-6, NKG7 6.4 x 10-19). To investigate whether response to CPB therapy for RT was associated with changes in the T cell receptor (TCR) repertoire, and to obtain protein-level validation of transcriptional signatures, we performed single-cell TCR sequencing with paired gene and protein expression (10x Genomics) on pre- and post-therapy samples from a RT responder and a non-responder. Indeed, we confirmed our gene expression findings, including validation of cytotoxic CD4 T cells and the enrichment of CD226 protein in E/EM CD8 T cells in the RT responder. TCR clonal expansion was observed in the RT responder at baseline with persistence of enriched clonotypes following CPB, suggesting the presence of tumor-reactive T cell clones. In contrast, the RT non-responder displayed higher TCR diversity with enriched clonotypes showing increased exhaustion post-CPB (p In conclusion, we identified marrow T cell populations enriched in RT patients and described distinct T cell transcriptional programs that delineate RT responders from non-responders. We have thus discovered candidate gene biomarkers that may identify patients likely to respond to CPB therapies and uncovered a CD8 E/EM T cell population that is likely to underlie response to PD-1 CPB. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Neuberg:Celgene: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Pharmacyclics: Research Funding. Rodig:Bristol Myers Squib: Consultancy, Honoraria, Other: Travel Expenses, Speakers Bureau; Kite, a Gilead Company: Research Funding; Affirmed: Research Funding; Merck: Research Funding. Wierda:KITE pharma: Research Funding; Gilead Sciences: Research Funding; AbbVie: Research Funding; Acerta Pharma Inc: Research Funding; Genentech: Research Funding; GSK/Novartis: Research Funding; Pharmacyclics LLC: Research Funding; Sunesis: Research Funding; Miragen: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Cyclcel: Research Funding; Loxo Oncology Inc.: Research Funding; Janssen: Research Funding; Xencor: Research Funding; Juno Therapeutics: Research Funding. Jain:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.
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- 2019
15. NK cell recovery after haploidentical HSCT with posttransplant cyclophosphamide: Dynamics and clinical implications
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Maria Teresa Lupo Stanghellini, Raffaella Greco, Sofia Berglund, Leo Luznik, Francesca Lorentino, Giacomo Oliveira, Fabio Giglio, Andrea Assanelli, Mara Morelli, Valentina Gambacorta, Simona Piemontese, Jacopo Peccatori, Antonio Russo, Luca Vago, Nicoletta Cieri, Fabio Ciceri, Chiara Bonini, Cristina Toffalori, Laura Zito, Russo, Antonio, Oliveira, Giacomo, Berglund, Sofia, Greco, Raffaella, Gambacorta, Valentina, Cieri, Nicoletta, Toffalori, Cristina, Zito, Laura, Lorentino, Francesca, Piemontese, Simona, Morelli, Mara, Giglio, Fabio, Assanelli, Andrea, Stanghellini, Maria Teresa Lupo, Bonini, Chiara, Peccatori, Jacopo, Ciceri, Fabio, Luznik, Leo, Vago, Luca, Russo, A, Oliveira, G, Berglund, S, Greco, R, Gambacorta, V, Cieri, N, Toffalori, C, Zito, L, Lorentino, F, Piemontese, S, Morelli, M, Giglio, F, Assanelli, A, Stanghellini, M, Bonini, C, Peccatori, J, Ciceri, F, Luznik, L, and Vago, L
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medicine.medical_specialty ,Cyclophosphamide ,medicine.medical_treatment ,Cell ,Immunology ,Hematopoietic stem cell transplantation ,Biology ,Biochemistry ,03 medical and health sciences ,Interleukin 21 ,0302 clinical medicine ,Internal medicine ,medicine ,Transplantation ,Hematology ,Hematopoietic Stem Cell Transplantation ,Cell Biology ,Killer Cells, Natural ,Haematopoiesis ,surgical procedures, operative ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Stem cell ,030215 immunology ,medicine.drug - Abstract
The use of posttransplant cyclophosphamide (PT-Cy) as graft-versus-host disease (GVHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (HSCT), allowing safe infusion of unmanipulated T cell-replete grafts. PT-Cy selectively eliminates proliferating alloreactive T cells, but whether and how it affects natural killer (NK) cells and their alloreactivity is largely unknown. Here we characterized NK cell dynamics in 17 patients who received unmanipulated haploidentical grafts, containing high numbers of mature NK cells, according to PT-Cy-based protocols in 2 independent centers. In both series, we documented robust proliferation of donor-derived NK cells immediately after HSCT. After infusion of Cy, a marked reduction of proliferating NK cells was evident, suggesting selective purging of dividing cells. Supporting this hypothesis, proliferating NK cells did not express aldehyde dehydrogenase and were killed by Cy in vitro. After ablation of mature NK cells, starting from day 15 after HSCT and favored by the high levels of interleukin-15 present in patients' sera, immature NK cells (CD62L+NKG2A+KIR-) became highly prevalent, possibly directly stemming from infused hematopoietic stem cells. Importantly, also putatively alloreactive single KIR+ NK cells were eliminated by PT-Cy and were thus decreased in numbers and antileukemic potential at day 30 after HSCT. As a consequence, in an extended series of 99 haplo-HSCT with PT-Cy, we found no significant difference in progression-free survival between patients with or without predicted NK alloreactivity (42% vs 52% at 1 year, P = NS). Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are lost upon PT-Cy administration, blunting NK cell alloreactivity in this transplantation setting.
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- 2018
16. Immune monitoring in allogeneic hematopoietic stem cell transplant recipients: a survey from the EBMT-CTIWP
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Fabio Ciceri, Jacopo Peccatori, Giacomo Oliveira, Jürgen Kuball, Maddalena Noviello, Ulrike Koehl, Antoine Toubert, Ebmt Cellular Therapy, Chiara Bonini, Katharina Fleischhauer, Christian Chabannon, Nicoletta Cieri, Francesco Dazzi, Attilio Bondanza, Dirk-Jan Eikema, Vanderson Rocha, Raffaella Greco, Steffie van der Werf, Annalisa Ruggeri, Luca Vago, Sofie Rosanne Terwel, Greco, Raffaella, Ciceri, Fabio, Noviello, Maddalena, Bondanza, Attilio, Vago, Luca, Oliveira, Giacomo, Peccatori, Jacopo, Cieri, Nicoletta, Ruggeri, Annalisa, Koehl, Ulrike, Fleischhauer, Katharina, Rocha, Vanderson, Dazzi, Francesco, van der Werf, Steffie Maria, Eikema, Dirk-Jan, Terwel, Sofie Rosanne, Kuball, Jürgen, Toubert, Antoine, Chabannon, Christian, and Bonini, Chiara
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0301 basic medicine ,Oncology ,medicine.medical_specialty ,Medizin ,MEDLINE ,Immune monitoring ,03 medical and health sciences ,0302 clinical medicine ,Monitoring, Immunologic ,Internal medicine ,Surveys and Questionnaires ,medicine ,Humans ,Transplantation, Homologous ,Transplantation ,Hematology ,business.industry ,Hematopoietic Stem Cell Transplantation ,Transplant Recipients ,030104 developmental biology ,Multicenter study ,Transplantation, Haploidentical ,Allogeneic hematopoietic stem cell transplant ,business ,Unrelated Donors ,030215 immunology - Published
- 2018
17. Exhausted Central Memory and Memory Stem T Cells Specific for Leukemia Infiltrate the Bone Marrow of AML Patients Relapsing after Allogeneic HSCT
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Giulia Casorati, Raffaella Greco, Nicoletta Cieri, Pantaleo De Simone, Eliana Ruggiero, Jacopo Peccatori, Luca Vago, Valentina Gambacorta, Attilio Bondanza, Paolo Dellabona, Tommaso Perini, Giacomo Oliveira, Maddalena Noviello, Francesco Manfredi, Monica Casucci, Fabio Ciceri, Filippo Cortesi, Chiara Bonini, and Cristina Toffalori
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business.industry ,medicine.medical_treatment ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Leukemia ,medicine.anatomical_structure ,Graft-versus-host disease ,Aldesleukin ,medicine ,Cytotoxic T cell ,Bone marrow ,business ,CD8 - Abstract
Background. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML); nonetheless, relapse remains the major cause of death after such therapeutic option. Patients and Methods . We investigated the expression of Inhibitory Receptors (IR; i.e. PD-1, CTLA-4, TIM-3, LAG-3 and KLRG1) on different T-cell subsets infiltrating the bone marrow (BM) of 8 healthy donors (HD) and 32 allogeneic HSCT recipients diagnosed with Acute Myeloid Leukemia, collected at relapse (median 251 days) or at complete remission (CR) 1 year after HSCT. Inclusion criteria were: a diagnosis of acute myeloid leukemia or myelodysplastic syndrome, a relapse-free survival of at least 4 months after allogenein HSCT, absence of active GvHD, CMV infections or other complications at the time of sampling. Samples were analysed by multi-parametric flow cytometry for the expression of inhibitory receptors on T-cell subsets and the results were validated with BH-SNE, an unbiased dimensionality reduction algorithm. We exploited HLA-mimicking fluorescent molecules loaded with a specific epitope to screen anti-tumour and anti-viral T cells whereas the T-cell receptor repertoire was assessed by TRAC and TRBC RNA sequencing and the relative frequency of each T-cell receptor calculated. To evaluate T-cell function and specificity, CD107a expression, cytokine profiles and killing of autologous blasts were quantified. Results. After Haploidentical-HSCT PD-1, CTLA-4, 2B4 and Tim-3 were expressed at higher percentage when compared to HD, independently from the clinical outcome. In contrast, after HLA-matched HSCT, patients who relapsed displayed a higher frequency of BM-infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To gain insights on the inhibited T-cell subpopulation identified in the BM of relapsing patients, we separately profiled the different T-cell memory subsets: in both HD and CR patients the IR expression was confined to effector memory and effectors whereas at relapse PD-1, 2B4, KLRG1 and Tim-3 were also expressed in BM-infiltrating central memory (TCM) and memory stem T cells (TSCM, p Interestingly, the TCR repertoire of BM-infiltrating T cells at relapse displayed a restricted clonality, suggesting that immune inhibitory signals are active on discrete and specific T-cell clones. To gain further insights on such clones, we assessed the IR expression profile on CD8 T cells specific for viral (CMV) and tumor-associated antigens (including peptides from WT1, EZH2 and PRAME). We observed a higher IR expression and co-expression on tumor-specific T cells when compared to viral-specific CD8 cells, particularly in case of patients who experienced post-transplant relapse. In accordance, IRpos sorted T cells harvested from relapsing patients showed a restricted TCR repertoire and, when challenged with autologous leukemic blasts, proved enriched in leukemic specificities as shown by higher expression of the activation marker HLA-DR (p Conclusion. These results highlight a wide, yet reversible, immunological dysfunction likely mediated by AML blasts in the BM of patients relapsing after allogeneic HSCT, that is particularly evident on memory T cells specific for tumor antigens. This suggest and open new therapeutic opportunities for AML. Figure. Figure. Disclosures Bondanza: Novartis: Employment. Vago:GENDX: Research Funding; Moderna TX: Research Funding. Bonini:Intellia Therapeutics: Research Funding.
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- 2018
18. Integrating a prospective pilot trial and patient-derived xenografts to trace metabolic changes associated with acute myeloid leukemia
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Alessandra Forcina, Laurette Tavel, Giacomo Oliveira, Matteo Carrabba, Fabio Ciceri, Cristina Tresoldi, Giovanna Musco, Alessandro Ambrosi, Giacomo Quilici, Luca Vago, Massimo Bernardi, Francesca Nardelli, Carrabba, Matteo G., Tavel, Laurette, Oliveira, Giacomo, Forcina, Alessandra, Quilici, Giacomo, Nardelli, Francesca, Tresoldi, Cristina, Ambrosi, Alessandro, Ciceri, Fabio, Bernardi, Massimo, Vago, Luca, and Musco, Giovanna
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0301 basic medicine ,Oncology ,Cancer Research ,medicine.medical_specialty ,Magnetic Resonance Spectroscopy ,Systems biology ,Metabolomic ,Pilot Projects ,Disease ,lcsh:RC254-282 ,Nuclear magnetic resonance ,Genetic Heterogeneity ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Metabolomics ,Patient-derived xenograft ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Animals ,Humans ,Longitudinal Studies ,Prospective Studies ,Letter to the Editor ,Molecular Biology ,Acute myeloid leukemia ,Hematology ,lcsh:RC633-647.5 ,business.industry ,Genetic heterogeneity ,Myeloid leukemia ,lcsh:Diseases of the blood and blood-forming organs ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,medicine.disease ,Clinical trial ,Patient-derived xenografts ,Leukemia, Myeloid, Acute ,Leukemia ,030104 developmental biology ,030220 oncology & carcinogenesis ,Immunology ,Heterografts ,business - Abstract
Despite the considerable progress in understanding the molecular bases of acute myeloid leukemia (AML), new tools to link disease biology to the unpredictable patient clinical course are still needed. Herein, high-throughput metabolomics, combined with the other “-omics” disciplines, holds promise in identifying disease-specific and clinically relevant features. In this study, we took advantage of nuclear magnetic resonance (NMR) to trace AML-associated metabolic trajectory employing two complementary strategies. On the one hand, we performed a prospective observational clinical trial to identify metabolic changes associated with blast clearance during the first two cycles of intensive chemotherapy in nine adult patients. On the other hand, to reduce the intrinsic variability associated with human samples and AML genetic heterogeneity, we analyzed the metabolic changes in the plasma of immunocompromised mice upon engraftment of primary human AML blasts. Combining the two longitudinal approaches, we narrowed our screen to seven common metabolites, for which we observed a mirror-like trajectory in mice and humans, tracing AML progression and remission, respectively. We interpreted this set of metabolites as a dynamic fingerprint of AML evolution. Overall, these NMR-based metabolomic data, to be consolidated in larger cohorts and integrated in more comprehensive system biology approaches, hold promise for providing valuable and non-redundant information on the systemic effects of leukemia. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0346-2) contains supplementary material, which is available to authorized users.
