20 results on '"Anczurowski M"'
Search Results
2. Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4 + T cells.
- Author
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Sugata K, Matsunaga Y, Yamashita Y, Nakatsugawa M, Guo T, Halabelian L, Ohashi Y, Saso K, Rahman MA, Anczurowski M, Wang CH, Murata K, Saijo H, Kagoya Y, Ly D, Burt BD, Butler MO, Mak TW, and Hirano N
- Subjects
- CD4 Antigens chemistry, CD4 Antigens metabolism, CD4-Positive T-Lymphocytes chemistry, CD4-Positive T-Lymphocytes cytology, CD4-Positive T-Lymphocytes metabolism, Cells, Cultured, Flow Cytometry, Humans, Protein Binding, HLA Antigens chemistry, HLA Antigens metabolism, Histocompatibility Antigens Class II chemistry, Histocompatibility Antigens Class II metabolism, Staining and Labeling methods
- Abstract
Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4
+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)- Published
- 2021
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3. Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.
- Author
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Kagoya Y, Guo T, Yeung B, Saso K, Anczurowski M, Wang CH, Murata K, Sugata K, Saijo H, Matsunaga Y, Ohashi Y, Butler MO, and Hirano N
- Subjects
- Allografts, Animals, Antigens, CD19 immunology, CRISPR-Cas Systems, Cells, Cultured, Disease Models, Animal, Histocompatibility Antigens Class I genetics, Histocompatibility Antigens Class II genetics, Humans, Leukocytes, Mononuclear, Lymphocyte Activation, Mice, Mice, Inbred NOD, Mice, SCID, Neoplasms immunology, Neoplasms metabolism, Receptors, Antigen, T-Cell antagonists & inhibitors, Receptors, Antigen, T-Cell genetics, Histocompatibility Antigens Class I chemistry, Histocompatibility Antigens Class II chemistry, Immunotherapy, Adoptive methods, Neoplasms therapy, Receptors, Antigen, T-Cell immunology, Receptors, Chimeric Antigen immunology
- Abstract
Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA , and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy., (©2020 American Association for Cancer Research.)
- Published
- 2020
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4. Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma.
- Author
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Murata K, Nakatsugawa M, Rahman MA, Nguyen LT, Millar DG, Mulder DT, Sugata K, Saijo H, Matsunaga Y, Kagoya Y, Guo T, Anczurowski M, Wang CH, Burt BD, Ly D, Saso K, Easson A, Goldstein DP, Reedijk M, Ghazarian D, Pugh TJ, Butler MO, Mak TW, Ohashi PS, and Hirano N
- Subjects
- Humans, Receptors, Antigen, T-Cell immunology, Antigens, Neoplasm immunology, Epitopes, T-Lymphocyte immunology, Histocompatibility Antigens Class I immunology, Lymphocytes, Tumor-Infiltrating immunology, Melanoma immunology
- Abstract
HLA-restricted T cell responses can induce antitumor effects in cancer patients. Previous human T cell research has largely focused on the few HLA alleles prevalent in a subset of ethnic groups. Here, using a panel of newly developed peptide-exchangeable peptide/HLA multimers and artificial antigen-presenting cells for 25 different class I alleles and greater than 800 peptides, we systematically and comprehensively mapped shared antigenic epitopes recognized by tumor-infiltrating T lymphocytes (TILs) from eight melanoma patients for all their class I alleles. We were able to determine the specificity, on average, of 12.2% of the TILs recognizing a mean of 3.1 shared antigen-derived epitopes across HLA-A, B, and C. Furthermore, we isolated a number of cognate T cell receptor genes with tumor reactivity. Our novel strategy allows for a more complete examination of the immune response and development of novel cancer immunotherapy not limited by HLA allele prevalence or tumor mutation burden., Competing Interests: KM The University Health Network has filed patent application related to this study on which Kenji Murata is named as an inventor (US16/095,913, US62/813,639, US62/813,642, US62/813,644, US62/813,645, US62/813,647, US62/813,650, US62/813,651, and US62/823,487). MN The University Health Network has filed patent application related to this study on which Munehide Nakatsugawa is named as an inventor (US16/095,913). MR The University Health Network has filed patent application related to this study on which Muhammed A. Rahman is named as an inventor (US16/095,913). LN, DM, DM, KS, HS, YM, YK, TG, MA, CW, BB, DL, AE, DG, MR, DG, TP, TM, PO No competing interests declared, KS The University Health Network has filed patent application related to this study on which Kayoko Saso is named as an inventor (US62/813,639, US62/813,642, US62/813,644, US62/813,645, US62/813,647, US62/813,650, US62/813,651, and US62/823,487). MB Marcus O. Butler has served on advisory boards for Merck, BMS, Novartis, GSK, Immunocore, immunovaccine, Sanofi, and EMD Serono and received research funding for investigator initiated clinical trials from Merck and Takara Bio. NH Naoto Hirano reports receiving a research grant from and is a consultant for Takara Bio and Otsuka Pharmaceutical and serving on an advisory board for F. Hoffmann-La Roche. The University Health Network has filed patent application related to this study on which Naoto Hirano is named as an inventor (US16/095,913, US62/813,639, US62/813,642, US62/813,644, US62/813,645, US62/813,647, US62/813,650, US62/813,651, and US62/823,487)., (© 2020, Murata et al.)
- Published
- 2020
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5. Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP 84GGPM87 .
- Author
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Anczurowski M, Sugata K, Matsunaga Y, Yamashita Y, Wang CH, Guo T, Murata K, Saijo H, Kagoya Y, Saso K, Butler MO, and Hirano N
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 3 genetics, ATP Binding Cassette Transporter, Subfamily B, Member 3 immunology, Antigens immunology, CD4-Positive T-Lymphocytes immunology, Calnexin genetics, Calnexin immunology, Calreticulin genetics, Calreticulin immunology, Cell Line, HEK293 Cells, Humans, Membrane Transport Proteins genetics, Membrane Transport Proteins immunology, Molecular Chaperones genetics, Proteasome Endopeptidase Complex metabolism, Protein Disulfide-Isomerases genetics, Protein Disulfide-Isomerases immunology, Antigen Presentation immunology, HLA-DP Antigens immunology, Histocompatibility Antigens Class I immunology, Molecular Chaperones immunology
- Abstract
Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPβ chain (DP
84GGPM87 ). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2019
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6. Arginine methylation of FOXP3 is crucial for the suppressive function of regulatory T cells.
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Kagoya Y, Saijo H, Matsunaga Y, Guo T, Saso K, Anczurowski M, Wang CH, Sugata K, Murata K, Butler MO, Arrowsmith CH, and Hirano N
- Subjects
- Animals, Biomarkers, Cell Line, Cytokines metabolism, Disease Models, Animal, Fluorescent Antibody Technique, Forkhead Transcription Factors genetics, Gene Expression Profiling, Graft vs Host Disease etiology, Graft vs Host Disease metabolism, Graft vs Host Disease mortality, Humans, Kaplan-Meier Estimate, Male, Methylation, Mice, Mutation, Protein Processing, Post-Translational, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism, T-Lymphocytes, Regulatory immunology, Arginine metabolism, Forkhead Transcription Factors metabolism, T-Lymphocytes, Regulatory metabolism
- Abstract
Forkhead box transcription factor 3 (FOXP3) plays a pivotal role in the suppressive function of regulatory T cells. In addition to mRNA levels, FOXP3 activity can also be controlled by posttranslational mechanisms, which have not been studied in a comprehensive manner. Through extensive screening using selective inhibitors, we demonstrate that the inhibition of type I protein arginine methytransferases (PRMTs) attenuates the suppressive functions of regulatory T cells. FOXP3 undergoes methylation on arginine residues at positions 48 and 51 by interacting with protein arginine methyltransferase 1 (PRMT1). The inhibition of arginine methylation confers gene expression profiles representing type I helper T cells to FOXP3
+ T cells, which results in attenuated suppressive activity. A methylation-defective mutant of FOXP3 displays less potent activity to suppress xenogeneic graft-versus-host disease in vivo. These results elucidate an important role of arginine methylation to enhance FOXP3 functions and are potentially applicable to modulate regulatory T cell functions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)- Published
- 2019
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7. Mechanisms of HLA-DP Antigen Processing and Presentation Revisited.
