scholarly journals Specific T Cell Receptor Gene Repertoire Profiles in Subgroups of CLL Patients with Distinct Genomic Aberrations

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3749-3749
Author(s):  
Elisavet Vlachonikola ◽  
Electra Sofou ◽  
Glykeria Gkoliou ◽  
Nikos Pechlivanis ◽  
Georgios Karakatsoulis ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Research Funding ◽  
Cell Receptor ◽  
Gene Repertoire ◽  
Genomic Aberration ◽  
Genomic Aberrations ◽  
Trbv Gene

Abstract Chronic lymphocytic leukemia (CLL) B cells engage in multifaceted bi-directional interactions with bystander cells, including T cells. Immunogenetic studies in CLL revealed clonal expansions of T cells and shared T cell clonotypes between different patients, strongly implying clonal selection by antigens. Although the exact nature of these antigens remains largely elusive, evidence exists that the clonotypic B cell receptor immunoglobulin (BcR IG) may serve as a source of antigenic epitopes for T cells. That said, recurrent genomic aberrations associated with distinct abnormal expression profiles could represent an alternative, non mutually exclusive, source of potent immunogenic onco-antigens that might shape the T cell repertoire in CLL. On these grounds, here we interrogated the T cell receptor (TR) gene repertoire of CLL patients with different genomic aberration profiles with the aim to identify unique signatures that would allude to distinct antigen selection pressures. The study group included 46 patients with CLL, sampled before treatment initiation, who were categorized in 5 subgroups defined by a unique genomic aberration, as follows: +12, n=18; del(11q), n=10; del(13q), n=7; del(17p)/TP53mut, n=6; NOTCH1mut, n=5. Confounding effects of multiple aberrations have been minimized, as we previously established through comprehensive characterization (including FISH, SNP arrays and gene panels) that the analyzed patients carried only one of the above aberrations. Starting material was RNA extracted from blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end next generation sequencing (NGS). Raw NGS reads (n=13,213,563| median: 294,757/sample) were processed through a purpose-built bioinformatics pipeline. Only productive rearrangements (n=9,249,546 | median=199,184/sample) were taken into consideration for the computation of clonotypes i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 (CDR3) sequence. Overall, 513,984 distinct clonotypes (median=10,304 clonotypes/sample) were assessed. The main measure of clonality employed in this study was the median cumulative frequency of the 10 most expanded T cell clonotypes/sample (MCF-10). For comparisons of the clonality profiles, a group of 17 aged-matched healthy individuals were used as controls. All patients displayed oligoclonal T cell expansions with the following MCF-10 values: del(11q): 21.6%, +12: 25%, del(13q): 20.6%, NOTCH1mut: 9.1%, del(17p)/TP53mut: 12.9%; the difference between the del(11q) and +12 groups versus the NOTCH1mut group was statistically significant (p<0.05). The MCF-10 value of the control group was estimated at 17.5%, supporting the notion of age-related decrease in TR repertoire diversity. However, the del(11q), +12 and del(13q) CLL groups displayed elevated clonality, reaching statistical significance (p<0.002) in the case of +12. TRBV gene repertoire analysis revealed that the TRBV12-3 gene predominated in most groups, except for the del(17p)/TP53mut, where the predominant gene was TRBV10-3. Clonotype comparisons disclosed the presence of shared TR clonotypes both within a particular group but also between groups. Overall, 446/513,984 clonotypes were found to be shared by at least two patients across all groups; the vast majority (392/446, 88%) of shared clonotypes appeared to be CLL-biased since they did not match entries in public databases of TR clonotypes from various contexts. Subgroup-specific clonotypes were identified for all aberrations examined; these emerged as unique to the particular subgroups, as revealed by extensive comparisons against both public databases but also a large TR clonotype database from CLL available to us from our previous studies. In conclusion, recurrent genomic aberrations, especially large chromosomal abnormalities, display an oligoclonal TR gene repertoire. The distinct immunogenetic profile of each group examined here and, most importantly, the existence of subgroup-specific clonotypes, suggest that abnormal protein expression and gene dosage effects likely represent a relevant source of CLL-specific selecting antigens. Disclosures Scarfo: Janssen: Honoraria, Other: Travel grants; Astra Zeneca: Honoraria; Abbvie: Honoraria. Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; ArQule/MSD: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Sunesis: Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Rosenquist: Roche: Honoraria; Janssen: Honoraria; Illumina: Honoraria; AstraZeneca: Honoraria; Abbvie: Honoraria. Stamatopoulos: Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Baliakas: Janssen: Honoraria; Gilead: Honoraria, Research Funding; Abbvie: Honoraria. Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