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- 2016
19. T-cell suicide gene therapy prompts thymic renewal in adults after hematopoietic stem cell transplantation
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Giacomo Oliveira, Chiara Bonini, Domenico Ghio, Catia Traversari, Sergio Fracchia, Alessandro Del Maschio, Alessandro Aiuti, Fabio Ciceri, Claudio Bordignon, Corrado Soldati, Jacopo Peccatori, Luca Vago, Maria Teresa Lupo Stanghellini, Raffaella Greco, Attilio Bondanza, Matteo Del Fiacco, Maddalena Noviello, Immacolata Brigida, Vago, L, Oliveira, G, Bondanza, Attilio, Noviello, M, Soldati, C, Ghio, D, Brigida, I, Greco, R, Stanghellini Mt, Lupo, Peccatori, J, Fracchia, S, Del Fiacco, M, Traversari, C, Aiuti, Alessandro, DEL MASCHIO, Alessandro, Bordignon, Claudio, Ciceri, Fabio, and Bonini, MARIA CHIARA
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Adult ,T-Lymphocytes ,T cell ,medicine.medical_treatment ,Immunology ,Recent Thymic Emigrant ,Gene Expression ,Thymus Gland ,Hematopoietic stem cell transplantation ,Thymidine Kinase ,Biochemistry ,Interleukin 21 ,Immune system ,medicine ,Humans ,Regeneration ,Lymphocyte Count ,Prospective Studies ,Reverse Transcriptase Polymerase Chain Reaction ,business.industry ,Interleukin-7 ,T-cell receptor ,Genes, Transgenic, Suicide ,Hematopoietic Stem Cell Transplantation ,Genetic Therapy ,Cell Biology ,Hematology ,Suicide gene ,Combined Modality Therapy ,Thymic Tissue ,Treatment Outcome ,medicine.anatomical_structure ,Hematologic Neoplasms ,Radiography, Thoracic ,Tomography, X-Ray Computed ,business - Abstract
The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+-cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31(+) recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution. (Blood. 2012;120(9):1820-1830) The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially-incompatible Hematopoietic Stem Cell Transplantation (HSCT). In the TK007 clinical trial 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the Herpes Simplex Virus Thymidine Kinase suicide gene (TK(pos) cells). After a first wave of circulating TK(pos) cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naïve lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK(pos) cell engraftment. Accordingly, after the infusions we documented an increase in circulating T cell Receptor Excision Circles and CD31+ recent thymic emigrants, and a substantial expansion of the active thymic tissue at chest tomography scans. Interestingly, a peak in the serum level of interleukin-7 was observed after each infusion of TK(pos) cells, anticipating the appearance of newly generated T cells. Taken together, our data show that the infusion of genetically modified donor T cells after transplantation can drive the recovery of thymic activity in adults, leading to immune reconstitution.
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- 2012
20. Erratum for the Research Article: 'Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory' by G. Oliveira, E. Ruggiero, M. T. L. Stanghellini, N. Cieri, M. D’Agostino, R. Fronza, C. Lulay, F. Dionisio, S. Mastaglio, R. Greco, J. Peccatori, A. Aiuti, A. Ambrosi, L. Biasco, A. Bondanza, A. Lambiase, C. Traversari, L. Vago, C. von Kalle, M. Schmidt, C. Bordignon, F. Ciceri, C. Bonini
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Chiara Bonini, Maria Teresa Lupo Stanghellini, C. Von Kalle, Raffaella Greco, Francesca Dionisio, Raffaele Fronza, Sara Mastaglio, Eliana Ruggiero, Catia Traversari, Christina Lulay, Fabio Ciceri, Attilio Bondanza, Mattia D'Agostino, Nicoletta Cieri, Alessandro Ambrosi, Luca Biasco, Manfred G. Schmidt, Antonio Lambiase, Jacopo Peccatori, Claudio Bordignon, Luca Vago, Giacomo Oliveira, and Alessandro Aiuti
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medicine.anatomical_structure ,Genetically engineered ,T cell ,Dynamics (mechanics) ,Translational medicine ,medicine ,General Medicine ,Immunological memory ,Biology ,Neuroscience ,Term (time) - Published
- 2015
21. Safety and Efficacy of Donor T Cells Engineered with Herpes Simplex Virus Thymidine-Kinase Suicide Gene (TK Cells) Given after T-Cell Depleted (TCD) Haploidentical Hematopoietic Transplantation (Haplo-HSCT): Results of a 14-Year Follow-Up in 45 Patients
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Eva M Weissinger, Maria Teresa Lupo Stanghellini, Fabio Ciceri, Raffaella Greco, John F. DiPersio, Myriam Labopin, Evangelia Yannaki, Chiara Bonini, Giacomo Oliveira, Arnon Nagler, Claudio Bordignon, Michele Donato, Attilio Bondanza, Michael Stadler, Dietger Niederwieser, Wolfgang Bethge, Donald Bunjes, Andrew L. Pecora, Lutz Uharek, Antonio Lambiase, Mohamad Mohty, Eduardo Olavarria, and Scialini Colombi
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Transplantation ,business.industry ,T cell ,Hematology ,Suicide gene ,medicine.disease_cause ,Virology ,Haematopoiesis ,Herpes simplex virus ,medicine.anatomical_structure ,Thymidine kinase ,medicine ,business - Published
- 2017
22. Tracking genetically engineered lymphocytes long-term reveals the dynamics of t cell immunological memory
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Eliana Ruggiero, Catia Traversari, Antonio Lambiase, Attilio Bondanza, Alessandro Ambrosi, Luca Biasco, Maria Teresa Lupo Stanghellini, Manfred Schmidt, Alessandro Aiuti, Nicoletta Cieri, Raffaella Greco, Francesca Dionisio, Claudio Bordignon, Jacopo Peccatori, Mattia D'Agostino, Sara Mastaglio, Chiara Bonini, Christof von Kalle, Raffaele Fronza, Giacomo Oliveira, Christina Lulay, Fabio Ciceri, Luca Vago, Oliveira, G, Ruggiero, E, Lupo Stanghellini, Mt, Cieri, N, D’Agostino, M, Fronza, R, Lulay, C, Dionisio, F, Mastaglio, S, Greco, R, Peccatori, J, Aiuti, Alessandro, Ambrosi, Alessandro, Biasco, L, Bondanza, Attilio, Lambiase, A, Traversari, C, Vago, L, Von Kalle, C, Schmidt, M, Bordignon, Claudio, Ciceri, Fabio, and Bonini, MARIA CHIARA
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Male ,Time Factors ,T-Lymphocytes ,Biochemistry ,Transgenic ,Interleukin 21 ,Cytotoxic T cell ,IL-2 receptor ,Genes, Transgenic, Suicide ,Hematopoietic Stem Cell Transplantation ,Hematology ,General Medicine ,Middle Aged ,Acquired immune system ,Tissue Donors ,Suicide ,medicine.anatomical_structure ,Phenotype ,Cell Tracking ,Female ,Genetic Engineering ,Adult ,T cell ,Immunology ,Streptamer ,Biology ,Thymidine Kinase ,Lymphocyte Depletion ,immunological memory ,Young Adult ,Immune system ,Antigen ,medicine ,Humans ,Lymphocyte Count ,Antigens ,Antigen-presenting cell ,Aged ,Cell Proliferation ,Clone Cells ,Genetic Therapy ,Immunologic Memory ,Cell Biology ,Suicide gene ,Genes ,Memory T cell ,CD8 - Abstract
BACKGROUND: Long-term T-cell survival is pivotal for the development of effective therapeutic approaches against pathogens and cancer, since the success of immunotherapy requires the generation of a robust, safe but also durable immune response. Even if it is established that memory cells can survive and persist for years, little is known about the requirements for their long-term persistence. Suicide gene therapy after T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) provides a unique model to study memory T cells. In this setting, patients receive the post-transplant infusion of donor-derived gene-modified memory T lymphocytes retrovirally transduced to express the Herpes Simples Virus Thymidine Kinase (TK) suicide gene and the DLNGFR selection marker. The presence of a safety switch allows the infusion into patients of a broad T-cell repertoire in the absence of immune suppression, while the surface marker enables unambiguous detection and close monitoring of gene-modified cells circulating in treated patients. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the fate of persisting TK cells to shed light on memory T cell dynamics in vivo and to unravel the requirements for long-term persistence directly in humans. RESULTS: We studied 10 adult patients who underwent haplo-HSCT and infusion of suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:1-39.5x106) for high-risk hematologic malignancies between 1995 and 2012. Three out of 10 patients (33%) experienced GvHD early after HSCT; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. At a median follow-up of 7 years (range 2-14), all patients were in complete remission and free of GvHD, and displayed a complete and broad donor-derived immune system characterized by physiological counts of NK cells, B lymphocytes, γδ T cells and naïve and memory CD4+ or CD8+ T cells. TK cells were detected in all patients, at low levels (median=4cells/uL), even in patients treated with GCV. Ex vivo selection of pure TK-cells confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Importantly, GCV sensitivity was preserved in long-term persisting TK cells, independently from their differentiation phenotype. Longitudinal follow up revealed that TK cells circulated in patients at stable levels and displayed a conserved phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells. The low level of Ki-67 positivity suggested the maintenance of a pool of gene-modified memory cells through homeostatic proliferation. Polyclonality was demonstrated by sequencing among TK cells of thousands of diverse TCRs with a broad usage of V and J alpha and beta genes. The number of TK cells persisting at the longest follow-up did not correlate with the amount of infused cells, but instead with the peak of TK cells measured within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Accordingly, we documented the persistence of CMV and Flu-specific TK cells only after post-transplant CMV reactivation or after Flu infection. Characterization of TK cell products infused to patients showed that the amount of infused TSCM cells positively correlates with early expansion and long-term persistence of gene-marked cells. By combining sorting of memory T-cell subsets with sequencing of integration sites, TCRα and TCRβ clonal markers, we longitudinally traced T-cell clones from infused products to late follow-up time-points. We showed that although T cells retrieved long-term are enriched in clones originally shared in different memory T-cell subsets, dominant long-term clonotypes preferentially originate from infused TSCM clones, suggesting that TSCM might play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: In a completely restored immune system, suicide gene-modified donor T cells persist for up to 14 years in treated patients. Long-term persistence of memory T cells is determined by antigen exposure, and by the original phenotype of infused cells, according to a hierarchical model in which TSCM are superior to TCM and TEM/EFF. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Membership on an entity's Board of Directors or advisory committees. Bonini:MolMed S.p.A: Consultancy.
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- 2015
23. Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors
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Jacopo Peccatori, Sven Olek, Luca Vago, A Lorusso, Lara Crucitti, Francesco Locatelli, Massimo Bernardi, Consuelo Corti, Maddalena Noviello, M. Tassara, Giacomo Oliveira, Sarah Marktel, Andrea Assanelli, Daniela Clerici, Fabio Giglio, Matteo Carrabba, Alessandra Forcina, Elena Guggiari, Maria Grazia Roncarolo, Francesca Patriarca, Katharina Fleischhauer, Marco Zecca, Alessandro Crotta, Sara Mastaglio, Alessandra Ferraro, Chiara Messina, Chiara Bonini, Fabio Ciceri, Roberto Crocchiolo, Manuela Battaglia, Attilio Bondanza, Maria Rosaria Carbone, Claudio Bordignon, Francesca Lunghi, Magda Marcatti, Silvano Rossini, Maria Teresa Lupo Stanghellini, Peccatori, J, Forcina, A, Clerici, D, Crocchiolo, R, Vago, L, Stanghellini, Mt, Noviello, M, Messina, C, Crotta, A, Assanelli, A, Marktel, S, Olek, S, Mastaglio, S, Giglio, F, Crucitti, L, Lorusso, A, Guggiari, E, Lunghi, F, Carrabba, M, Tassara, M, Battaglia, MARCO MARIA, Ferraro, A, Carbone, Mr, Oliveira, G, Roncarolo, Mg, Rossini, S, Bernardi, M, Corti, C, Marcatti, M, Patriarca, F, Zecca, M, Locatelli, F, Bordignon, Claudio, Fleischhauer, K, Bondanza, Attilio, Bonini, MARIA CHIARA, and Ciceri, Fabio
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Male ,Cancer Research ,Transplantation Conditioning ,Neutrophils ,medicine.medical_treatment ,T-Lymphocytes ,Medizin ,Administration, Oral ,Graft vs Host Disease ,Hematopoietic stem cell transplantation ,T-Lymphocytes, Regulatory ,Antibodies, Monoclonal, Murine-Derived ,immune system diseases ,HLA Antigens ,Prospective Studies ,Child ,Hematology ,Middle Aged ,Tissue Donors ,Fludarabine ,surgical procedures, operative ,medicine.anatomical_structure ,sirolimus ,Treatment Outcome ,Settore MED/38 - PEDIATRIA GENERALE E SPECIALISTICA ,Oncology ,Female ,Rituximab ,Immunosuppressive Agents ,Vidarabine ,medicine.drug ,Adult ,Blood Platelets ,Platelet Engraftment ,Adolescent ,T cells ,graft-versus-host ,chemical and pharmacologic phenomena ,Treosulfan ,Young Adult ,medicine ,Humans ,Busulfan ,Aged ,Antilymphocyte Serum ,Sirolimus ,Peripheral Blood Stem Cell Transplantation ,business.industry ,Mycophenolic Acid ,medicine.disease ,Graft-versus-host disease ,Immunology ,Bone marrow ,business - Abstract
Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts. Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients’ bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II–IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transplant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus– mycophenolate–ATG-F–rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.