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Anczurowski M and Hirano N
- Subjects
- CD4-Positive T-Lymphocytes immunology, HLA-DP Antigens genetics, Humans, Peptides immunology, Polymorphism, Genetic genetics, Polymorphism, Genetic immunology, Antigen Presentation immunology, HLA-DP Antigens immunology
- Abstract
Polymorphisms in HLA-DP can modulate interactions with the invariant chain chaperone, contributing independently to differences in the peptide repertoire presented on DP. The resulting presentation of intracellular antigens directly to CD4
+ T cells may partly explain genetic and clinical studies describing previously unexplained links between polymorphism in DP and disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)- Published
- 2018
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8. Two Weeks' Notice from Allogeneic Sources.
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Anczurowski M and Hirano N
- Subjects
- Antigens, Neoplasm, Female, Humans, Receptors, Antigen, T-Cell, T-Lymphocytes, Cytotoxic, Hematopoietic Stem Cell Transplantation, Ovarian Neoplasms
- Abstract
A novel pipeline for neoantigen-specific T-cell receptor (TCR) identification has been validated in ovarian cancer, making use of HLA-matched allogeneic healthy donor T cells. This workflow allows for the identification of tumor-specific TCRs 2 weeks after antigen-specific stimulation and eliminates problematic patient-to-patient variation in the selection of neoantigen-specific TCRs. Clin Cancer Res; 24(21); 5195-7. ©2018 AACR See related article by Matsuda et al., p. 5357 ., (©2018 American Association for Cancer Research.)
- Published
- 2018
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9. DOT1L inhibition attenuates graft-versus-host disease by allogeneic T cells in adoptive immunotherapy models.
- Author
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Kagoya Y, Nakatsugawa M, Saso K, Guo T, Anczurowski M, Wang CH, Butler MO, Arrowsmith CH, and Hirano N
- Subjects
- Animals, Disease Models, Animal, Female, Graft vs Host Disease genetics, Histone-Lysine N-Methyltransferase, Humans, Lymphocyte Activation, Male, Methyltransferases genetics, Mice, Inbred BALB C, MicroRNAs genetics, MicroRNAs immunology, Allogeneic Cells immunology, Graft vs Host Disease immunology, Graft vs Host Disease therapy, Immunotherapy, Adoptive, Methyltransferases immunology, T-Lymphocytes immunology
- Abstract
Adoptive T-cell therapy is a promising therapeutic approach for cancer patients. The use of allogeneic T-cell grafts will improve its applicability and versatility provided that inherent allogeneic responses are controlled. T-cell activation is finely regulated by multiple signaling molecules that are transcriptionally controlled by epigenetic mechanisms. Here we report that inhibiting DOT1L, a histone H3-lysine 79 methyltransferase, alleviates allogeneic T-cell responses. DOT1L inhibition reduces miR-181a expression, which in turn increases the ERK phosphatase DUSP6 expression and selectively ameliorates low-avidity T-cell responses through globally suppressing T-cell activation-induced gene expression alterations. The inhibition of DOT1L or DUSP6 overexpression in T cells attenuates the development of graft-versus-host disease, while retaining potent antitumor activity in xenogeneic and allogeneic adoptive immunotherapy models. These results suggest that DOT1L inhibition may enable the safe and effective use of allogeneic antitumor T cells by suppressing unwanted immunological reactions in adoptive immunotherapy.
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- 2018
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10. Mechanisms underlying the lack of endogenous processing and CLIP-mediated binding of the invariant chain by HLA-DP 84Gly .