scholarly journals High-Throughput T Cell Receptor (TR) Repertoire Analysis of Virus-Specific T Cells: Implications for T Cell Immunotherapy and Viral Infection Risk Stratification

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2057-2057 ◽  
Author(s):  
Chrysi Galigalidou ◽  
Anastasia Papadopoulou ◽  
Evangelia Stalika ◽  
Andreas Agathangelidis ◽  
Elisavet Vlachonikola ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Risk Stratification ◽  
T Cell Receptor ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Research Funding ◽  
Viral Infections ◽  
Cell Receptor ◽  
Gene Repertoire

Abstract Viral infections, mainly by cytomegalovirus (CMV), Epstein Barr virus (EBV) and polyomavirus type I (BKV), are major causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As effective immune responses against human viruses rely on an armamentarium of T-cell receptor (TR) repertoire capable of recognizing a broad range of antigenic peptides of those pathogens, reconstitution of antiviral immunity, either by spontaneous generation of endogenous virus-specific T cells (VSTs) or by adoptive immunotherapy with VSTs, plays a critical role to fight infections. We here evaluated the diversity and clonality of TR repertoire of functional tri-virus-specific T cell products generated from immunocompetent donors (n=10) and compared their TR gene repertoire to that of peripheral blood mononuclear cells (PBMCs) from patients who had undergone allo-HSCT (n=5). To generate tri-VSTs, PBMCs derived from 15-20ml of peripheral blood of normal donors, were exposed to EBV, CMV and BKV overlapping peptides and cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Specificity of donor-derived VSTs and patient-derived PBMCs was measured by IFN-γElispot. TR diversity was investigated by next-generation sequencing on a MiSeq Sequencer, after amplification of TR beta chain gene rearrangements by RT-PCR with the BIOMED-2 protocol. Raw NGS reads were filtered based on their length and quality and the filtered-in sequences were submitted to IMGT/HighVQUEST. Metadata analysis and clonotype computation were performed using a validated in-house bioinformatics platform. As clonotype we defined sequences carrying the same TRBV gene and identical CDR3 amino acid sequence. Tri-VSTs provided 947,298 productive TRBV-TRBD-TRBJ rearrangements and a polyclonal and highly diverse TR gene repertoire, consisting of a total of 169,502 unique clonotypes (average: 16,950/sample, range 4,057-45,602), 64,971 (38.3%) of which were expanded (corresponding to more than one sequence). In terms of clonality, the mean relative frequency of the major clonotype in all tri-VSTs was 12.6% (range 3.3-29.2%). Interestingly, among tri-VST cell lines, 637 clonotypes were shared (present in >2/10 samples), 80 were highly shared (present in >3/10 samples) while 7 were present in 6-8 different VST lines and largely expanded, accounting for up to 29.2% of all sequences. Importantly, there were 65 of 96 major VST clonotypes shared, thus suggesting that they were potentially associated with recognition of the targeted viruses. Given that 4/10 VSTs cell lines were not specific for CMV, while being EBV-and BKV-specific, dominant TRs in those 4 cell lines can potentially be associated with EBV- or BKV-activity. By searching a public database of TR clonotypes with known reactivity against EBV and/or CMV (ShugayM, Nucleic Acids Research, 2018), we found 8 shared EBV-specific and 4 shared CMV-specific clonotypes among our VSTs and the 499 public clonotypes. When we compared the produced VSTs with PBMCs from 3 allo-grafted patients with circulating CMV-, BKV- and EBV-specific T cells and previous viral reactivation, we detected 163 shared clonotypes. Likewise, we observed 21 and 23 shared clonotypes in similar frequencies, between VSTs and PBMCs from 2 patients with CMV- or BKV-specific T cell immunity. These data identify clones that potentially expand in vivo and protect patients from viral infections. Overall, our findings reveal high levels of TR clonality in cell lines enriched for T cells reactive against EBV and/or CMV and/or BKV and provide insights into the TR repertoire of ex vivo- or endogenously-generated VSTs. Our approach may help to identify optimal TRs for immunotherapy as well as TRs which can be used as a tool for risk stratification of viral infections. Disclosures Agathangelidis: Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Abbvie: Research Funding; Janssen: Honoraria, Research Funding.