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- 2015
24. Generation of human memory stem T cells after haploidentical T-replete hematopoietic stem cell transplantation
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Chiara Bonini, Raffaella Greco, Francesca Lunghi, Maddalena Noviello, Beatrice Claudia Cianciotti, Sarah Marktel, Giacomo Oliveira, Fabio Ciceri, Veronica Valtolina, Jacopo Peccatori, Nicoletta Cieri, Luca Vago, Attilio Bondanza, Laura Bellio, Mattia Forcato, Cristian Taccioli, Claudio Bordignon, Silvio Bicciato, Cieri, N., Oliveira, G., Greco, R., Forcato, M., Taccioli, C., Cianciotti, B., Valtolina, V., Noviello, M., Vago, L., Bondanza, Attilio, Lunghi, F., Marktel, S., Bellio, L., Bordignon, Claudio, Bicciato, S., Peccatori, J., Ciceri, Fabio, and Bonini, MARIA CHIARA
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Homologous ,Adult ,medicine.medical_treatment ,Cellular differentiation ,T-Lymphocytes ,Immunology ,Blood Donors ,T-Cell Antigen Receptor Specificity ,Hematopoietic stem cell transplantation ,Biology ,Biochemistry ,Immune system ,Antigen ,medicine ,Humans ,Transplantation, Homologous ,Lymphopoiesis ,Antigens ,Cell Proliferation ,Transplantation ,Medicine (all) ,T-cell receptor ,Hematopoietic Stem Cell Transplantation ,Interleukin ,Cell Differentiation ,Hematology ,Cell Biology ,Antigens, CD31 ,Haplotypes ,Immunologic Memory ,Platelet Endothelial Cell Adhesion Molecule-1 ,hematopoietic stem cell transplantation ,CD31 ,memory stem T cell - Abstract
Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to posttransplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naive T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT. Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to post transplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen specific and clonal level that TSCM lymphocytes can differentiate directly from naïve precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naïve T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT.
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- 2015
25. Haploidentical HSCT: a 15-year experience at San Raffaele
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Claudio Bordignon, Catia Traversari, Nicoletta Cieri, Maria Teresa Lupo Stanghellini, Consuelo Corti, Attilio Bondanza, Massimo Bernardi, Raffaella Greco, Fabio Ciceri, Luca Vago, Elisabetta Zappone, Jacopo Peccatori, Giacomo Oliveira, Chiara Bonini, Bonini, MARIA CHIARA, Peccatori, J, Stanghellini, Mtl, Vago, L, Bondanza, Attilio, Cieri, N, Greco, R, Bernardi, M, Corti, C, Oliveira, G, Zappone, E, Traversari, C, Bordignon, Claudio, and Ciceri, Fabio
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Oncology ,Male ,medicine.medical_specialty ,Myeloid ,Lymphocyte Transfusion ,medicine.medical_treatment ,T-Lymphocytes ,Graft vs Host Disease ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,law.invention ,haploidentical HSCT ,Randomized controlled trial ,law ,Internal medicine ,Medicine ,Humans ,Transplantation ,business.industry ,Hematopoietic Stem Cell Transplantation ,Hematology ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,medicine.disease ,Allografts ,Surgery ,Clinical trial ,Calcineurin ,Leukemia ,Leukemia, Myeloid, Acute ,surgical procedures, operative ,medicine.anatomical_structure ,Female ,business ,Genetic Engineering - Abstract
Hematopoietic SCT (HSCT) from HLA haploidentical family donors is a promising therapy for high-risk hematological malignancies. In the past 15 years at San Raffaele Scientific Institute, we investigated several transplant platforms and post transplant cellular-based interventions. We showed that T cell-depleted haploidentical transplantation followed by the infusion of genetically modified donor T cells (TK007 study, Eudract-2005-003587-34) promotes fast and wide immune reconstitution and GvHD control. This approach is currently tested in a phase III multicenter randomized trial (TK008 study, NCT00914628). We targeted patients with advanced leukemia with a sirolimus-based, calcineurin inhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated PBSCs from haploidentical family donors (TrRaMM study, Eudract 2007-5477-54). Results of these approaches are summarized and discussed. Hematopoietic SCT (HSCT) from HLA haploidentical family donors is a promising therapy for high-risk hematological malignancies. In the past 15 years at San Raffaele Scientific Institute, we investigated several transplant platforms and post transplant cellular based interventions. We showed that T cell-depleted haploidentical transplantation followed by the infusion of genetically modified donor T cells (TK007 study, Eudract-2005-003587-34) promotes fast and wide immune reconstitution and GvHD control. This approach is currently tested in a phase III multicenter randomized trial (TK008 study, NCT00914628). We targeted patients with advanced leukemia with a sirolimus-based, calcineurin inhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated PBSCs from haploidentical family donors (TrRaMM study, Eudract 2007-5477-54). Results of these approaches are summarized and discussed.
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- 2015
26. Adoptive immunotherapy with genetically modified lymphocytes in allogeneic stem cell transplantation
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Nicoletta Cieri, Attilio Bondanza, Chiara Bonini, Giacomo Oliveira, Monica Casucci, Sara Mastaglio, Cieri, N, Mastaglio, S, Oliveira, G, Casucci, M, Bondanza, Attilio, and Bonini, MARIA CHIARA
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Genetic enhancement ,medicine.medical_treatment ,T-Lymphocytes ,Immunology ,Receptors, Antigen, T-Cell ,Graft vs Host Disease ,Hematopoietic stem cell transplantation ,Biology ,Immunotherapy, Adoptive ,Transduction, Genetic ,Neoplasms ,medicine ,Immunology and Allergy ,Animals ,Humans ,Transplantation, Homologous ,Cancer immunology ,Gene Transfer Techniques ,Hematopoietic Stem Cell Transplantation ,Genetic Therapy ,Suicide gene ,medicine.disease ,Transplantation ,surgical procedures, operative ,Graft-versus-host disease ,Cellular Microenvironment ,Cancer cell ,Stem cell ,Genetic Engineering - Abstract
Hematopoietic stem cell transplantation from a healthy donor (allo-HSCT) represents the most potent form of cellular adoptive immunotherapy to treat malignancies. In allo-HSCT, donor T cells are double edge-swords: highly potent against residual tumor cells, but potentially highly toxic, and responsible for graft versus host disease (GVHD), a major clinical complication of transplantation. Gene transfer technologies coupled with current knowledge on cancer immunology have generated a wide range of approaches aimed at fostering the immunological response to cancer cells, while avoiding or controlling GVHD. In this review, we discuss cell and gene therapy approaches currently tested in preclinical models and in clinical trials.
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- 2014
27. Backtracking Leukemia Clonal Evolution from PostTransplantation Relapse to Initial Diagnosis to Identify Founder Mutations and Mechanisms of Immune Evasion
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Nicoletta Cieri, Vito Lampasona, Cristina Tresoldi, Elia Stupka, Giacomo Oliveira, Jacopo Peccatori, Bernhard Gentner, Chiara Brambati, Fabio Ciceri, Dejan Lazarevic, Matteo Carrabba, Lara Crucitti, Massimo Bernardi, Benedetta Mazzi, Lorenza Chiesa, Gabriele Bucci, Katharina Fleischhauer, Luca Vago, Chiara Bonini, Cristina Toffalori, Davide Cittaro, Vago, L, Cittaro, D, Toffalori, C, Lazarevic, D, Brambati, C, Oliveira, G, Bucci, G, Crucitti, L, Cieri, N, Chiesa, L, Mazzi, B, Tresoldi, C, Gentner, B, Carrabba, Mg, Bernardi, M, Peccatori, J, Bonini, C, Lampasona, V, Fleischhauer, K, Ciceri, F, and Stupka, E
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Mutation ,medicine.medical_treatment ,Immunology ,Medizin ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,Biology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,Somatic evolution in cancer ,Transplantation ,Leukemia ,medicine ,Digital polymerase chain reaction ,Exome sequencing - Abstract
INTRODUCTION: Although allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) can accomplish apparent leukemia eradication in most patients, residual tumor cells can persist over time and eventually outgrow, resulting in clinical relapse. The genetic landscape of relapsing leukemia is often markedly different from its counterpart at diagnosis, due to clonal evolution (Ding et al, Nature, 2012) and selection of treatment-resistant variants (Vago et al, N Engl J Med, 2009). A potential solution to treat, or even prevent, relapse is to identify and specifically target mutations occurring very early in the leukemic transformation process, and thus putative hallmarks of cancer progenitors. Next-Generation Sequencing (NGS) provides the opportunity to track disease subclones during the clinical history of patients, pinpoint founder alterations and identify the mechanisms by which leukemia evades elimination. METHODS: In the present study, we combined immunogenetic analyses and NGS to detail the complex clinical history of a patient with therapy-related myelodysplasia (t-MDS), diagnosed years after sequential intensive chemotherapy for B cell Acute Lymphoblastic Leukemia (B-ALL). Leukemic cells collected at serial time-points during the patient disease history (initial diagnosis of B-ALL, first presentation of t-MDS, relapse after first allo-HSCT, relapse after second allo-HSCT) were characterized by means of genomic HLA typing, HLA allele-specific quantitative PCR and whole exome sequencing, with a minimum depth of coverage of 70x. Patient fibroblasts and peripheral blood mononuclear cells (PBMCs) collected before the occurrence of t-MDS, as well as PBMCs from the two allogeneic HSC donors, served as controls and reference exomes. Targeted resequencing of mutations of interest was performed using the Fluidigm Access Array system. Gene segments encompassing newly-identified mutations in TP53, NHEJ1 and BTNL8and their respective wild-type counterparts were cloned into plasmids, and used to design and validate specific droplet digital PCR assays, using the Bio-Rad QX100 instrument. RESULTS: A 54-year-old patient with high-risk t-MDS received two subsequent allo-HSCTs from partially-incompatible family donors, and after each transplant experienced disease recurrences. Genomic HLA typing and HLA allele-specific qPCR demonstrated that both relapses were due to mutant variants of the original leukemia that had evaded immune pressure through genomic loss of the HLA haplotype targeted by the respective donor’s T cells. Immunogenetic studies ruled out any linear relationship between the two relapses, suggesting that both might have derived from a common HLA-heterozygous progenitor. Accordingly, whole exome sequencing demonstrated direct clonal evolution from the initial presentation of t-MDS to first relapse, whereas the large majority of mutations present at second relapse were unique to the sample. Only five non-synonymous coding mutations were present in all three t-MDS samples, comprising disrupting mutations in TP53, in NHEJ1 (a key factor in DNA double strand-break repair), and in the T cell costimulatory receptor BTNL8. Of notice secondary mutations, detected only in one or two of the three disease presentations, were predicted to result in partially overlapping functional effects, and could be modeled in a conjoint DNA damage repair network, centered around the putative founder mutation in TP53. By ultra-sensitive droplet digital PCR, the same TP53 mutation was backtracked throughout the whole nine years of patient clinical history, including the phases of apparent complete remission, up to a preleukemic progenitor present in the patient bone marrow at the time of B-ALL initial diagnosis. CONCLUSIONS: Our results demonstrate that therapies, and the immune pressure of allo-HSCT in particular, can have a dramatic effect in shaping leukemia clonal evolution. Importantly, by identifying leukemia founder mutations, we might further our insights into leukemogenesis, and identify novel targets for post-transplantation diagnostics and leukemia-eradicating therapies. Disclosures Bonini: MolMed S.p.A.: Consultancy.
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- 2014
28. How to Monitor Immune Reconstitution Following Allogeneic Hematopoietic Stem Cell Transplantation: A Survey from the EBMT- Cellular Therapy & Immunobiology Working Party
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Raffaella Greco, Christian Chabannon, Fabio Ciceri, Steffie van der Werf, Vanderson Rocha, Jacopo Peccatori, Luca Vago, Sofie Rosanne Terwel, Maddalena Noviello, Antoine Toubert, Annalisa Ruggeri, Giacomo Oliveira, Attilio Bondanza, Katharina Fleischhauer, Francesco Dazzi, Ulrike Koehl, Chiara Bonini, and Nicoletta Cieri
- Subjects
Oncology ,medicine.medical_specialty ,biology ,business.industry ,T cell ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,Biochemistry ,Clinical trial ,medicine.anatomical_structure ,Antigen ,Cord blood ,Internal medicine ,biology.protein ,Medicine ,Antibody ,business ,B cell - Abstract
Background: Post transplant immune reconstitution plays a major role in determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is currently monitored with different techniques in different Centers, with the aim of identifying clinically relevant immunological biomarkers. However, it is unclear which and how many of these immunological tests are currently performed on a routine basis, and which ones have the potential to predict patient outcome, and possibly guide patient care after allo-HSCT. Methods: The EBMT Cellular Therapy & Immunobiology Working Party (CTIWP) conducted a survey to identify current policies to monitor immune reconstitution in patients undergoing allo-HSCT and possibly reach a general consensus. This study followed the EBMT study guidelines. All EBMT Centers were invited to participate. Each participating Center received a questionnaire on the availability of specific immunomonitoring assays, specifying the use in clinical practice and/or within investigational trials. Assays were based on relatively simple and readily available parameters such as absolute lymphocyte counts (ALC) to more complex cellular and molecular tests. Moreover, the Centers were asked to define the transplant platform (HLA-identical sibling, matched unrelated donor, haploidentical and/or cord blood) on which each test is generally performed. Results: Policies for post-transplant immunomonitoring have been reported by 35 participating EBMT Centers active in 14 Countries and performing allo-HSCT from HLA identical related (35 centers), matched unrelated (33), haploidentical (34), unrelated cord blood (27). Complete blood counts and immunoglobulins are routinely tested for patients' care by all centers. Relative proportions of T cell subsets are currently tested by flow-cytometry as "standard of care" or "investigational" by 82% and 17% of centers respectively. B cell and NK cell counts are quantified routinely by 46% and 23% of Centers, and investigationally by 40% of Centers. The availability of molecular tests (STR, qPCR, Fish) to measure post-transplant engraftment are reported by all Centers, except two, as a standard of care measure. T cell receptor-expressing circles (TRECs) and/or K-deleting recombination excision circles (KRECs) are quantified within selected clinical trials by 37% of Centers. Interestingly, 60% of Centers evaluate, mostly as an investigational measure, antigen specific T cell responses by: proliferation assays (49%), interferon-gamma enzyme-linked immunospot-Elispot (49%), intracellular cytokine staining (46%) and tetramer/dextramer staining (37%). Most of these Centers test responses to Cytomegalovirus and Epstein Barr Virus, and 5 Centers use at least one of these assays on a routine basis. About half of the participating Centers (43%) commonly test antigen-specific antibodies, mainly as responses to vaccines, and not routinely. T-cell receptors (TCR) and B-cell receptors (BCR) repertoires are measured by spectratyping in 14 out of 35 Centers (4 as clinical practice and 10 in selected trials), or, in selected trials, by next generation sequencing (in 11 out of 35 the participating Centers). Conclusions: Results of this survey indicate that country- and center expertise are associated with heterogeneous and distinct protocols, and underline the clinical need to harmonize methods and to provide practical recommendations for monitoring post-transplant immune reconstitution, both for routine purposes and investigational studies. Adequate reporting and connection between individual Centers exploiting these data will foster collaborative and comparative research studies, with the ultimate goals of improving patient care and refining our understanding of the immunological correlates to clinical outcome. Acknowledgments: R. Ram, M. A. Diaz, G. McQuaker, D. Russo, E. Faber, P. Chiusolo, C. Rössig, S. M. Martin, A. Anagnostopoulos, M. Stelljes, K. Orchard, P. Jindra, A. Sampol, K. Patrick, M. A. Bekadja, J. Gayoso, A. Olivieri, J. Passweg, E. Jost, H Labussiere-Wallet, Y Koc, A. Lange, I. Garcia Cadenas, N. Kröger, A. Biondi, N. Milpied, D. Olive, E. Lanino, G. Stuhler, J.H. Dalle, J.R. Cabrera Marín, F. Ciceri, D. Uckan-Cetinkaya, R. Parody Porras, G. Kriván. Disclosures Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.