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Anczurowski M, Yamashita Y, Nakatsugawa M, Ochi T, Kagoya Y, Guo T, Wang CH, Rahman MA, Saso K, Butler MO, and Hirano N
- Subjects
- Antigens, Differentiation, B-Lymphocyte genetics, Antigens, Differentiation, B-Lymphocyte immunology, HLA-D Antigens genetics, HLA-D Antigens immunology, HLA-D Antigens metabolism, HLA-DP Antigens genetics, HLA-DP Antigens immunology, Histocompatibility Antigens Class II genetics, Histocompatibility Antigens Class II immunology, Humans, Lymphocyte Activation, Peptide Fragments immunology, Antigen Presentation immunology, Antigens, Differentiation, B-Lymphocyte metabolism, HLA-DP Antigens metabolism, Histocompatibility Antigens Class II metabolism, Peptide Fragments metabolism, T-Lymphocytes immunology
- Abstract
While the principles of classical antigen presentation via MHC class II are well-established, the mechanisms for the many routes of cross-presentation by which endogenous antigens become associated with class II molecules are not fully understood. We have recently demonstrated that the single amino acid polymorphism HLA-DPβ
84Gly (DP84Gly ) is critical to abrogate class II invariant chain associated peptide (CLIP) region-mediated binding of invariant chain (Ii) to DP, allowing endoplasmic reticulum (ER)-resident endogenous antigens to constitutively associate with DP84Gly such as DP4. In this study, we demonstrate that both the CLIP and N-terminal non-CLIP Ii regions cooperatively generate an Ii conformation that cannot associate with DP84Gly via the CLIP region. We also demonstrate the ability of DP4 to efficiently process and present antigens encoded in place of CLIP in a chimeric Ii, regardless of wild type Ii and HLA-DM expression. These data highlight the complex interplay between DP polymorphisms and the multiple Ii regions that cooperatively regulate this association, ultimately controlling the presentation of endogenous antigens on DP molecules. These results may also offer a mechanistic explanation for recent studies identifying the differential effects between DP84Gly and DP84Asp as clinically relevant in human disease.- Published
- 2018
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11. A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects.
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Kagoya Y, Tanaka S, Guo T, Anczurowski M, Wang CH, Saso K, Butler MO, Minden MD, and Hirano N
- Subjects
- Animals, Antigens, CD19 therapeutic use, CD28 Antigens genetics, CD28 Antigens therapeutic use, Cell Differentiation genetics, Cell Line, Tumor, Cell Proliferation genetics, Humans, Immunotherapy, Adoptive, Interleukin-2 Receptor beta Subunit therapeutic use, Janus Kinases genetics, Lymphocyte Activation genetics, Neoplasms genetics, Neoplasms pathology, Receptors, Antigen, T-Cell therapeutic use, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen therapeutic use, STAT3 Transcription Factor therapeutic use, Signal Transduction, T-Lymphocytes metabolism, Xenograft Model Antitumor Assays, Antigens, CD19 genetics, Interleukin-2 Receptor beta Subunit genetics, Neoplasms therapy, Receptors, Antigen, T-Cell genetics, STAT3 Transcription Factor genetics
- Abstract
The adoptive transfer of T cells engineered with a chimeric antigen receptor (CAR) (hereafter referred to as CAR-T cells) specific for the B lymphocyte antigen CD19 has shown impressive clinical responses in patients with refractory B cell malignancies. However, the therapeutic effects of CAR-T cells that target other malignancies have not yet resulted in significant clinical benefit. Although inefficient tumor trafficking and various immunosuppressive mechanisms can impede CAR-T cell effector responses, the signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals, including T cell receptor (TCR) engagement (signal 1), co-stimulation (signal 2) and cytokine engagement (signal 3). However, CAR constructs currently being tested in the clinic contain a CD3z (TCR signaling) domain and co-stimulatory domain(s) but not a domain that transmits signal 3 (refs. 13, 14, 15, 16, 17, 18). Here we have developed a novel CAR construct capable of inducing cytokine signaling after antigen stimulation. This new-generation CD19 CAR encodes a truncated cytoplasmic domain from the interleukin (IL)-2 receptor β-chain (IL-2Rβ) and a STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif, together with the TCR signaling (CD3z) and co-stimulatory (CD28) domains (hereafter referred to as 28-ΔIL2RB-z(YXXQ)). The 28-ΔIL2RB-z(YXXQ) CAR-T cells showed antigen-dependent activation of the JAK kinase and of the STAT3 and STAT5 transcription factors signaling pathways, which promoted their proliferation and prevented terminal differentiation in vitro. The 28-ΔIL2RB-z(YXXQ) CAR-T cells demonstrated superior in vivo persistence and antitumor effects in models of liquid and solid tumors as compared with CAR-T cells expressing a CD28 or 4-1BB co-stimulatory domain alone. Taken together, these results suggest that our new-generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicity in the clinic and that clinical translation of this novel CAR is warranted.