scholarly journals Characterization of Changes in the T-Cell Receptor Repertoire in Patients with Acute Myeloid Leukemia with Durable Remission Following Allogeneic Stem Cell Transplant

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5186-5186
Author(s):  
Ronald M. Paranal ◽  
Hagop M. Kantarjian ◽  
Alexandre Reuben ◽  
Celine Kerros ◽  
Priya Koppikar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Receptor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Donor T Cells ◽  
Durable Remission

Introduction: Allogeneic hematopoietic stem-cell transplantation (HSCT) is curative for many patients with advanced hematologic cancers, including adverse-risk acute myeloid leukemia (AML). This is principally through the induction of a graft-versus-leukemia (GVL) immune effect, mediated by donor T-cells. The incredible diversity and specificity of T-cells is due to rearrangement between V, D, and J regions and the random insertion/deletion of nucleotides, taking place in the hypervariable complementarity determining region 3 (CD3) of the T-cell receptor (TCR). Massively parallel sequencing of CDR3 allows for a detailed understanding of the T-cell repertoire, an area relatively unexplored in AML. Therefore, we sought out to characterize the T-cell repertoire in AML before and after HSCT, specifically for those with a durable remission. Methods: We identified 45 bone marrow biopsy samples, paired pre- and post-HSCT, from 14 patients with AML in remission for > 2 years as of last follow-up. We next performed immunosequencing of the TCRβ repertoire (Adaptive Biotechnologies). DNA was amplified in a bias-controlled multiplex PCR, resulting in amplification of rearranged VDJ segments, followed by high-throughput sequencing. Resultant sequences were collapsed and filtered in order to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region. We next employed various metrics to characterize changes in the TCR repertoire: (1) clonality (range: 0-1; values closer to 1 indicate a more oligoclonal repertoire), it accounts for both the number of unique clonotypes and the extent to which a few clonotypes dominate the repertoire; (2) richness with a higher number indicating a more diverse repertoire with more unique rearrangements); (3) overlap (range: 0-1; with 1 being an identical T-cell repertoire). All calculations were done using the ImmunoSeq Analyzer software. Results: The median age of patients included in this cohort was 58 years (range: 31-69). Six patient (43%) had a matched related donor, and 8 (57%) had a matched unrelated donor. Baseline characteristics are summarized in Figure 1A. Six samples were excluded from further analysis due to quality. TCR richness did not differ comparing pre- and post-HSCT, with a median number pre-HSCT of 3566 unique sequences (range: 1282-22509) vs 3720 (range: 1540-12879) post-HSCT (P = 0.7). In order to assess whether there was expansion of certain T-cell clones following HSCT, we employed several metrics and all were indicative of an increase in clonality (Figure 2B). Productive clonality, a measure of reactivity, was significantly higher in post-transplant samples (0.09 vs 0.02, P = 0.003). This is a measure that would predict expansion of sequences likely to produce functional TCRs. The Maximum Productive Frequency Index was higher post-HSCT indicating that the increase in clonality was driven by the top clone (most prevalent per sample). Similarly for the Simpson's Dominance index, another marker of clonality which was higher post-HSCT (0.01 vs 0.0009, P = 0.04). In order to determine whether this clonal expansion was driven by TCR clones shared among patients, we compared the degree of overlap in unique sequences among pre and post-HSCT samples. We found there was very little overlap between samples in the pre and the post-transplant setting and no change in the Morisita and Jaccard Overlap Indices. Conclusions: In conclusion, we show in this analysis an increase in clonality of T-cells following HSCT in patients with AML. This is likely related to the GVL effect after recognition of leukemia antigens by donor T cells and subsequent expansion of these T-cells. These expanded T-cell clonotypes were unlikely to be shared by patients in this cohort, likely reflecting the variety of antigens leading to the GVL effect. This could have direct implications on TCR-mediated immune-therapies given the likely need for a personalized, patient-specific design for these therapies. Figure 1 Disclosures Kantarjian: BMS: Research Funding; Novartis: Research Funding; AbbVie: Honoraria, Research Funding; Jazz Pharma: Research Funding; Astex: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Takeda: Honoraria; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Short:Takeda Oncology: Consultancy, Research Funding; AstraZeneca: Consultancy; Amgen: Honoraria. Cortes:Takeda: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Sun Pharma: Research Funding; BiolineRx: Consultancy; Novartis: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding. Molldrem:M. D. Anderson & Astellas Pharma: Other: Royalties.