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- 2016
29. Clinical and Biological Features Associated with Engraftment of Acute Myeloid Leukemia Patient-Derived Xenografts
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Fabio Ciceri, Chiara Bonini, Cristina Toffalori, Lucia Zanotti, Raffaella Greco, Attilio Bondanza, Lara Crucitti, Dejan Lazarevic, Matteo Carrabba, Laura Zito, Gabriele Bucci, Barbara Camisa, Massimo Bernardi, Giacomo Oliveira, Jose Manuel Garcia-Manteiga, Francesca Biavasco, Carolina Caserta, and Luca Vago
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FLT3 Internal Tandem Duplication ,Myeloid ,Microarray ,medicine.diagnostic_test ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Flow cytometry ,Gene expression profiling ,Transplantation ,Leukemia ,medicine.anatomical_structure ,medicine ,Cancer research - Abstract
Background: Patient-derived xenografts (PDXs) are key models for interrogating the biology of tumor cells that poorly survive in vitro. In particular, over the last decade, immunodeficient mouse models have been extensively used to assess the in vivo growth potential of human leukemia, to provide insights into its biology, and to perform preclinical validation of therapies. Still, only a fraction of the cases of acute myeloid leukemia (AML) are able to engraft into mice, and the biological and clinical correlates of the ability to generate PDXs are unknown. Methods: Primary AML harvested from 52 patients at diagnosis (n=37, 71%), at relapse after treatments (n=15, 29%), or both (n=6) were purified and infused into non-irradiated NOD-SCID γ-chain null (NSG) mice. Upon leukemia engraftment, assessed by multiparametric flow cytometry, mice were sacrificed and leukemic cells were isolated, characterized, and reinfused in serial recipients, in up to four serial passages. Gene expression profile was analyzed using Illumina microarray, and deregulated genes and processes identified by pairwise LIMMA analysis and classified using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) curated databases. The mutational asset of infused AML was assessed through targeted resequencing, using a custom panel comprising 192 targets and based on the Agilent Haloplex HS technology. Results: Twenty-six out of 52 primary AML samples (50%) generated xenografts. Engraftment and growth kinetics of the human leukemic cells were highly consistent among littermates, and specific for each tested leukemia. Circulating leukemic cells were firstly detected in the peripheral blood of animals at a median time of 22.5 days (range 14 - 150). In vivo growth allowed expansion of infused AMLs in bone marrows and spleens of the animal, with a median fold increase of 3.5 (range 0.1 - 351.4). The gene expression profile of xenografts was reproducible amongst littermates and recapitulated the features of parental AML: genes deregulated in xenografts accounted for 9.1% of the transcript assessed, with substantial overlap in the genes and processes deregulated in each of the studied cases. GO and GSEA demonstrated the selective deregulation of genes involved in cell proliferation (CDC20, AURKA), syster chromatyde organization (CENPF CEP170) and myeloid differentiation (AZU1, MPO, MYADM, CTSG). Of note, the ability to generate xenografts was conserved when AML cells were challenged at different time-points during the clinical history of the patients, with leukemia harvested at relapse after transplantation displaying a more aggressive behavior. Similarly, upon serial transfer AML exhibited an accelerated growth kinetic. Engraftment in mice significantly correlated with poor patient prognosis: AML engrafters had dramatically lower leukemia free-survival rates compared to non-engrafters (median 5.9 vs. 21.8 months after induction chemotherapy, p=0.0022, Fig. 1A), confirmed also by multivariate analysis (p=0.002). Also the mutational profile differed greatly between engrafters and non-engrafters, as summarized in Fig. 1B. In particular, while the presence of an aberrant karyotype was not associated with PDX generation, FLT3 internal tandem duplication, DNMT3A and NPM1 mutation were all significantly associated to engraftment (p=0.0244, p=0.009 and p=0.0437 respectively). In particular the co-occurrence of mutations in these three genes, recently reported to confer very poor prognosis to AML patients (Papaemmanuil et al, NEJM 2016), markedly enhanced the ability to generate PDXs (Fig.1C). Conclusion: These data show that engraftment into immunodeficient mice mirrors the biology of primary human leukemia, providing a proxy to select cases with a higher chance to generate PDXs. Further comparisons between AML capable or not to generate PDXs might provide novel markers of leukemia aggressiveness and rationales for targeted therapies. Figure 1 Figure 1. Disclosures Bonini: TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Ciceri:MolMed SpA: Consultancy.
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- 2016
30. Multiple Inhibitory Receptors Are Expressed on Central Memory and Memory Stem T Cells Infiltrating the Bone Marrow of AML Patients Relapsing after Allo-HSCT
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Francesco Manfredi, Nicoletta Cieri, Paolo Dellabona, Maddalena Noviello, Giacomo Oliveira, Giulia Casorati, Fabio Ciceri, Luca Vago, Jacopo Peccatori, Filippo Cortesi, Raffaella Greco, Attilio Bondanza, Valentina Gambacorta, Tommaso Perini, Chiara Bonini, and Cristina Toffalori
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business.industry ,medicine.medical_treatment ,Immunology ,FOXP3 ,Cell Biology ,Hematology ,Human leukocyte antigen ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,03 medical and health sciences ,Leukemia ,0302 clinical medicine ,medicine.anatomical_structure ,Aldesleukin ,030220 oncology & carcinogenesis ,medicine ,Bone marrow ,IL-2 receptor ,business ,CD8 ,030215 immunology - Abstract
Background:Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse still remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse. Patients and Methods: To this,we longitudinally analyzed the T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA identical (HLAid, 20 pts) or HLA haploidentical (haplo, 12 pts) donors. Samples were analysed by multi-parametric flow cytometry to investigate the expression of inhibitory receptors (IRs) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression, and to assess the proportion of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Results were also analyzed with the BH-SNE algorithm, an unbiased computational method for the analysis of FACS data. To evaluate T-cell effector functions, the CD107a degranulation assay was performed and the production of cytokines (IL-2, IFNg and TNFa) was measured by intracellular staining. BM and PB were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained (CR; 16 pts), at 1 year. Samples from 8 healthy donors (HD) were used as controls. Results:After transplant, BM and PB T cells showed a lower CD4/CD8 ratio (p We next investigated the expression of several IRs as T-cell exhaustion markers. After haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were significantly upregulated in BM and PB T cells at all time-points, compared to HD and independently from the clinical outcome. Conversely, after HLAid-HSCT, at the late time-point, patients who relapsed, displayed a higher frequency of BM infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To verify whether the T-cell exhaustion phenotypic profile at relapse associates with functional impairment, we evaluated T-cell effector functions upon polyclonal stimulation. Strikingly, we observed a lower degranulation ability of CD8 cells at relapse when compared to CR (p Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing pts includes PD-1, CTLA-4, 2B4 and Tim-3. The expression of IRs on early differentiated central memory and memory stem T cells at relapse suggests a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Disclosures Bondanza: TxCell: Research Funding; MolMed SpA: Research Funding; Formula Pharmaceuticals: Honoraria. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.
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- 2016
31. Natural Killer Cell Reconstitution after Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplant Cyclophosphamide: Elimination of Donor-Derived Mature Alloreactive NK Cells, but Favorable Conditions for Adoptive Immunotherapy
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Valentina Gambacorta, Simona Piemontese, Chiara Bonini, Jacopo Peccatori, Cristina Toffalori, Fabio Ciceri, Mara Morelli, Leo Luznik, Andrea Assanelli, Antonio Russo, Maria Teresa Lupo Stanghellini, Fabio Giglio, Raffaella Greco, Nicoletta Cieri, Laura Zito, Luca Vago, Sofia Berglund, and Giacomo Oliveira
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business.industry ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Natural killer cell ,Transplantation ,Graft-versus-host disease ,medicine.anatomical_structure ,Interleukin 15 ,medicine ,Cytotoxic T cell ,Bone marrow ,Stem cell ,business - Abstract
INTRODUCTION The use of high-dose cyclophosphamide as post-transplant Graft versus Host Disease (GvHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (haplo-HSCT), allowing the safe infusion of T cell replete grafts. The efficacy of post-transplant cyclophosphamide (PT-Cy) has its basis in its capacity to selectively eliminate proliferating cells, including alloreactive T cells. It is however to date unknown whether PT-Cy affects the reconstitution of Natural Killer (NK) cells, whose alloreactivity is known to play a major role in T cell-depleted haplo-HSCT. PATIENTS AND METHODS We analyzed the grafts and serial peripheral blood (PB) and bone marrow (BM) samples from 14 patients who received T cell replete haplo-HSCT followed by PT-Cy at the San Raffaele Scientific Institute, Milan (n=10, OSR) or the Johns Hopkins University, Baltimore (n=4, JHU). OSR patient received a myeloablative conditioning, PB stem cell grafts, and sirolimus and mycophenolate as pharmacological GvHD prophylaxis. JHU patients received the "classical" Baltimore nonmyeloablative conditioning, unmanipulated BM grafts, and tacrolimus and mycophenolate as pharmacological GvHD prophylaxis. To characterize NK cells reconstitution, we monitored absolute counts and employed a 27-marker flow cytometry panel with high dimensional single-cell analysis using the bh-SNE algorithm. We used intracellular staining to determine the frequency of Ki67+ proliferating cells and expression of Aldehyde Dehydrogenase (ALDH), known to confer resistance to PT-Cy. Interleukin-15 (IL-15) serum concentration was quantified using the Bio-Plex Pro Human Cytokine 4-plex assay. To directly assess the effect of PT-Cy on proliferating NK cells we exposed graft NK cells to IL-15 and mafosfamide, a cyclophosphamide analogue active in vitro. Functional assays against leukemic cell lines and primary AML blasts were performed measuring CD107A degranulation on NK cells and Annexin V on targets. RESULTS All patients received high numbers of mature donor NK cells as part of the graft (OSR median 17x106/kg, JHU median 7.25x106/kg ), and donor-derived NK cells were detectable as early as day 3 after HSCT and throughout the entire follow-up. At day 3 after HSCT, all subsets of NK cells, including single KIR+ alloreactive cells, were actively proliferating (mean 61.23% of Ki-67+ cells for OSR patients, and 58% for JHU patients), possibly driven by the high levels of IL-15 detected in patient serum after conditioning (Fig.1A). After PT-Cy, a marked reduction in the frequency and counts of proliferating NK cells was evident irrespectively of the transplantation platform, suggesting selective killing of dividing cells by PT-Cy. In line with this hypothesis, NK cells from the graft and from patient PB at day 3 after HSCT showed no detectable ALDH expression, and NK cells prompted to proliferate in vitro were killed in a dose-dependent manner by mafosfamide (Fig.1B). The phenotype of NK cells also changed upon PT-Cy: whereas before the infusion they resembled their mature counterparts from the graft, after PT-Cy an immature phenotype, CD62L+NKG2A+KIR-, became prevalent, suggesting derivation from donor HSCs rather than from infused NK cells (Fig.1C). Accordingly, bhSNE maps demonstrated differential clustering of NK cells from the graft and analyzed 30 days after HSCT (Fig.1D). In line with these features, we detected very low numbers of putatively alloreactive single KIR+ NK cells both in the PB and in the BM of patients at day 30 after HSCT, and these NK cells displayed impaired cytotoxic potential against leukemic targets. Finally, consistent with these observations, when we analyzed the impact of predicted NK alloreactivity in an extended series of 99 patients who received myeloablative haplo-HSCT with PT-Cy, we detected no significant difference in progression-free survival (Fig.1E). CONCLUSION Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are eliminated upon PT-Cy administration and that in this transplantation platform NK cell alloreactivity might be blunted by the elimination of donor single KIR+ NK cells and by the competition between reconstituting NK and T cells. Still, the high levels of IL-15 detected in patients' sera at early time-points might provide a biological rationale for the infusion of mature donor NK cells early after PT-Cy administration. Figure 1 Figure 1. Disclosures Bonini: TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Ciceri:MolMed SpA: Consultancy.
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- 2016
32. IL-7 and IL-15 instruct the generation of human memory stem T cells from naïve precursors
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Elena Provasi, Jacopo Peccatori, Fulvio Mavilio, Mattia Forcato, Silvio Bicciato, Barbara Camisa, Claudio Bordignon, Fabio Ciceri, Anna Mondino, Chiara Bonini, Nicoletta Cieri, Attilio Bondanza, Giacomo Oliveira, Alessandra Recchia, Maria Teresa Lupo-Stanghellini, Fabienne Cocchiarella, Cieri, N, Camisa, B, Cocchiarella, F, Forcato, M, Oliveira, G, Provasi, E, Bondanza, Attilio, Bordignon, Claudio, Peccatori, J, Ciceri, Fabio, Lupo Stanghellini, Mt, Mavilio, F, Mondino, A, Bicciato, S, Recchia, A, and Bonini, MARIA CHIARA
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phenotypic and functional profile of Tcell ,Cell Survival ,Cellular differentiation ,CD3 ,Immunology ,Population ,Mice, SCID ,Biology ,Biochemistry ,Immunophenotyping ,Cell therapy ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Antigen ,Mice, Inbred NOD ,T-Lymphocyte Subsets ,medicine ,Animals ,Cluster Analysis ,Humans ,fas Receptor ,L-Selectin ,education ,030304 developmental biology ,Cell Proliferation ,Interleukin-15 ,0303 health sciences ,education.field_of_study ,Precursor Cells, T-Lymphoid ,Gene Expression Profiling ,Interleukin-7 ,CD28 ,Cell Differentiation ,Cell Biology ,Hematology ,medicine.disease ,Graft-versus-host disease ,biology.protein ,Leukocyte Common Antigens ,Female ,Immunologic Memory ,CD8 ,030215 immunology - Abstract
Long-living memory stem T cells (T-SCM) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T-SCM lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7R alpha(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T-SCM accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T-SCM prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T-SCM are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T-SCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy. (Blood. 2013; 121(4): 573-584) Long-living memory stem T cells (TSCM) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here we show that it is possible to differentiate in vitro, expand and gene modify in clinically compliant conditions CD8+ TSCM lymphocytes starting from naïve precursors. Requirements for the generation of this T-cell subset, described as CD62L+CCR7+CD45RA+CD45R0+IL-7Rα+CD95+, are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly TSCM accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T lymphocyte subset, intermediate between naïve and central memory cells. When transplanted in immunodeficient mice, gene-modified naïve-derived TSCM prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GvHD. Furthermore, gene-modified TSCM are the only T-cell subset able to expand and mediate GvHD upon serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for TSCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy. "Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3\/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy."