- Published
- 2018
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12. A Subset of Human Autoreactive CD1c-Restricted T Cells Preferentially Expresses TRBV4-1 + TCRs.
- Author
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Guo T, Koo MY, Kagoya Y, Anczurowski M, Wang CH, Saso K, Butler MO, and Hirano N
- Subjects
- Antigen Presentation immunology, Antigen-Presenting Cells immunology, Antigen-Presenting Cells metabolism, Arginine genetics, Biomarkers, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes metabolism, Cell Line, Clonal Evolution genetics, Clonal Evolution immunology, Codon, Complementarity Determining Regions genetics, Humans, Immunophenotyping, Lymphocyte Activation, Phenotype, Tyrosine genetics, Antigens, CD1 immunology, Antigens, CD1 metabolism, Autoimmunity, Gene Expression, Genes, T-Cell Receptor beta, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism
- Abstract
In humans, a substantial portion of T cells recognize lipids presented by the monomorphic CD1 proteins. Recent studies have revealed the molecular basis of mycobacterial lipid recognition by CD1c-restricted T cells. Subsets of CD1c-restricted T cells recognize self-lipids in addition to foreign lipids, which may have implications in human diseases involving autoimmunity and malignancy. However, the molecular identity of these self-reactive T cells remains largely elusive. In this study, using a novel CD1c
+ artificial APC (aAPC)-based system, we isolated human CD1c-restricted autoreactive T cells and characterized them at the molecular level. By using the human cell line K562, which is deficient in MHC class I/II and CD1 expression, we generated an aAPC expressing CD1c as the sole Ag-presenting molecule. When stimulated with this CD1c+ aAPC presenting endogenous lipids, a subpopulation of primary CD4+ T cells from multiple donors was consistently activated, as measured by CD154 upregulation and cytokine production in a CD1c-specific manner. These activated CD4+ T cells preferentially expressed TRBV4-1+ TCRs. Clonotypic analyses of the reconstituted TRBV4-1+ TCR genes confirmed CD1c-restricted autoreactivity of this repertoire, and the strength of CD1c reactivity was influenced by the diversity of CDR3β sequences. Finally, alanine scanning of CDR1 and CDR2 sequences of TRBV4-1 revealed two unique residues, Arg30 and Tyr51 , as critical in conferring CD1c-restricted autoreactivity, thus elucidating the molecular basis of the observed V gene bias. These data provide new insights into the molecular identity of human autoreactive CD1c-restricted T cells., (Copyright © 2018 by The American Association of Immunologists, Inc.)- Published
- 2018
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13. HLA-DP 84Gly constitutively presents endogenous peptides generated by the class I antigen processing pathway.