First Clinical Application of Talen Engineered Universal CAR19 T Cells in B-ALL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2046-2046 ◽  
Author(s):  
Waseem Qasim ◽  
Persis Jal Amrolia ◽  
Sujith Samarasinghe ◽  
Sara Ghorashian ◽  
Hong Zhan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Research Funding ◽  
Cell Receptor ◽  
Receptor Expression ◽  
Molecular Remission ◽  
Equity Ownership

Abstract Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self inactivating lentiviral vector encoding a 4g7 CAR19 (CD19 scFv- 41BB- CD3ζ) linked to RQR8, an abbreviated sort/suicide gene encoding both CD34 and CD20 epitopes. Cells were then electroporated with two pairs of TALEN mRNA for multiplex targeting of both the T cell receptor alpha constant chain locus, and the CD52 gene locus. Following ex-vivo expansion, cells still expressing TCR were depleted using CliniMacs alpha/beta TCR depletion, yielding a T cell product with <1% TCR expression, 85% of which expressed CAR19, and 64% becoming CD52 negative. This universal CAR19 (UCART19) cell bank has been characterized in detail, including sterility, molecular and cytometric analyses and human/murine functional studies ahead of submissions for regulatory approvals and Phase 1 testing in trials for relapsed B cell leukaemia. In the interim we received a request for therapy on a compassionate basis for an infant with refractory relapsed B-ALL, and with the agreement of Cellectis, we treated this first patient under UK special therapy regulations. An 11 month girl with high risk CD19+infant ALL (t(11;19) rearrangement) relapsed in bone marrow 3 months after a myeloablative 8/10 mismatched unrelated donor transplant. Leukaemic blasts expressed CD19 but were CD52negative. Her disease progressed despite treatment with Blinatumomab (70% blasts in marrow) and we were unable to generate donor-derived CAR19 T cells on an existing study. Following institutional ethics review, detailed counseling, and parental consent, the patient received cytoreduction with Vincristine, Dexamethasone and Asparaginase followed by lymphodepleting conditioning with Fludarabine 90mg/m2, Cyclophosphamide 1.5g/m2 and Alemtuzumab 1mg/kg. Immediately prior to infusion of UCART19 cells, the bone marrow showed persisting disease (0.5% FISH positive). She received a single dose (4.5x106/kg) of UCART19 T cells without any significant toxicity. To date there has been no significant perturbation of cytokine levels in peripheral blood, and no indication of cytokine release syndrome. Although profoundly lymphopenic, UCART19 T cells were detectable by qPCR in the circulation by day 14 and at increased levels in both blood (VCN 0.35) and marrow (VCN 0.22) on day 28. The patient exhibited signs of count recovery and the bone marrow, while hypoplastic, was in cytogenetic and molecular remission. Chimerism was 90% donor, and a clearly demarcated population (7%) of third party cells indicated persistence of UCART19. A residual persistence of 3% recipient cells in the marrow suggests that leukemic clearance was not mediated by transplant mediated alloreactivity. Within the short period of follow up available, our intervention comprising lymphodepletion and infusion of UCART19 T cells has induced molecular remission where all other treatments had failed. This first-in-man application of TALEN engineered cells provides early proof of concept evidence for a ready-made T cell strategy that will now be tested in early phase clinical trials. Disclosures Qasim: CATAPULT: Research Funding; CELLMEDICA: Research Funding; CALIMMUNE: Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding; CELLECTIS: Research Funding. Off Label Use: UCART19 T Cells are an unlicensed investigational medicinal product and in this case were used under MHRA special licence arrangements. Stafford:CELLECTIS: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Thrasher:CATAPULT: Patents & Royalties, Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding. Pule:AUTOLUS: Employment, Equity Ownership, Research Funding; CELLECTIS: Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties.