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- 2013
33. Use of TK-cells in haploidentical hematopoietic stem cell transplantation
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Chiara Bonini, Luca Vago, Raffaella Greco, Giacomo Oliveira, and Maria Teresa Lupo-Stanghellini
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medicine.medical_treatment ,T-Lymphocytes ,Graft vs Host Disease ,Disease ,Hematopoietic stem cell transplantation ,Haploidy ,medicine.disease_cause ,Thymidine Kinase ,medicine ,Animals ,Humans ,business.industry ,Genes, Transgenic, Suicide ,Hematopoietic Stem Cell Transplantation ,Herpes Simplex ,Hematology ,Genetic Therapy ,Suicide gene ,medicine.disease ,Genetically modified organism ,Clinical trial ,surgical procedures, operative ,Graft-versus-host disease ,Herpes simplex virus ,Thymidine kinase ,Immunology ,business - Abstract
PURPOSE OF REVIEW Preserving the beneficial effects of donor T cells against tumor and pathogens while avoiding noxious graft-versus-host disease (GvHD) is the 'holy grail' of allogeneic hematopoietic stem cell transplantation (HSCT). The suicide gene strategy allows the selective elimination of genetically modified donor T cells during GvHD. This review summarizes the results obtained in recent years in the clinical trials of suicide gene therapy using the paradigmatic herpes simplex virus thymidine kinase (TK) suicide gene. RECENT FINDINGS T cells genetically modified to express the TK suicide gene, TK-cells, are safe and preserve most of their functional features; when infused into patients they are capable of conferring substantial protection against infections and tumor recurrence, and are promptly eliminated in the case of GvHD, with complete resolution of the adverse reaction in all treated cases. Unexpectedly, TK-cells also have the indirect effect of promoting patient thymopoiesis, contributing to the renewal of a host-tolerant immune repertoire. SUMMARY Suicide gene therapy with TK-cells is a promising approach to overcome the risk of GvHD in allogeneic HSCT, especially from partially incompatible donors, and is currently under evaluation in a multicentric phase III clinical trial.
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- 2012
34. HLA Loss Leukemia Relapses after Partially-Incompatible Allogeneic HSCT As a Prototypical System to Investigate Natural Killer Cell Dynamics
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Maria Teresa Lupo Stanghellini, Matteo Carrabba, Raffaella Greco, Benedetta Mazzi, Lara Crucitti, Jacopo Peccatori, Fabio Ciceri, Maddalena Noviello, Laura Zito, Luca Vago, Massimo Bernardi, Giacomo Oliveira, Valentina Gambacorta, Katharina Fleischhauer, Chiara Bonini, Cristina Toffalori, Gambacorta, V, Oliveira, G, Zito, L, Toffalori, C, Crucitti, L, Noviello, M, Mazzi, B, Greco, R, Stanghellini, Mtl, Carrabba, Mg, Bernardi, M, Peccatori, J, Fleischhauer, K, Bonini, C, Ciceri, F, and Vago, L
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medicine.medical_treatment ,Immunology ,Medizin ,Myeloid leukemia ,Context (language use) ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,CD16 ,Biology ,medicine.disease ,NKG2D ,Biochemistry ,Natural killer cell ,Leukemia ,medicine.anatomical_structure ,medicine - Abstract
Background Despite the constant improvement in the outcome of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Acute Myeloid Leukemia (AML), relapses remain frequent, warranting investigation on their biological bases. After haploidentical HSCT up to one third of AML relapses feature selective genomic loss of the HLA haplotype targeted by alloreactive donor T cells (Vago, N Engl J Med, 2009; Crucitti, Leukemia, 2015), evading their control and gaining the ability to outgrow. Yet, Natural Killer (NK) cells mediate alloreactivity in response to loss of specific HLA allotypes from target cells: thus, in theory, HLA loss relapses should represent excellent targets for donor NK cell recognition. Here we investigated the dynamics of NK cells in this unique immunogenetic context, to understand the biological bases of their failure in preventing the emergence of HLA loss variants. Methods We took into consideration 23 patients who after T cell replete haploidentical HSCT experienced HLA loss relapses. NK cell alloreactivity was predicted according to the Perugia algorithm (Ruggeri, Science, 1999). Killer Cell Immunoglobulin-like Receptor (KIR) typing was performed using a commercially-available kit, and KIR B-content estimated using the EMBL-EBI calculator. The phenotypic features of peripheral blood NK cells were assessed by multiparametric flow cytometry, and high dimensional single-cell analysis was performed using the viSNE bioinformatic tool. Results Based on donor-recipient HLA typing, at the time of HSCT NK cell alloreactivity in the graft-versus-leukemia direction was predicted in 10/23 patients who experienced HLA loss relapses (43.5%). In 7/23 additional patients (30.4%), conditions for predicted NK cell alloreactivity were fulfilled at time of relapse, upon genomic loss of the mismatched HLA haplotype from AML blasts. In all cases KIR genotyping confirmed the presence in the donor repertoire of the necessary KIR genes. Only 3/17 HSC donors were homozygous for KIR A haplotypes, encoding preferentially inhibitory KIR genes, and most carried equal or higher numbers of activating KIR genes than expected (Cooley, Blood, 2010). Thus, the absence of NK cell-mediated control of HLA loss variants can not be explained by an unfavorable immunogenetic asset of HSC donors. Therefore we characterized the phenotypic features of NK cells circulating in the peripheral blood of 7 patients at the time of HLA loss relapse (median time after HSCT 307 days, range 147-703), and compared them to their counterparts in healthy individuals (n=6), and matched-paired transplanted patients in remission (n=6), or at the time of non HLA loss ("classical") relapse (n=7). We analyzed a total of 27 markers involved in NK cell target recognition (KIRs, NKG2A, NKG2C, SIGLEC7, SIGLEC9), activation (NKp30, NKp44, NKp46, NKG2D, 2B4, DNAM1), maturation (CD57, CD16, CD62L), and exhaustion (PD1, TIM3, KLRG1). At the time of HLA loss relapse, NK cells had recovered a mature phenotype, although with a slightly higher frequency of CD56bright cells. In all cases in which NK cell alloreactivity had been predicted we detected the single-KIR+ NK cells of interest, without significant differences between patients and controls. However NK cells from transplanted patients expressed lower levels of the SIGLEC9 (p Conclusions Even at late timepoints after partially-incompatible allo-HSCT, when HLA loss relapses typically occur, the reconstituted NK cell repertoire displays profound differences from its counterpart in healthy subjects, hinting for defective immunosurveillance. Therapeutic protocols employing freshly isolated mature donor NK cells should thus be further investigated for the prevention and treatment of HLA loss relapses. Figure 1. The same viSNE map, obtained by analysis of the entire dataset, is differentially colored to evidence the spatial distribution, and thus phenotypic similarity, of NK cells from each cohort (upper row) or the intensity of expression of the indicated markers (lower row). Figure 1. The same viSNE map, obtained by analysis of the entire dataset, is differentially colored to evidence the spatial distribution, and thus phenotypic similarity, of NK cells from each cohort (upper row) or the intensity of expression of the indicated markers (lower row). Disclosures No relevant conflicts of interest to declare.
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- 2015
35. Mechanism of thymic renewal after infusion of suicide gene-modified donor T cells after hematopoietic stem cell transplantation (HSCT) in adult patients
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Fabio Ciceri, Alessandro Aiuti, Immacolata Brigida, Jacopo Peccatori, Maria Teresa Lupo-Stanghellini, Chiara Bonini, Domenico Ghio, Luca Vago, Corrado Soldati, Giacomo Oliveira, Antonio Lambiase, Claudio Bordignon, Maddalena Noviello, Attilio Bondanza, A. Del Maschio, Bordignon, Claudio, Vago, L., Oliveira, G., Noviello, M., Soldati, C., Ghio, D., Brigida, I., Aiuti, A., Lupo-stanghellini, M. T., Peccatori, J., Lambiase, A., Bondanza, A., DEL MASCHIO, Alessandro, Ciceri, Fabio, and Bonini, MARIA CHIARA
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Cancer Research ,business.industry ,Mechanism (biology) ,viruses ,medicine.medical_treatment ,chemical and pharmacologic phenomena ,Hematopoietic stem cell transplantation ,Suicide gene ,medicine.disease_cause ,Genetically modified organism ,Interleukin 21 ,surgical procedures, operative ,Herpes simplex virus ,Oncology ,immune system diseases ,Thymidine kinase ,Immunology ,medicine ,Cytotoxic T cell ,business - Abstract
6526 Background: In haploidentical HSCT, the infusion of donor T cells genetically modified to express the Herpes Simplex Virus Thymidine kinase (HSV-Tk) suicide gene allows GvHD control, while rap...
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- 2011
36. Revealing the Generation of Human Memory Stem T Cells in Haploidentical T-Replete Hematopoietic Stem Cell Transplantation
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Claudio Bordignon, Silvio Bicciato, Chiara Bonini, Jacopo Peccatori, Luca Vago, Veronica Valtolina, Giacomo Oliveira, Nicoletta Cieri, Maddalena Noviello, Cristian Taccioli, Sarah Marktel, Mattia Forcato, Raffaella Greco, Francesca Lunghi, Fabio Ciceri, Attilio Bondanza, and Laura Bellio
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biology ,CD3 ,T cell ,medicine.medical_treatment ,Immunology ,T-cell receptor ,Priming (immunology) ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Graft-versus-host disease ,medicine.anatomical_structure ,Immune system ,Antigen ,biology.protein ,medicine - Abstract
Introduction: The T cell memory compartment is multi-faceted and encompasses multiple subsets with divergent properties. In addition to central memory (TCM) and effector memory (TEM) cells, the spectrum of immunological memory has been recently extended with the identification of memory stem T cells (TSCM). Gene expression profiling, corroborated by in vitro and in vivo experimental results, posits TSCM upstream TCM and TEM in T-cell ontogeny. However, TSCM role in immune reconstitution (IR) following allogeneic HSCT remains largely unknown. Here, we tracked TSCM dynamics in patients undergoing haploidentical HSCT. By this process, we unconventionally exploited HSCT as model system to provide novel insights into the contribution of TSCM to a mounting immune response. Patients and Methods: We characterized T cell dynamics during the first month post HSCT in 20 patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft, and GvHD prophylaxis consisting of cyclophosphamide (PT-Cy day 3,4), MMF and sirolimus from day 5. Results: Upon infusion, naïve T cells (TN) cells gradually disappeared from circulation, and by day 8 post HSCT the peripheral compartment was composed only by memory lymphocytes. At day 8, TSCM cells were the most represented circulating subset, and their frequency was significantly higher than that of the infused graft (LK). To investigate whether such high TSCM frequency was due to expansion of TSCM cells infused or to their direct in vivo generation, T cell subset proliferation was analyzed. At day 3 upon infusion (in the absence of immunosuppression), all memory subsets robustly proliferated, with TSCM cells displaying significantly higher frequencies of Ki-67+ cells compared to all other subsets. In sharp contrast, TN cells were scantly Ki-67+, in line with the longer timeframe required for their priming. PT-Cy abrogated proliferation in all subsets. T cell counts however did not drop between day 5 and 8 post HSCT, and TSCM cells increased in percentages and absolute numbers. We thus explored whether TSCM cells were resistant to PT-Cy, but failed to record ALDH activity, the major mechanism of Cy inactivation, in CD3+ cells. neither within LK nor upon infusion. Accordingly, we detected high percentages of apoptotic cells within all memory subsets at day 5 post HSCT. Nonetheless, at day 8 significantly lower percentages of TSCM cells were Annexin V+ compared to TCM/TEM, while by day 15 post-HSCT all memory subsets displayed very low levels of apoptosis. Thus, we reasoned that direct conversion of TN into TSCM could be the preferential mechanism underlying TSCM expansion. To prove this, we exploited the TCR sequences harbored by each individual T cell as surrogate clonal markers. Purified T cell subsets from LK and harvested 30 days post HSCT in 3 consecutive patients were subjected to TCR sequencing. Sequences harbored by LK-TN were retrieved in all memory subsets at day 30 after HSCT, showing that TN were able to generate the complete spectrum of immunological memory, including TSCM. Quantitative analysis revealed that only clonotypes originally harbored by LK-TN were significantly increased in counts at day 30 post-HSCT. In contrast, sequences unique to LK-TSCM, LK-TCM or LK-TEM were similar or reduced in counts at day 30 post-HSCT compared to LK, indicating that either PT-Cy efficiently dampened their expansion and/or that such memory lymphocytes persisted unchallenged upon in vivo infusion. Overall, in vivo fate mapping through TCR sequencing allowed defining the in vivo differentiation landscapes of human naïve T cells upon HSCT, highlighting TSCM as privileged players in the diversification of immunological memory. We next validated these results at the antigen-specific level. In suitable 5 patients, we recorded the presence of WT1 or PRAME-specific TN cells within donor LK. Upon HSCT, tumor-specific T cells increased in frequency and displayed a memory phenotype, comprising TSCM. Finally, by quantifying patient serum cytokines, we found that the degree of IL-7 availability at day 1 post HSCT correlated with the extent of TSCM expansion at day 8. Conclusions: These data provide novel insights into TSCM biology and ongoing analyses will define correlations with clinical events. Longer immunological follow-up will advance our understanding of the contribution of early TSCM generation to the overall success of post HSCT IR. Disclosures Bordignon: MolMed: Employment. Bonini:MolMed S.p.A.: Consultancy.