- Author
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Yamashita Y, Anczurowski M, Nakatsugawa M, Tanaka M, Kagoya Y, Sinha A, Chamoto K, Ochi T, Guo T, Saso K, Butler MO, Minden MD, Kislinger T, and Hirano N
- Subjects
- Animals, Endoplasmic Reticulum immunology, Endoplasmic Reticulum metabolism, HEK293 Cells, HLA-DP beta-Chains genetics, HLA-DP beta-Chains metabolism, Histocompatibility Antigens Class I metabolism, Humans, K562 Cells, Lysosomes immunology, Lysosomes metabolism, Mice, Inbred NOD, Mice, Knockout, Mice, SCID, Peptides metabolism, Proteasome Endopeptidase Complex immunology, Proteasome Endopeptidase Complex metabolism, Protein Transport immunology, Antigen Presentation immunology, HLA-DP beta-Chains immunology, Histocompatibility Antigens Class I immunology, Peptides immunology
- Abstract
Classical antigen processing leads to the presentation of antigenic peptides derived from endogenous and exogenous sources for MHC class I and class II molecules, respectively. Here we show that, unlike other class II molecules, prevalent HLA-DP molecules with β-chains encoding Gly84 (DP
84Gly ) constitutively present endogenous peptides. DP84Gly does not bind invariant chain (Ii) via the class II-associated invariant chain peptide (CLIP) region, nor does it present CLIP. However, Ii does facilitate the transport of DP84Gly from the endoplasmic reticulum (ER) to the endosomal/lysosomal pathway by transiently binding DP84Gly via a non-CLIP region(s) in a pH-sensitive manner. Accordingly, like class I, DP84Gly constitutively presents endogenous peptides processed by the proteasome and transported to the ER by the transporter associated with antigen processing (TAP). Therefore, DP84Gly , found only in common chimpanzees and humans, uniquely uses both class I and II antigen-processing pathways to present peptides derived from intracellular and extracellular sources.- Published
- 2017
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14. Correction: Key Residues at Third CDR3β Position Impact Structure and Antigen Recognition of Human Invariant NK TCRs.
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Chamoto K, Guo T, Scally SW, Kagoya Y, Anczurowski M, Wang CH, Rahman MA, Saso K, Butler MO, Chiu PPL, Julien JP, and Hirano N
- Published
- 2017
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15. Key Residues at Third CDR3β Position Impact Structure and Antigen Recognition of Human Invariant NK TCRs.
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Chamoto K, Guo T, Scally SW, Kagoya Y, Anczurowski M, Wang CH, Rahman MA, Saso K, Butler MO, Chiu PP, Julien JP, and Hirano N
- Subjects
- Antigens, CD1d immunology, Complementarity Determining Regions chemistry, Humans, Molecular Dynamics Simulation, Antigens immunology, Natural Killer T-Cells immunology, Receptors, Antigen, T-Cell immunology
- Abstract
The human invariant NK (iNK) TCR is largely composed of the invariant TCR Vα24-Jα18 chain and semivariant TCR Vβ11 chains with variable CDR3β sequences. The direct role of CDR3β in Ag recognition has been studied extensively. Although it was noted that CDR3β can interact with CDR3α, how this interaction might indirectly influence Ag recognition is not fully elucidated. We observed that the third position of Vβ11 CDR3 can encode an Arg or Ser residue as a result of somatic rearrangement. Clonotypic analysis of the two iNK TCR types with a single amino acid substitution revealed that the staining intensity by anti-Vα24 Abs depends on whether Ser or Arg is encoded. When stained with an anti-Vα24-Jα18 Ab, human primary invariant NKT cells could be divided into Vα24 low- and high-intensity subsets, and Arg-encoding TCR Vβ11 chains were more frequently isolated from the Vα24 low-intensity subpopulation compared with the Vα24 high-intensity subpopulation. The Arg/Ser substitution also influenced Ag recognition as determined by CD1d multimer staining and CD1d-restricted functional responses. Importantly, in silico modeling validated that this Ser-to-Arg mutation could alter the structure of the CDR3β loop, as well as the CDR3α loop. Collectively, these results indicate that the Arg/Ser encoded at the third CDR3β residue can effectively modulate the overall structure of, and Ag recognition by, human iNK TCRs., (Copyright © 2017 by The American Association of Immunologists, Inc.)
- Published
- 2017
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16. Transient stimulation expands superior antitumor T cells for adoptive therapy.