Clonality Analysis as a Tool To Study the Immunologic Reconstitution with Leukemia Patients Following Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5020-5020
Author(s):  
Xin Du ◽  
Yangqiu Li ◽  
Jianyu Weng ◽  
Zesheng Lu ◽  
Rong Xie ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Gene Repertoire ◽  
Hematopoietic Stem ◽  
Receptor Repertoire

Abstract Introduction The extensive diversity of the mature T-cell receptor(TCR) is determined primarily by the complementarity-determining regions (CDR3) of the TCR. The CDR3 of both TCRα and TCRβ genes is generated by extensive rearrangement and fusion between the V,D,and J segments and by random insertion and deletion of junctional nucleotides, which yields final products that are quite heterogeneous in size. As a result of these gene rearrangements, each T cell has a unique TCR and the diversity of the T-cell repertoire at any specific time can be characterized by the examination of CDR3 within that population. Using CDR3 spectratying technique, normal individuals demonstrate a highly diverse and polyclonal The aim of our study was to evaluate to investigate restricted expansion of TCR Vβ gene repertoire and the reconstitution of T cell receptor repertoire following allogeneic hematopoietic stem cell transplantation. Methods Patients Ten patients(9 males, 1 females; median age 31 years,range18–45) with 6 chronic myeloid leukemia-chronic phase and 4 cases of acute myelogenous lenkemia(CR1) who underwent HLA-matching sibling or unrelated BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department between July 1999 and May 2000 were considered evaluable restricted expansion of TCR Vβ gene repertoire, the reconstitution of T cell receptor repertoire and oligoclonal T Cell Expansion in Chronic Graft-Versus-Host Disease. RT-PCR and Genes scan analysis (CDR 3 length analysis). Results Only 2-18Vβ genes were found in samples from these ten patients within one year, and there are different distribution in different patients. TCR repertoire complexity was abnormal in all patients, parts of the genes were expansion and part of them were suppressed. Samples from 9 patients with GVHD show V β3 in 7 cases, V β 8 and V β 23 in 6 patients. The results of genescan show that the PCR production of peripheral blood samples from these patients disply oligoclonal. Only 5–22Vβ subfamily T cells were found in samples from these patients whose transplantation more than one year. TCR repertoire complexity was abnormal in all patients. Discussion Following allogeneic BMT, regeneration of T-cell populations with a diverse repertoire can occure by at least two mechanisms: One mechanism is a thymic-dependent pathway, which presumably involves both negative and positive selection and recapitulates fetal ontogeny. Alternatively, regeneration of peripheral T cells may occur through thymic-independent mechanisms. All patients had marked abnormalities in their spectratypes, only 5-22Vβ subfamily T cells were found in samples from these patients, most of it was influenced after transplant, although the number of circulating CD3+ T lymphocytes in these patients have restored at normal lever by flow cytometic analysis, but the CD4+ T cell subset returned slowly in these patients resulting in an inversion of the normal CD4/CD8 ratio for more than 1 year after tuansplantation. Therefore, the analysis of TCRVβ subfamily is a usuaful methods and techniques for monitoring immune reconstitution after transplant.

scholarly journals High-Throughput T-Cell Receptor Gene Repertoire Profiling in Chronic Lymphocytic Leukemia Reveals a Molecular Signature of Antigen Selection