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- 2014
37. Infusion of Donor Lymphocytes Genetically Engineered to Express the Herpes Simplex Virus Thymidine Kinase (HSV-TK) Suicide Gene after Haploidentical Hematopoietic Stem Cell Transplantation (HSCT): Preliminary Efficacy Data from the Randomized TK008 Study
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Donald Bunjes, Attilio Bondanza, Dietger Niederwieser, Fabio Ciceri, Michele L. Donato, Michael Stadler, Maria Teresa Lupo Stanghellini, Evangelia Yannaki, Raffaella Greco, Lutz Uharek, Jacopo Peccatori, Claudio Bordignon, John F. DiPersio, Arnon Nagler, Wolfgang Bethge, Scialini Colombi, Giacomo Oliveira, Andrew L. Pecora, Lambiase Antonio, Eva Mischak-Weissinger, Chiara Bonini, Eduardo Olovarria, and Luca Vago
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Ganciclovir ,Oncology ,medicine.medical_specialty ,Acute leukemia ,Cyclophosphamide ,business.industry ,Surrogate endpoint ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Donor Lymphocytes ,Biochemistry ,Surgery ,Leukemia ,Internal medicine ,medicine ,Cumulative incidence ,business ,medicine.drug - Abstract
Background: Haploidentical family donors represent the ideal solution to offer a potential cure for every high-risk leukemia patient undergoing HSCT. Widespread use of haploidentical HSCT has been historically limited by high rates of late non-relapse mortality (NRM) and relapse, which are associated with the delayed immune reconstitution (IR) due to either ex vivo T-cell depletion or in vivo post-HSCT cyclophosphamide given as graft-vs-host disease (GvHD) prevention. We have previously shown in a phase 2 trial (TK007, Lancet Oncol 2009;10:489) that TK cells (donor T cells genetically modified to express the HSV-TK suicide gene) can safely induce an early IR when given after T-cell depleted haploidentical HSCT Methods: In a confirmatory, ongoing phase 3 trial (TK008, NCT00914628), up to 4 monthly infusions of TK cells are given at 1x107for kg of patient body weight, starting 21 to 49 days after T-cell depleted haploidentical HSCT in the experimental arm. Control arm consists of either T-cell depleted or post-HSCT cyclophosphamide haploidentical HSCT, at physician discretion. High-risk acute leukemia patients lacking an HLA-matched donor are included. So far, 34 patients have been enrolled from eight EU and US sites, with 19 being in complete remission, 18 presenting with AML and 22 having PS of 0 at HSCT. Hypothesis testing for primary efficacy endpoint: 1-year disease-free survival (DFS) of 30% (control arm) vs 52% (TK arm). Secondary endpoints include overall survival (OS), NRM and relapse incidence, proportion and timing to IR, incidence and control of GvHD by suicide-gene induction with ganciclovir Results: Data refer only to 24 patients randomly assigned to experimental arm. Median follow-up was 1.2 years. TK cells were timely given at a median of 28 days after HSCT (95% CI, 25 to 35). IR (defined as a CD3+ cell count > 100/µL) was achieved by 15 of 19 patients (79%) who received TK cells, after a median number of 2 doses (range, 1 to 4) and a median cumulative TK-cell dose of 2.4x107 (range, 1.0 to 3.9). The median time to reach IR computed from HSCT and last TK-cell infusion was 105 days (95% CI, 68 to 125) and 27 days (95% CI, 23 to 33), respectively. No patient received post-HSCT immune-suppressive therapy as GvHD prophylaxis. Acute GvHD developed in 7 patients (6 grade II and 1 grade III) for a 1-year cumulative incidence of 33% (±10) and was rapidly abrogated by suicide-gene induction with ganciclovir administered for a median of 14 days. None of the patients who experienced GvHD subsequently received prophylaxis with long-term immune-suppressive therapy (IST). No progression from acute to chronic GvHD and no GvHD-related death occurred. TK-cell-induced IR was characterized by a wide T-cell repertoire and high frequency of T cells specific for opportunistic pathogens and was associated with high survival rates. By ITT analysis at 1 year, OS was 85% (±8), DFS and IST-free survival were both 74% (±10) and NRM was 10% (±7). For patients achieving IR, the corresponding values of OS, DFS and NRM were 100%, 86% and 0%, respectively. Relapse incidence at 1 year was 16% (±8) for all patients and resulted related to the cumulative TK-cell dose, being 0% for patients who had received higher doses (≥3x107) compared to 20% for those treated with lower doses. By pooling data from TK007 and TK008 trial, these dose-related outcomes were confirmed in a landmark analysis set at 100 days after HSCT to mitigate the guarantee-time bias, with 5-years relapse incidence being inversely related to the TK-cell dose received (0%, 19% and 64% for doses ≥ 3x107, 1.1-2.9x107 and ≤ 1.0x107, respectively; p=0.008) Conclusions: Preliminary results of this ongoing phase 3 trial confirm the potential clinical benefit of T-cell gene transfer technology integrated with T-cell depleted haploidentical HSCT, and highlight the role of early IR as surrogate endpoint for survival outcomes and the dose-related antileukemic effects of TK cells Disclosures Niederwieser: Novartis, Gentium, Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Colombi:MolMed: Employment. Antonio:MolMed: Employment. Bordignon:MolMed: Employment.
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- 2014
38. GvHD Kinetics after Haploidentical TK-Cells: In-Vivo HSV-TK Suicide Machinery Is Effective in GvHD Control and Provide a Long-Term Immune-Suppressive Treatment-Free Survival
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Attilio Bondanza, Luca Vago, Giacomo Oliveira, Claudio Bordignon, Chiara Bonini, Antonio Lambiase, Raffaella Greco, Scialini Colombi, Jacopo Peccatori, Maria Teresa Lupo-Stanghellini, and Fabio Ciceri
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Ganciclovir ,medicine.medical_specialty ,business.industry ,Immunology ,Valganciclovir ,Cell Biology ,Hematology ,Suicide gene ,medicine.disease ,Donor Lymphocytes ,Biochemistry ,Gastroenterology ,Transplantation ,Leukemia ,Prednisone ,Concomitant ,Internal medicine ,medicine ,business ,medicine.drug - Abstract
Introduction Extensive application of haploidentical SCT (haplo-SCT) is limited by high rate of late transplant mortality and relapse incidence associated with the delayed immune-reconstitution (IR) secondary to the procedures for severe graft-versus-host-disease (GvHD) prevention and treatment. In the past 20 years we deeply investigate the application of the paradigmatic herpes simplex virus thymidine kinase (TK) suicide gene strategy to allow the selective elimination of genetically modified donor T cells during GvHD, while sparing IR with effective graft-versus-leukemia and graft-versus-infectious effects. Aim of the study Here we report the incidence, characterization, stratification, treatment and outcome for both acute (a-) and chronic (c-) GvHD in haplo-SCT after TK-cells infusion. Methods We included for analysis 57 adult patients (pts, median age 53 years – r 17-66) who underwent an haplo-SCT according to TK-trial (Ciceri, Bonini et al, Lancet Oncol 2009; Phase III TK008, NCT0091462), between 2002 and 2014 at our Center. Data were collected from our Institutional database. A written consent was given by pts allowing the use of medical records for research in accordance with the Declaration of Helsinki. All consecutive pts receiving graft after selection of peripheral CD34+ cells - CliniMacs one-step procedure - were selected. No immune-suppression was introduced after SCT as GvHD prophylaxis. In vivo T-cell depletion with ATG (Fresenius) was administered in all pts. Donor lymphocytes genetically engineered to express the TK gene were infused in 34/57 pts (median 2 infusion/pts), 25/34 achieved IR (median time from SCT 84 days – r 18/182; median time from last TK-cells infusion 27 days – r 13/42). Results Twelve of 25 immune-reconstituted pts developed a-GvHD (grade I–IV; median time of onset 84 days post SCT – r 20/162; 19 days post last TK-infusion – r 8/54) and one developed c-GVHD. Direct association of TK-cells and GvHD was confirmed by vector-encoded protein immunostaining of lymphocytes infiltrating affected lesions. Eleven pts needed GvHD treatment: 4 pts received ganciclovir iv (GCV 5 mg/Kg/12h/14 days), 7 pts valganciclovir per os (VGCV 900 mg/12h/14 days). Both GCV and VGCV were effective in control clinical manifestations of GvHD in a median of 14 days (see figures 1. and 2.) and resulted in a significant reduction in numbers of circulating TK-cells, without reduction of CD3+ TK-negative lymphocytes maintaining long-term IR (see table). In 5 pts additional concomitant treatment with low-dose steroid (prednisone A pt who presented severe gut and liver GvHD and one who received at SCT an high dose of unmanipulated lymphocytes (5.4x105/Kg) – were successfully treated with a combined therapy of prednisone and cyclosporine or rapamicine in association with GCV. Patient TK44 developed a severe classic de novo c-GVHD, with sclerodermatous lichenoid skin and mouth features plus moderate dry-eye symptoms that was successfully treated with VGCV and a transient course of mycophenolate mofetil (2 g per day) over a 2 months period. No cases of quiescent or progressive c-GvHD was observed after a median follow-up of 679 days (r 139/4035). Conclusion In our 12-years experience we can confirm that infusion of TK-cells is effective in accelerating IR while controlling GvHD, providing a long-term immunosuppressive therapy free survival in absence of GvHD related deaths or long-term complications. Abstract 548 Despite a consistent reduction in TK-cell numbers, the GCV/VGCV treatment of GVHD did not impair or prevent long-term IR. TK-cells infused / Kg x107 Time from SCT to GvHD (days) Time from last TK-cells to GvHD (days) Description (acute – chronic, grade) Before GCV/VGCV After GCV/VGCV GvHD outcome ,days after 1st dose of GCV/VGCV TK+ cells/mcl TK-cells/mcl % of TK+,tot CD3+cells TK+ cells/mcl TK-cells/mcl % of TK+, tot CD3+cells TK5 1 91 15 A II 36 248 12.7 7 378 1.8 CR 21 TK6 1 98 51 A I 213 270 44.1 / / / CR NA TK8 10 20 17 A IV 207 224 57.9 24 36 40.2 CR 20 TK16 2.2 20 8 A II 90 99 47.6 25 69 26.6 CR 4 TK20 2.4 30 28 A II 22 139 13.7 13 129 9.2 CR 29 TK25 0.4 162 14 A II 23 123 16 13 184 6.8 CR 7 TK38 1 110 54 A III 99 436 15.6 15 503 2.0 CR 10 TK44 1 159 146 C severe 12 399 2.9 0 303 0 CR 84 TK47 1 90 19 A II 351 422 45.4 34 222 13.3 CR 3 TK50 1 66 41 A II 355 238 59.9 26 88 22.8 CR 18 TK1007A 1.2 117 30 A I 20 146 12 / / / CR NA TK1011A 1.06 78 20 A II 11 97 11.3 7 131 5 CR 90 TK1014A 0.96 15 10 A II 11 41 26.8 2 85 2.3 CR 8 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Bonini: MolMed S.p.A.: Consultancy. Colombi:MolMed: Employment. Lambiase:MolMed S.p.A: Employment. Bordignon:MolMed: Employment.
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- 2014
39. Long-Term Immunological Profile of Patients Treated with Haploidentical HSCT and TK-Cells to Study the Requirements of Memory T Cell Persistence
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Chiara Bonini, Fabio Ciceri, Giacomo Oliveira, Catia Traversari, Nicoletta Cieri, Christof von Kalle, Alessandro Aiuti, Manfred Schmidt, Maddalena Noviello, Maria Teresa Lupo Stanghellini, Sara Mastaglio, Luca Vago, Claudio Bordignon, Antonio Lambiase, Raffaella Greco, Mattia D'Agostino, Eliana Ruggiero, Luca Biasco, and Attilio Bondanza
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business.industry ,Genetic enhancement ,T cell ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Suicide gene ,medicine.disease ,Biochemistry ,Immune system ,medicine.anatomical_structure ,Graft-versus-host disease ,Medicine ,business ,Memory T cell ,CD8 - Abstract
BACKGROUND: Suicide gene therapy applied to haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. RESULTS: We studied 9 adult patients who underwent haplo-HSCT and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-39.5x106) for high-risk hematologic malignancies between 1995 and 2010 (TK patients). At a median follow-up of 7,4 years (range 3.2-12.3), all patients are in complete remission. Two out of 9 patients (22%) experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. A complete recovery of NK cells, B lymphocytes and αβ or γδ T cells was observed. The CD8+ and CD4+ T cell compartment of TK patients were characterized by level of naïve and memory cell comparable to age and sex matched healthy controls. The quantification of CD4+ CD31+ CD62L+ CD45RA+ CD95- recent thymic emigrants and measure of single joint T-cell receptor excision circles demonstrated that the normalization of the T cell compartment was supported by a completely recovered thymic output. TK-cells were detected in all patients (100%), at low levels (median=4cells/uL). Ex vivo selection of pure TK-cells after polyclonal stimulation and LNGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Of notice TK-cells could be retrieved also in patients successfully treated with GCV for GvHD, thus confirming the selective action of GCV only on proliferating TK-cells. Accordingly, GCV sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. TK-cells circulating in patients displayed a memory phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells and exhibited a low level of Ki-67 positivity, thus suggesting the maintenance of a pool of gene modified memory cells through homeostatic proliferation. The number of TK-cells circulating at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. We evaluated whether the phenotype of infused TK-cells was able to affect the long-term fate of gene-modified memory T cells. We observed that the number of infused TSCM cells positively correlated with early TK-cell expansion and with their long-term persistence, suggesting that TSCMmight play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: These data show that a complete and physiological donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 12 years in treated patients. This setting can be exploited to investigate the requirements at the basis of the generation of a long-lasting immunological memory in vivo. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Chairman and CEO Other. Bonini:MolMed S.p.A: Consultancy.