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Kagoya Y, Nakatsugawa M, Ochi T, Cen Y, Guo T, Anczurowski M, Saso K, Butler MO, and Hirano N
- Subjects
- Animals, Antigen-Presenting Cells, CD28 Antigens immunology, CD3 Complex immunology, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes transplantation, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes transplantation, Cell Line, Tumor, Cell Proliferation, Cytokines immunology, Graft vs Host Disease, Humans, Immunologic Memory immunology, K562 Cells, Lymphocyte Activation immunology, Male, Mice, T-Lymphocytes immunology, Xenograft Model Antitumor Assays, Immunization methods, Immunotherapy, Adoptive methods, Neoplasms therapy, T-Lymphocytes transplantation
- Abstract
Adoptive cell therapy is a potentially curative therapeutic approach for patients with cancer. In this treatment modality, antitumor T cells are exponentially expanded in vitro prior to infusion. Importantly, the results of recent clinical trials suggest that the quality of expanded T cells critically affects their therapeutic efficacy. Although anti-CD3 mAb-based stimulation is widely used to expand T cells in vitro, a protocol to generate T cell grafts for optimal adoptive therapy has yet to be established. In this study, we investigated the differences between T cell stimulation mediated by anti-CD3/CD28 mAb-coated beads and cell-based artificial antigen-presenting cells (aAPCs) expressing CD3/CD28 counter-receptors. We found that transient stimulation with cell-based aAPCs, but not prolonged stimulation with beads, resulted in the superior expansion of CD8
+ T cells. Transiently stimulated CD8+ T cells maintained a stem cell-like memory phenotype and were capable of secreting multiple cytokines significantly more efficiently than chronically stimulated T cells. Importantly, the chimeric antigen receptor-engineered antitumor CD8+ T cells expanded via transient stimulation demonstrated superior persistence and antitumor responses in adoptive immunotherapy mouse models. These results suggest that restrained stimulation is critical for generating T cell grafts for optimal adoptive immunotherapy for cancer., Competing Interests: The authors have declared that no conflict of interest exists.- Published
- 2017
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17. Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain.
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Guo T, Ochi T, Nakatsugawa M, Kagoya Y, Anczurowski M, Wang CH, Rahman MA, Saso K, Butler MO, and Hirano N
- Subjects
- Antigens, Neoplasm, Epitopes, Humans, Immunotherapy, Retroviridae, T-Lymphocytes, Receptors, Antigen, T-Cell
- Abstract
T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCRα or TCRβ hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.
- Published
- 2016
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18. BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models.
- Author
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Kagoya Y, Nakatsugawa M, Yamashita Y, Ochi T, Guo T, Anczurowski M, Saso K, Butler MO, Arrowsmith CH, and Hirano N
- Subjects
- Animals, CD8-Positive T-Lymphocytes metabolism, Cell Differentiation, Cell Proliferation, Disease Models, Animal, Epigenesis, Genetic, HEK293 Cells, Humans, Male, Mice, Nuclear Proteins metabolism, Oligonucleotide Array Sequence Analysis, Protein Domains, RNA, Small Interfering metabolism, Receptors, Antigen, T-Cell immunology, Signal Transduction, Transcription Factors metabolism, Immunotherapy, Adoptive methods, T-Lymphocytes cytology
- Abstract
Adoptive immunotherapy is a potentially curative therapeutic approach for patients with advanced cancer. However, the in vitro expansion of antitumor T cells prior to infusion inevitably incurs differentiation towards effector T cells and impairs persistence following adoptive transfer. Epigenetic profiles regulate gene expression of key transcription factors over the course of immune cell differentiation, proliferation, and function. Using comprehensive screening of chemical probes with defined epigenetic targets, we found that JQ1, an inhibitor of bromodomain and extra-terminal motif (BET) proteins, maintained CD8+ T cells with functional properties of stem cell-like and central memory T cells. Mechanistically, the BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8+ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQ1-treated T cells showed enhanced persistence and antitumor effects in murine T cell receptor and chimeric antigen receptor gene therapy models. Furthermore, we found that histone acetyltransferase p300 supported the recruitment of BRD4 to the BATF promoter region, and p300 inhibition similarly augmented antitumor effects of the adoptively transferred T cells. These results demonstrate that targeting the BRD4-p300 signaling cascade supports the generation of superior antitumor T cell grafts for adoptive immunotherapy.
- Published
- 2016
- Full Text
- View/download PDF
19. Mouse and Human CD1d-Self-Lipid Complexes Are Recognized Differently by Murine Invariant Natural Killer T Cell Receptors.