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1950-1950
Author(s):  
Anna Vardi ◽  
Evangelia Stalika ◽  
Athanasios Gkoufas ◽  
Maria Karypidou ◽  
Vasilis Bikos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Median Frequency ◽  
Gene Repertoire ◽  
Gene Usage ◽  
Healthy Control ◽  
Trbv Gene

Abstract The role of antigen(s) in shaping the T-cell repertoire in chronic lymphocytic leukemia (CLL) is largely unexplored, though highly relevant in light of the interactions of the CLL B cells with T cells, effectively inducing tolerance to the latter. Our recent classic subcloning/Sanger sequencing studies of the T-cell receptor beta chain (TRB) gene repertoire in CLL indicated repertoire restriction, pointing to antigenic selection. However, due to the inherent limitations of low-throughput analysis, definitive conclusions were not possible. Here, we sought to advance the analytical depth of our approach by employing high-throughput, next generation sequencing (NGS) for exploring the TRB gene repertoire in CLL. Our study included 9 untreated CLL cases assigned to two paradigmatic stereotyped subsets, namely clinically indolent subset #4 (n=7) and clinically aggressive subset #1 (n=2). RNA was isolated from peripheral blood mononuclear cells (n=7 cases) or purified CD4+ and CD8+ T cells (n=2, both subset #4). TRBV-TRBD-TRBJ gene rearrangements were amplified on cDNA according to the BIOMED2 protocol and were subjected to NGS (MiSeq Illumina Platform). The paired-end Illumina protocol allowed sequencing of the complementarity determining region 3 (CDR3) twice/read, thus increasing the accuracy of results. Still, considering the inherent limitations of PCR-based NGS, the experimental setup included many internal controls: (i) replicate samples of the same patient at the same timepoint; (ii) samples of the same patient at sequential time points (two-timepoint longitutinal analysis for 1 case); (iii) replicate cDNA samples for PCR amplification; and, (iv) analysis of a healthy individual. A bioinformatics pipeline was developed for raw NGS data processing, performing: (i) quality filtering of reads; (ii) merging of paired-end reads via local alignment; (iii) preparation of filtered-in fasta sequences for submission to the IMGT/HighV-QUEST tool; and, (iv) IMGT/HighV-QUEST metadata clustering, analysis and interpretation. Overall, 19 samples were analyzed, producing 7,920,136 TRBV-TRBD-TRBJ reads (median 359,957 reads/sample, median Q-score 38.3). Poor quality, incomplete, out-of-frame and unproductive rearrangements were filtered out (median 2.1% of reads/sample). For repertoire analyses, clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid CDR3 sequence) rather than single rearrangement reads were considered, so as to avoid possible biases due to clonal expansion following antigenic stimulation (median 56194 distinct clonotypes/sample, 33619 singletons versus 13725 expanded). Among the 53 functional TRBV genes identified, the following 5 predominated: TRBV12-3/12-4 (7.5%), TRBV19 (6.1%), TRBV5-1 (5.2%), TRBV29-1 (4.9%) and TRBV27 (4.8%), collectively accounting for 28.5% of the TRBV repertoire. Comparison of the TRBV gene repertoire of CD8+ vs CD4+ cells in subset #4 CLL cases showed that TRBV19 was overrepresented in the CD4+ compartment (9.4% versus 6.9%, p<0.001). Comparison between subset #4 versus subset #1 cases revealed significant overrepresentation of TRBV12-3/12-4 in subset #4 (8.6% versus 4.1%, p<0.001). The TRB repertoire was significantly more oligoclonal in CLL compared to the healthy control (median frequency of the predominant clonotype: 7.3% versus 0.47%, respectively, p<0.001), and this skewing stemmed mainly from the CD8+ rather than the CD4+ compartment (median frequency of the predominant clonotype 10.7% versus 1.0%, respectively, p<0.001). Cluster analysis of all CLL cases identified 11281 different clonotypes (excluding singletons) shared by different patients and not present in the healthy control. Of these, 10670 and 12 were exclusively found in subset #4 and subset #1 cases, respectively. The longitudinal analysis of one case identified 14.6% of all expanded clonotypes persisting over time. Moreover, comparison of TRBV gene usage and clonotype repertoire among replicate samples revealed high reproducibility of results. Overall, our study provides large-scale, reproducible evidence of TR repertoire skewing and oligoclonality in CLL, mainly derived from the CD8+ T cell compartment, strongly supporting antigenic selection. The functional role of clonally expanded T cells remains to be elucidated. Disclosures No relevant conflicts of interest to declare.