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- 2014
40. Efficacy of HSV-TK+ suicide gene donor lymphocytes after haploidentical transplantation (haplo-HSCT): Preliminary results of randomized TK008 study
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Giacomo Oliveira, Chiara Bonini, Lutz Uharek, Arnon Nagler, Maria Teresa Lupo Stanghellini, Dietger Niederwieser, Raffaella Greco, Wolfgang Bethge, Fabio Ciceri, Andrew L. Pecora, Eduardo Olavarria, Michael Stadler, John F. DiPersio, Eva M Weissinger, Attilio Bondanza, Claudio Bordignon, Michele Donato, Evangelia Yannaki, Donald Bunjes, and Antonio Lambiase
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Cancer Research ,Leukemia ,Oncology ,Haploidentical transplantation ,business.industry ,medicine.medical_treatment ,Immunology ,Medicine ,Hematopoietic stem cell transplantation ,Suicide gene ,business ,medicine.disease ,Donor Lymphocytes - Abstract
7003 Background: Haploidentical family donors represent the ideal solution to offer for every patient with high risk leukemia the potential cure of hematopoietic stem cell transplantation. Extensiv...
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- 2014
41. Long-Term Immunological Profile and T Cell Dynamics In Patients Treated With Allogeneic Transplantation and TK-Cells For Hematological Malignancies
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Chiara Bonini, Giacomo Oliveira, Catia Traversari, Maddalena Noviello, Maria Teresa Lupo Stanghellini, Eliana Ruggiero, Nicoletta Cieri, Veronica Valtolina, Raffaella Greco, Luca Vago, Christof von Kalle, Manfred Schmidt, Claudio Bordignon, Fabio Ciceri, and Anna Paruzynski
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business.industry ,medicine.medical_treatment ,T cell ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Suicide gene ,medicine.disease ,Biochemistry ,Immune system ,medicine.anatomical_structure ,Graft-versus-host disease ,medicine ,Cytotoxic T cell ,business ,Memory T cell ,CD8 - Abstract
Background Suicide gene therapy applied to allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. Results We studied 14 adult patients who underwent allo-HSCT (haploidentical HSCT: n=11; HLA-identical HSCT n=3) and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-2.8x108) for high-risk hematologic malignancies between 1995 and 2010. At a median follow-up of 8,7 years (range 3-17), all patients are in complete remission. Five out of 14 patients experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. We observed a complete recovery of NK cells, comprising of mature (CD56+CD16+) and immature (CD56+CD16-) NK cells. Interestingly the proportion of B cells circulating long-term in patients was significantly higher than that observed in age-related healthy controls (p TK-cells were detected in the majority of analyzed patients (90%), at low levels (median=0,43%±6,9%). Ex vivo selection of pure TK-cells after polyclonal stimulation and NGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. The proportion of TK-cells detectable at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first 3 months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Of notice TK-cells could be retrieved also in patients successfully treated with ganciclovir for GvHD, thus confirming the selective action of ganciclovir only on proliferating TK-cells. Accordingly, ganciclovir sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. While infused TK-cells displayed a predominant effector memory phenotype, gene modified T cells persisting long-term were enriched for central memory (CD45RA-CD62L+) and stem memory (CD45RA+CD62L+CD95+) phenotypes, suggesting the higher ability of these T cell subsets to persist and shape the immunological profile long-term in treated patients. Conclusion These data show that a complete donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 14 years in treated patients. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures: Valtolina: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Employment. Bonini:MolMed S.p.A: Consultancy.
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- 2013
42. Modeling Antileukemic Adoptive Immunotherapy In Mouse-Humans Chimeras To Identify Novel Mechanisms Of Cancer Immunoediting
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Dejan Lazarevic, Chiara Bonini, Cristina Toffalori, Claudio Bordignon, Giacomo Oliveira, Jose Manuel Garcia-Manteiga, Barbara Camisa, Fabio Ciceri, Elia Stupka, Lara Crucitti, Massimo Bernardi, Attilio Bondanza, Luca Vago, Katharina Fleischhauer, and Jacopo Peccatori
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T cell ,Immunology ,Antigen presentation ,Cell Biology ,Hematology ,Human leukocyte antigen ,Suicide gene ,Biology ,medicine.disease ,Biochemistry ,Leukemia ,medicine.anatomical_structure ,Immune system ,Immunoediting ,medicine ,Bone marrow - Abstract
Introduction In spite of the documented efficacy of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) in the cure of Acute Myeloid Leukemia (AML), post-transplantation relapse remains an unmet clinical need. According to the “leukemia immunoediting” hypothesis relapse may be due to the outgrowth of immune-resistant leukemic variants upon the selective pressure of the transplanted immune system. We provided a clinically relevant proof-of-principle of this model in transplanted patients (Vago et al, N Engl JMed, 2009), but ex vivo studies often lack mechanistic insights, due to high inter-individual variability and lack of suitable controls. Conversely, “mouse-in-mouse” models of cancer immunoediting can provide precious and reproducible insights into molecular mechanisms, but those results are often difficult to translate into clinical practice due to species-specific factors. To overcome these limitations, here we set up a novel mouse-human chimeric model of antileukemic adoptive immunotherapy, to dissect how T cell immune pressure can sculpt leukemia gene expression profile. Methods Purified primary human AML blasts were infused into non-irradiated immunodeficient NOD/SCID γ-chain null (NSG) mice. Upon documentation of leukemia engraftment, mice received serial infusions of human T cells, either autologous or allogeneic (HLA-identical, HLA-haploidentical or HLA-disparate) to the leukemic cells to mimic immune pressure. Absolute counts of human leukemic and T cells were monitored weekly in mice peripheral blood. At sacrifice, leukemic cells were FACS-purified, total RNA was extracted and gene expression profile was analyzed using Illumina microarray. Deregulated genes and signatures were identified by pairwise LIMMA analysis. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) curated databases were interrogated to identify deregulated processes. Results Infused leukemic cells stably and reproducibly engrafted into the murine bone marrow, and could be detected circulating in the peripheral blood of treated mice from three weeks after infusion. Leukemic cells exponentially expanded, could be transferred to secondary and tertiary recipients, and, in the absence of immune pressure, displayed a stable gene expression profile amongst littermates and upon serial transfer. HLA-disparate and HLA-haploidentical T cells eradicated AML from 10/10 treated mice, and complete eradication was confirmed by no disease recurrence in second transplant recipients. HLA-identical T cells granted only temporary control in 6/6 mice, while autologous T cells were completely inefficacious in 6/6 mice. Leukemic blasts subjected to T cell-mediated immune pressure showed a specific and reproducible gene signature. GO and GSEA demonstrated the selective deregulation of genes involved in immune processes. Among the top-ranked upregulated genes we identified genes related to response to interferons, comprising proteasome and immunoproteasome subunits, as well as molecules and receptors involved in antigen processing and presentation, comprising classical and non-classical HLA Class I and II molecules (HLA-DMA, HLA-DRA, CD74, B2M, TAPBP, TAPBPL). Conclusions Our findings provide further proof of the leukemia immunoediting hypothesis, demonstrating that leukemic cells modify their expression profile, and their immunogenicity, in response to T cell-mediated immune pressure, and that antigen presentation pathways represent key targets in these processes. The model we set up provides a novel and valuable tool to investigate these mechanisms in detail. Experiments with T cells genetically modified to express a suicide gene are currently ongoing, for a time-wise control of alloreactivity aimed at modeling a longer phase of equilibrium between adoptively transferred immune cells and leukemia. Disclosures: Bordignon: MolMed SpA: Employment. Bonini:MolMed SpA: Consultancy.
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- 2013
43. Tracking T Cell Dynamics In The First Month After Haplo-HSCT With Post-Transplant Cyclophosphamide Reveals a Predominant Contribution Of Memory Stem T Cells To The Early Phase Of Immune Reconstitution
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Jacopo Peccatori, Fabio Ciceri, Chiara Bonini, Sarah Marktel, Giacomo Oliveira, Nicoletta Cieri, Francesca Lunghi, and Raffaella Greco
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medicine.medical_treatment ,T cell ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Biology ,medicine.disease ,Biochemistry ,Transplantation ,Graft-versus-host disease ,medicine.anatomical_structure ,Immune system ,medicine ,Cytotoxic T cell ,Bone marrow ,CD8 - Abstract
Haploidentical hematopoietic stem cell transplantation (HSCT) with T–replete grafts and post-transplant cyclophosphamide (PT-Cy) has gained much interest in the transplantation community for the low rates of GvHD, non-relapse mortality and opportunistic infections. This platform, devoid of anti-thymocyte globulins, allows a thorough analysis of circulating cells in the early phase post-HSCT. Indeed, several biological events that play a critical role for transplant outcome occur within the first month after HSCT; while engraftment and hematological reconstitution are carefully monitored, the shape of T cell dynamics within this timeframe remains largely unknown. We characterized immune reconstitution (IR) during the first month post HSCT in 18 high-risk leukemia patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft (PBSCs), and GvHD prophylaxis consisting of PT-Cy (day 3, 4), followed by mycophenolate mofetil and sirolimus from day 5. Infused PBSCs and blood samples harvested at day 1, 3, 5, 8, 15 and 30 post HSCT were analyzed by multiparametric flow cytometry. T cells were infused in the absence of immunosuppressive agents, and by day 3, prior to PT-Cy, a large fraction of memory lymphocytes, possibly enriched for allo-specificities, proliferated (assessed by Ki-67 staining). Conversely, naïve T cells (TN) were scantly Ki-67+ (P< 0.001). A high CD4:CD8 ratio was observed at this time-point (10). PT-Cy efficiently abated T cell proliferation and appeared to affect CD4 more than CD8 T cells. Nevertheless, T cell numbers progressively increased (mean CD3 counts, day 5: 19 cells/µL; day 8: 27 cells/µL; day 15: 97 cells/µL), suggesting that residual proliferation in extravascular sites was likely to fuel the surge in circulating T cells. Consistently, we observed an expansion of antigen-experienced T cells including central memory (TCM), effector memory (TEM), effectors (TEFF) and the recently described stem memory T cells (TSCM). TSCM are a subset of memory cells hierarchically superior to TCM and TEM, for self-renewal, long-term persistence and functional capacity. Similarly to TN, TSCM coexpress CD45RA and CD62L but differently from TN, TSCM express CD95, a marker of memory cells. As early as day 8 post HSCT, the T cell compartment was predominantly composed by TSCM cells (P < 0.01 compared to all other subsets). Such enrichment in TSCM was not due to a selective resistance to PT-Cy, as suggested by the lack of activity of the ALDH enzyme, which converts Cy to a non-toxic metabolite, in TSCM infused with the graft. Rather, we hypothesized that TSCM expansion came directly from the differentiation of TN infused within the graft, which escaped the purging effect of PT-Cy thanks to a delayed activation kinetics compared to alloreactive memory T cells. We demonstrated the in vivo differentiation of WT1 and PRAME specific TN cells, present in the graft, into memory lymphocytes, comprising TSCM cells, in 4/7 patients suitable for dextramer tracking. Such tumor specific T cells were detected in the peripheral blood and bone marrow of treated patients, suggesting that PT-Cy did not hamper GvL players. Of note in the remaining patients for whom tumor-specific TN were not detectable in the graft, no tumor response could be documented in vivo. From day 15 post HSCT, TSCM were outnumbered by other memory subsets, suggesting their differentiation into more committed TCM TEM and TEFF. The quality of IR correlated with clinical events. The percentage of circulating Ki-67+ CD8 TEMcells at day 8 post HSCT accurately predicted the occurrence of periengraftment syndrome, observed in 4 patients with a median time to onset of 15 days. Acute GvHD (Grade I/II in 6 patients, grade III/IV in 4) was accompanied by a rise in circulating Ki-67+ CD8 cells, and response to therapy resulted in a drop in Ki-67 expression. No immunological parameter correlated with chronic GvHD, observed in 2 patients. In all patients with a CMV-seropositive donor, CMV-specific T cells were tracked in the graft, at early time-points and up to 180 days post HSCT, indicating that virus-specific T cells escaped PT-Cy. These results suggest that PT-Cy acts mainly on alloreactive memory T cells infused within the graft, while sparing infused virus-specific, non cross-reactive, memory cells and TN, which can differentiate predominantly in TSCM, but also in TCM TEM and TEFF, thus promoting a rapid and broad IR. Disclosures: Bonini: MolMed SpA: Consultancy.
