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The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

The prediction of peptide-MHC (pMHC) recognition by αβ T-cell receptors (TCRs) remains a major biomedical challenge. Here, we develop STAPLER (Shared TCR And Peptide Language bidirectional Encoder Representations from transformers), a transformer language model that uses a joint TCRαβ- peptide input to allow the learning of patterns within and between TCRαβ and peptide sequences that encode recognition. First, we demonstrate how data leakage during negative data generation can confound performance estimates of neural network-based models in predicting TCR – pMHC specificity. We then demonstrate that, because of its pre-training and fine-tuning masked language modeling tasks, STAPLER outperforms both neural network-based and distance-based ML models in predicting the recognition of known antigens in an independent dataset, in particular for antigens for which little related data is available. Based on this ability to efficiently learn from limited labeled TCR- peptide data, STAPLER is well-suited to utilize growing TCR – pMHC datasets to achieve accurate prediction of TCR – pMHC specificity.

Although most characterized tumor antigens are encoded by canonical transcripts (such as differentiation or tumor-testis antigens) or mutations (both driver and passenger mutations), recent results have shown that noncanonical transcripts including long noncoding RNAs and transposable elements (TEs) can also encode tumor-specific neo-antigens. Here, we investigate the presentation and immunogenicity of tumor antigens derived from noncanonical mRNA splicing events between coding exons and TEs. Comparing human non–small cell lung cancer (NSCLC) and diverse healthy tissues, we identified a subset of splicing junctions that is both tumor specific and shared across patients. We used HLA-I peptidomics to identify peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides were immunogenic in vitro, and CD8 + T cells specific for junction-encoded epitopes were present in tumors and tumor-draining lymph nodes from patients with NSCLC. We conclude that noncanonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in patients with NSCLC.

Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse. Significance: Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397

T cell receptor (TCR) sequencing has been used to characterize the immune response to cancer. However, most analyses have been restricted to quantitative measures such as clonality that do not leverage the complementarity-determining region 3 (CDR3) sequence. We use DeepTCR, a framework of deep learning algorithms, to reveal sequence concepts that are predictive of response to immunotherapy. We demonstrate that DeepTCR can predict response and use the model to infer the antigenic specificities of the predictive signature and their unique dynamics during therapy. The predictive signature of nonresponse is associated with high frequencies of TCRs predicted to recognize tumor-specific antigens, and these tumor-specific TCRs undergo a higher degree of dynamic changes on therapy in nonresponders versus responders. These results are consistent with a biological model where the hallmark of nonresponders is an accumulation of tumor-specific T cells that undergo turnover on therapy, possibly because of the dysfunctional state of these T cells in nonresponders.

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.

Within the tumour microenvironment, CD4(+) T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules(1,2), but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4(+) T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4(+) T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4(+) T regulatory (T(Reg)) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4(+) T(Reg) clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4(+) T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4(+) T(Reg) cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.

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.

After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P

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.

Background: Evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, up to 40% of AML relapses display complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods: Starting from primary AML samples pairwise collected from five patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches and in the different patients were integrated by Multi-Omics Factor Analysis (MOFA) and Gene Set Enrichment Analysis (GSEA). Finally, we tested the immunological effects of epigenetic drugs on AML cells and on their recognition by T cells in ex-vivo short-term cultures and in PDXs. Results: We verified that PDXs faithfully recapitulate immune-related differences between AML diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules. Differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility, and largely unrelated to the DNA methylation profile. In particular, in all five patients analyzed, we documented genomewide chromatin compaction at time of relapse, that was particularly evident for HLA class II genes and their master regulator CIITA, and not detected in relapses after sole chemotherapy. Integration of all the high-throughput technologies by MOFA, and of results from different patients by GSEA, pointed to the Polycomb Repressive Complex 2 (PRC2) as the main candidate mediator of HLA class II silencing.Pharmacological inhibition of PRC2 subunits rescued HLA class II expression in AML relapses ex vivo and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Conclusions: Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML post-transplantation relapse. Citation Format: Valentina Gambacorta, Stefano Beretta, Martina Ciccimarra, Laura Zito, Kety Giannetti, Angela Andrisani, Daniela Gnani, Lucia Zanotti, Giacomo Oliveira, Matteo G. Carrabba, Davide Cittaro, Ivan Merelli, Fabio Ciceri, Raffaella Di Micco, Luca Vago. Integrated multiomic profiling identifies the epigenetic regulator PRC2 as a therapeutic target to counteract leukemia immune escape and relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB563.

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.

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.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses

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.

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.

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.

Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.

rhTCRseq (RNase H-dependent PCR-enabled T cell receptor sequencing) is a procedure that can be used to determine paired alpha/beta T cell receptor (TCR) clonotypes in single cells or perform both alpha and beta TCR repertoire analysis in bulk RNA samples. Relying on the enhanced specificity of RNase H-dependent PCR (rhPCR), it achieves TCR-specific amplification and addition of dual index barcodes in a single PCR step. For single cells, the stages of the protocol are sorting single cells into 96- or 384-well plates, generation of full-length cDNA libraries, the single TCR-specific amplification step, a second PCR on a pooled sample to generate a sequencing library, and sequencing on the MiSeq platform. In the bulk RNA method, the sorting and cDNA library steps are replaced with a reverse transcriptase reaction that add a Unique Molecular Identifier (UMI) to each cDNA molecule in order to improve the accuracy of repertoire frequency measurements. Compared to other multiplex PCR methods for TCR sequencing, rhTCRseq has a streamlined workflow and the ability to analyze single cells in 384-well plates. Compared to TCR reconstruction from single-cell transcriptome sequencing data, it improves the success rate for obtaining paired alpha/beta information and ensures recovery of complete CDR3 sequences, which is a prerequisite for the cloning and expression of discovered TCRs. Although it does not have the throughput of droplet-based methods, rhTCRseq is well-suited to the analysis of small sorted populations, especially cases where analysis of 96 or 384 single cells is sufficient to identify predominant T cell clones. For single cells, sorting typically requires two to four hours and can be performed days, or even months, before library processing. The remainder of the single cell protocol takes on the order of four days, including data processing. For bulk RNA, the overall time is about three days, including data processing.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.