- Author
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Guo T, Chamoto K, Nakatsugawa M, Ochi T, Yamashita Y, Anczurowski M, Butler MO, and Hirano N
- Subjects
- Animals, Cells, Cultured, HEK293 Cells, Humans, Jurkat Cells, Lipid Droplet Associated Proteins immunology, Lipid Droplet Associated Proteins metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Natural Killer T-Cells metabolism, Antigen Presentation genetics, Antigen Presentation immunology, Antigens, CD1d genetics, Antigens, CD1d immunology, Antigens, CD1d metabolism, Autoantigens genetics, Autoantigens immunology, Autoantigens metabolism, Natural Killer T-Cells immunology, Receptors, Antigen, T-Cell metabolism
- Abstract
Invariant natural killer T (iNKT) cells recognize self-lipids presented by CD1d through characteristic TCRs, which mainly consist of the invariant Vα14-Jα18 TCRα chain and Vβ8.2, 7 or 2 TCRβ chains with hypervariable CDR3β sequences in mice. The iNKT cell-CD1d axis is conserved between humans and mice, and human CD1d reactivity of murine iNKT cells have been described. However, the detailed differences between the recognition of human and mouse CD1d bound to various self-lipids by mouse iNKT TCRs are largely unknown. In this study, we generated a de novo murine iNKT TCR repertoire with a wider range of autoreactivity compared with that of naturally occurring peripheral iNKT TCRs. Vβ8.2 mouse iNKT TCRs capable of recognizing the human CD1d-self-lipid tetramer were identified, although such clones were not detectable in the Vβ7 or Vβ2 iNKT TCR repertoire. In line with previously reports, clonotypic Vβ8.2 iNKT TCRs with unique CDR3β loops did not discriminate among lipids presented by mouse CD1d. Unexpectedly, however, these iNKT TCRs showed greater ligand selectivity toward human CD1d presenting the same lipids. Our findings demonstrated that the recognition of mouse and human CD1d-self-lipid complexes by murine iNKT TCRs is not conserved, thereby further elucidating the differences between cognate and cross-species reactivity of self-antigens by mouse iNKT TCRs.
- Published
- 2016
- Full Text
- View/download PDF
20. CD4(+) and CD8(+) TCRβ repertoires possess different potentials to generate extraordinarily high-avidity T cells.
- Author
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Nakatsugawa M, Rahman MA, Yamashita Y, Ochi T, Wnuk P, Tanaka S, Chamoto K, Kagoya Y, Saso K, Guo T, Anczurowski M, Butler MO, and Hirano N
- Subjects
- Amino Acid Sequence, CD4-Positive T-Lymphocytes cytology, CD8-Positive T-Lymphocytes cytology, Cross Reactions, Gene Expression, HLA-A2 Antigen genetics, HLA-A2 Antigen immunology, Humans, MART-1 Antigen genetics, MART-1 Antigen immunology, Primary Cell Culture, Receptors, Antigen, T-Cell, alpha-beta genetics, Transduction, Genetic, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, MART-1 Antigen metabolism, Receptors, Antigen, T-Cell, alpha-beta immunology
- Abstract
Recent high throughput sequencing analysis has revealed that the TCRβ repertoire is largely different between CD8(+) and CD4(+) T cells. Here, we show that the transduction of SIG35α, the public chain-centric HLA-A*02:01(A2)/MART127-35 TCRα hemichain, conferred A2/MART127-35 reactivity to a substantial subset of both CD8(+) and CD4(+) T cells regardless of their HLA-A2 positivity. T cells individually reconstituted with SIG35α and different A2/MART127-35 TCRβ genes isolated from CD4(+) or CD8(+) T cells exhibited a wide range of avidity. Surprisingly, approximately half of the A2/MART127-35 TCRs derived from CD4(+) T cells, but none from CD8(+) T cells, were stained by A2/MART127-35 monomer and possessed broader cross-reactivity. Our results suggest that the differences in the primary structure of peripheral CD4(+) and CD8(+) TCRβ repertoire indeed result in the differences in their ability to form extraordinarily high avidity T cells which would otherwise have been deleted by central tolerance.
- Published
- 2016
- Full Text
- View/download PDF
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