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- 2013
44. Non-Linear Clonal Evolution Of Leukemia Driven By Selective Immune Pressure and Revealed By HLA Typing and High-Depth Exome Sequencing
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Maurilio Ponzoni, Chiara Bonini, Cristina Toffalori, Matteo Carrabba, Benedetta Mazzi, Claudio Bordignon, Lorenza Chiesa, Jacopo Peccatori, Davide Cittaro, Fabio Ciceri, Dejan Lazarevic, Katharina Fleischhauer, Giacomo Oliveira, Massimo Bernardi, Luca Vago, and Elia Stupka
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medicine.medical_treatment ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Human leukocyte antigen ,Hematopoietic stem cell transplantation ,Biology ,medicine.disease ,Biochemistry ,Somatic evolution in cancer ,Leukemia ,medicine ,Clone (B-cell biology) ,Exome ,Exome sequencing - Abstract
Introduction Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the best curative option for many patients with Acute Myeloid Leukemia and related malignancies, mostly due to the antileukemic activity of immune cells contained in the graft. Although in most of the cases HSCT can accomplish apparent disease eradication, frequently residual leukemic cells are able to evade elimination, eventually outgrowing and resulting in clinical relapse. Apparently indistinguishable from disease at diagnosis, relapses commonly display a more aggressive behavior, and most of the available therapeutic options are ineffective. Next-generation sequencing provides the opportunity to track specific alterations and disease subclones during the clinical history of patients, and from this information to identify mechanisms by which leukemic cells evade elimination. Methods Serial disease samples collected longitudinally during the complex clinical history of a patient with high-risk myelodysplastic syndrome were analyzed by genomic HLA typing and by high-depth exome sequencing (minimum depth of coverage 70x). Patient fibroblasts and mononucleated peripheral blood cells harvested at remission served as reference for germline HLA typing and exome sequence. Whenever possible, leukemic blast were FACS-purified. Sequencing data were aligned to the reference genome using the BWA aligner and analyzed using the tools developed from the Cancer Genome Analysis group at the Broad institute to identify newly acquired mutations, clonal segregation of novel and pre-existing mutations and quantitative clonal dynamics. Results A 54 year old patient with high-risk refractory anemia with excess blasts (pancytopenia, blasts 19%, adverse cytogenetics) underwent unmanipulated T cell-repleted HLA-haploidentical HSCT in the presence of active disease. After one year of remission a first relapse occurred. HLA typing of the leukemic blasts harvested at relapse demonstrated the selective genomic loss of the mismatched HLA haplotype targeted by donor T cells, a frequent mechanism of leukemia immune evasion after haploidentical HSCT, first described by our group (Vago et al, N Engl J Med, 2009). The patient was re-transplanted from a different haploidentical donor mismatched for the HLA alleles retained by the mutated variants; accordingly, T cells from this donor were expectedly alloreactive against the relapsed leukemia. The patient re-obtained complete remission, which was maintained for five years, before the occurrence of a second relapse. Unexpectedly, at this relapse leukemic cells had lost the HLA molecules mismatched with the second donor, and expressed those that had gone amiss at first relapse. Since at both relapses HLA haplotype loss was due to a stable genomic alteration, chromosome 6p acquired uniparental disomy (aUPD), any linear relation between the first and second relapse could be ruled out (Figure 1). Leukemic samples at diagnosis, first relapse and second relapse were further characterized by high-depth exome sequencing, revealing that most of the mutations present in each of the three samples were not found in the other two: only a minute fraction of the mutations could be identified in all three disease presentations, possibly comprising the “driver mutations” harbored by the original leukemic stem cell clone. Amongst these shared mutations we are currently validating the functional and epidemiological relevance of missense alterations in the TP53 tumor suppressor gene, in the NHEJ1 gene (related to chromosomal instability), and in the BTNL8 gene, encoding the costimulatory receptor B7-H5, which may putatively play a role in leukemia recognition by T cells. Conclusions Our results demonstrate that antileukemic immunity has a profound impact on leukemia clonal evolution, driving the selection of immuno-privileged subclones. Importantly, the genetic alterations shared between the leukemic variants that emerged at years of distance during the clinical history of our patient suggest the long-term persistence of a reservoir of leukemic stem cells which are resistant to, or in stable equilibrium with, the immune pressure that sculpted their progeny. Finally, the longitudinal approach adopted in this study holds promise for the identification of leukemia “driver” mutations, to provide new insights into leukemogenesis and new rationales for targeted eradicating therapies. Disclosures: Bordignon: MolMed SpA: Employment. Bonini:MolMed SpA: Consultancy.
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- 2013
45. Long-term safety and survival outcomes after TK-expressing donor lymphocyte infusion (TK-DLI) in allogeneic hematopoietic stem cell transplantation (HSCT)
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Chiara Bonini, Giacomo Oliveira, Fabio Ciceri, Raffaella Greco, Claudio Bordignon, Attilio Bondanza, Antonio Lambiase, Luca Vago, and Maria Teresa Lupo-Stanghellini
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Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,medicine.medical_treatment ,Hematopoietic stem cell transplantation ,Suicide gene ,medicine.disease ,Tumor response ,Donor lymphocyte infusion ,Leukemia ,surgical procedures, operative ,Immune system ,Internal medicine ,Allogeneic hsct ,medicine ,Long term safety ,business - Abstract
7007 Background: Suicide gene therapy (SGT) was firstly applied to allogeneic HSCT, addressing the need for modulation of graft vs host disease (GvHD) reactions while preserving graft vs leukemia (GvL) effect of alloreactive T cells. HSV-TK gene insertion in donor T-cells modulates alloreactivity by selectively destroying dividing alloreactive cells involved in GvHD. Methods: Long-term safety and survival was assessed in 128 pts entering worldwide 10 phase I-II trials that used TK-DLI to improve GvL, immune reconstitution (IR) and GvHD control. In all, 57 pts received TK DLI at our Institution: 23 to treat relapse after HLA-identical HSCT (Ciceri, 2007) and 34 to improve IR after haploidentical HSCT (Ciceri, Bonini, 2009). Results: SGT was feasible, safe and effective in promoting a dynamic and specific modulation of alloreactivity. TK-DLI clinical benefit, defined by chimerism, tumor response and/or IR, was achieved by 65 pts (51%). Grade 2 to 4 GvHD (n=28, 22%) was fully controlled by SGT. TK-DLI engrafted in 51 pts (90%) and, being detectable at low frequency up to 14 yrs, no SGT-related adverse events occurred. In HLA-identical setting (n=23; median follow-up, 15 yrs), 11 pts (48%) had disease response and 2 pts (9%) were alive in complete response (CR). In haploidentical setting (n=34; median follow-up, 7 yrs), 25 pts (73%) had IR and 9 pts (26%) were alive in CR. All pts were monitored according to guidelines on long-term survivors (Majhail, 2012). There were no major infections, while 3 pts had a second tumor. Immunity against TK-DLI was reported exclusively after HLA-identical allo-HSCT indicating that TK-DLI is not limited by SGT-specific immunity after haploidentical HSCT. Conclusions: Long-term follow-up confirms the high benefit to risk ratio of TK-DLI. A phase III trial is ongoing in haploidentical HSCT (NCT00914628). Clinical trial information: NCT00423124.
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- 2013
46. T Cell Suicide Gene Therapy Prompts Thymic Renewal in Adults After Haploidentical Hematopoietic Stem Cell Transplantation in the Absence of Post-Transplant Immunesuppression
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Fabio Ciceri, Sergio Fracchia, Claudio Bordignon, Catia Traversari, Giacomo Oliveira, Chiara Bonini, Maria Teresa Lupo Stanghellini, Domenico Ghio, Attilio Bondanza, Raffaella Greco, Maddalena Noviello, Immacolata Brigida, Alessandro Aiuti, Jacopo Peccatori, Matteo Del Fiacco, Luca Vago, Alessandro Del Maschio, Corrado Soldati, and Antonio Lambiase
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business.industry ,T-cell receptor excision circles ,ELISPOT ,T cell ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Suicide gene ,Donor Lymphocytes ,Biochemistry ,Transplantation ,medicine.anatomical_structure ,Immune system ,Medicine ,business - Abstract
Abstract 1968 BACKGROUND: The genetic modification of T cells with a suicide gene grants a mechanism of control of Graft-versus-Host Disease (GvHD), allowing safe infusion of donor lymphocytes after partially HLA-incompatible Hematopoietic Stem Cell Transplantation (HSCT). In the TK007 phase I-II clinical trial, which enrolled a total of 54 adults with hematologic malignancies, 22 of the 28 treated patients experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the Herpes Simplex Virus Tymidine Kinase suicide gene (TK cells; Ciceri and Bonini et al., Lancet Oncology, 2009). In these patients, after a first wave of circulating TK cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naïve lymphocytes, leading us to hypothesize a thymus-dependent development of T cells, occurring only upon TK cell engraftment. METHODS: Thymic function was investigated in a total of 31 patients enrolled in the TK007 trial (median age 55 years), which were compared to a cohort of adult patients receiving non T cell-depleted haploidentical transplantation (n=31), and to healthy pediatric and adult subjects. T cell subsets and the proportion of CD31+ recent thymic emigrants amongst CD4 naïve T cells were measured by immunophenotypic analysis. Single joint T cell Receptor Excision Circles (sjTREC) were quantified by qPCR. The volume of the biologically active thymus was assessed by chest CT scans. Serum concentration of cytokines was assessed by a multiplex luminex-based assay. Pathogen-specific immunity was quantified by interferon-γ ELISpot. RESULTS: After the infusion of TK cells we documented a significant increase in peripheral blood sjTRECs as compared to the pre-HSCT determination (p = 0.02), suggesting an improved thymic output. Importantly, in line with that, only in TK007 patients almost the totality of CD4 naïve T cells circulating after transplantation were CD31+, thus bona fide recent thymic emigrants (89.54±9.55% at immune reconstitution, 81.84±15.9% at 6 months after HSCT, and 79.55±16.66% at 12 months after HSCT). Accordingly, a substantial expansion of the active thymic tissue was observed at chest tomography scans as compared to the pre-HSCT counterparts (p < 0.0001). A peculiar observation, possibly linked to the renewal of thymic activity and unique to the TK007 patients who achieved immune reconstitution, was the documentation of a peak in the serum level of interleukin-7, reproducibly occurring after each infusion of suicide gene-modified cells and anticipating the appearance of the newly generated T cells. Ultimately, the development of a wide repertoire of T cells in the patient thymus from donor precursors ensured a long-term protective immunity against pathogens, as exemplified by the preservation of a physiological and protective response against viruses both ex vivo and in vivo, even after the elimination of the infused TK cells in case of GvHD. CONCLUSIONS: Our data from TK007 patients show that the infusion of genetically modified donor T cells after transplantation can drive the recovery of thymic activity in adults, leading to long-term immune reconstitution. On the lead of the encouraging biological and clinical results of the phase I-II clinical trial, demonstrating a dramatic decrease in late infectious mortality, a multicenter, phase III clinical trial (TK008 study) to assess the efficacy of TK cells in the context of haploidentical HSCT for leukemia started in 2010 at the San Raffaele Institute, and is currently expanding to multiple centers throughout Europe and US. Main endpoints of this randomized phase III trial are disease free survival and overall survival. The first TK008 patients randomized to receive suicide gene-modified cells showed recovery of thimyc activity and concomitantly achieved a rapid and robust T cell immune reconstitution. Disclosures: Bonini: MolMed SpA: Consultancy.
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- 2011
47. Thymic Renewal and Anti-Leukemic Effect In Adults After Haploidentical Transplantation and Donor T Cell Suicide Gene Therapy
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Luca Vago, Maria Teresa Lupo Stanghellini, Raffaella Greco, Giacomo Oliveira, Claudio Bordignon, Fabio Ciceri, Jacopo Peccatori, Maddalena Noviello, Sara Mastaglio, Chiara Bonini, Domenico Ghio, Roberto Nicoletti, Attilio Bondanza, Alessandro Aiuti, Corrado Soldati, Antonio Lambiase, Immacolata Brigida, and Katharina Fleischhauer
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business.industry ,Lymphocyte ,medicine.medical_treatment ,T cell ,Immunology ,Cell Biology ,Hematology ,Human leukocyte antigen ,Hematopoietic stem cell transplantation ,Suicide gene ,medicine.disease ,Donor Lymphocytes ,Biochemistry ,Leukemia ,medicine.anatomical_structure ,Immune system ,medicine ,business - Abstract
Abstract 833 Introduction: Hematopoietic Stem Cell Transplantation (HSCT) from partially HLA-matched (haploidentical) family donors represents a promising therapy for high-risk leukemia, but requires appropriate strategies to control the adverse reactions mediated by the partially incompatible, transplanted immune system. In a recent phase II study (TK007 study), we demonstrated that the infusion of donor lymphocytes transduced with the Herpes Simplex Virus Thymidine kinase (HSV-Tk) suicide gene allows to control Graft-versus-Host Disease (GvHD) and to rapidly provide an effective and polyclonal anti-infective T cell repertoire (Ciceri and Bonini et al., Lancet Oncology, 2009). Even though their engraftment is necessary to achieve these effects, HSV-Tkpos cells represent the minority of lymphocytes circulating in treated patients. Therefore, in the present study, we investigated the putative role of HSV-Tkpos cells in promoting thymic activity and T cell development from graft progenitors. Methods: Twenty-eight adult patients underwent haploidentical HSCT and infusion of purified suicide gene-modified donor T cells for high-risk hematologic malignancies in the TK007 study. Thymic function was investigated in a selected cohort of this study (n=14) and in a control group who underwent unselected T cell-replete haploidentical transplantation with an ATG-rapamycin-mycophenolate-based GvHD prophylaxis (n=31), after validation in healthy pediatric and adult controls. T cell subsets and the proportion of CD31+ recent thymic emigrants (RTEs) amongst CD4+ naïve T cells were measured by immunophenotypic analysis. Single joint T cell Receptor Excision Circles (sjTREC) were quantified by qPCR. Thymic output was correlated with thymic volume, as assessed by CT scans. Post-transplantation pathogen-specific immune response was quantified by ELISpot. Alloreactivity against leukemic blasts was studied by mixed lymphocyte cultures. Results: Post-transplantation recovery of naïve CD45RA+CD62L+ T cells occurred in patients treated with gene modified T cells, reaching values of healthy controls in approximately one year. At the time of immune reconstitution (median 76 days after HSCT, defined as CD3+ cells > 100/ml peripheral blood), 76.5% of circulating T cells did not carry the HSV-Tk suicide gene, and the CD4+ naïve subset was largely comprised of cells recently originated from the thymus (90.5±3.2%). This observed frequency of CD31+ RTEs in these patients was significantly higher than that measured in the same patients before HSCT (60.7±6%, p=0,0087) or in patients analyzed 90 days after T cell-replete haploidentical HSCT (31.0±6.3%, p Conclusions: These data show that the infusion of suicide gene-modified T cells prompts the renewal of thymic activity, which contributes to the recovery of a polyclonal T cell repertoire protective against pathogens. Contextually, the infused transduced cells mediate also a direct antitumor effect through their recognition of allogeneic determinants on leukemic cells. A phase III clinical trial (TK008 study) to assess the efficacy of HSV-Tkpos cells in the context of haploidentical HSCT for leukemia started in 2010 in Italy, and is currently expanding to multiple centers throughout Europe. Disclosures: Bonini: MolMed S.p.A.: Consultancy.
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- 2010
48. Thymic renewal and antileukemic effect in adults after haploidentical transplantation and suicide gene therapy
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Jacopo Peccatori, Attilio Bondanza, Antonio Lambiase, Giacomo Oliveira, Luca Vago, Fabio Ciceri, Chiara Bonini, Domenico Ghio, Claudio Bordignon, and M.T. Lupo Stanghellini
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endocrine system ,Cancer Research ,Hematopoietic cell ,Haploidentical transplantation ,business.industry ,Suicide gene ,Donor Lymphocytes ,medicine.disease ,Transplantation ,Leukemia ,surgical procedures, operative ,Oncology ,hemic and lymphatic diseases ,Immunology ,Medicine ,business - Abstract
6534 Background: Hematopoietic Cell Transplantation from partially-HLA matched family donors (haplo-HCT) represents a promising therapy for high-risk leukemia. The infusion of donor lymphocytes tra...
- Published
- 2010
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