T Cell Receptor Gene Repertoire Restriction in Chronic Lymphocytic Leukemia with Stereotyped IGHV4–34/IGKV2–30 Antigen Receptors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3908-3908 ◽  
Author(s):  
Anna Vardi ◽  
Andreas Agathangelidis ◽  
Evangelia Stalika ◽  
Millaray Marincevic ◽  
Maria Karypidou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Clonal Selection ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Gene Repertoire ◽  
Time Points ◽  
Selection Of ◽  
Antigen Selection

Abstract Abstract 3908 Chronic lymphocytic leukemia (CLL) exhibits a remarkably skewed immunoglobulin (IG) gene repertoire mainly evident in the existence of subsets of patients with quasi-identical IGs in their B cell receptors (BcRs), collectively accounting for one-third of CLL patients. BcR stereotypy is strongly suggestive of clonal selection by a restricted set of antigens. However, it is not yet clear at which phase of clonal evolution these antigens act, or whether the stimulation is persistent. Furthermore, the possible role of antigens in the selection and activation of cognate T lymphocytes remains obscure yet highly relevant, given recent data about T cell interactions with CLL B cells and their tolerized behavior. Here, we analyzed the repertoire of T cell receptor β chain genes (TRB) in CLL expressing stereotyped IGHV4–34/IGKV2–30 BcR IGs (subset #4), which exhibit a series of immunogenetic features, such as pronounced intraclonal diversification of IG genes, suggestive of ongoing interactions with (auto)antigens. Furthermore, subset #4 CLL cells have distinctive functional responses to BcR and/or Toll-like receptor triggering, rendering this subset a paradigmatic example for seeking evidence of antigen selection also within the T cell population. We analyzed 18 peripheral blood samples of 12 untreated subset #4 patients (samples from different time points were analyzed in 4 cases). No case had evidence of infection at sampling. PCR amplicons for TRBV-TRBD-TRBJ gene rearrangements (BIOMED2 protocol) were subcloned by transformation into E. coli/TOP10F bacteria and randomly chosen individual colonies were subjected to Sanger sequencing. Only productive rearrangements (n=320, ranging from 14–52/case) were analyzed. All cases were found to carry clusters of identical rearrangements (≥2) corresponding to distinct clonotypes; the number of expanded clonotypes/case ranged from 1–13 (median 5). The relative frequency of each clonotype/case was determined by dividing the number of the corresponding identical sequences by the total number of subcloned sequences analyzed. The frequency of the most expanded (immunodominant) clonotype/case ranged from 8.1–70.4%. Collectively, the frequency of all expanded clonotypes/case ranged from 29.7–93.3%. In 2/4 cases that were analyzed at different time points, at least one clonotype was found to persist. Importantly, cluster analysis of the TRB CDR3 sequences of all cases identified ‘public’ clonotypes: 2 identical clonotypes (TRBV15*02/TRBD1*01/TRBJ2–2*01 and TRBV30*01/TRBD1*01/TRBJ2–2*01) each shared by a pairs of different patients and a highly similar clonotype shared by an additional pair of patients. In conclusion, the present study provides clear evidence of repertoire skewing among T cells in CLL patients belonging to subset #4, strongly supporting antigen selection. The finding of ‘public’ clonotypes raises the possibility that shared antigenic epitopes may be relevant for clonal selection of T cells in different subset #4 cases. Whether the antigens that drive T cell repertoire restriction are identical/related to those implicated in the selection of CLL progenitors of subset #4 or even the malignant cells themselves or whether they are tumor-associated antigens remains to be clarified. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of Specificity Groups in Myeloma Patients T Cell Receptor (TCR) Repertoire through Single Cell TCR Sequencing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4459-4459 ◽  
Author(s):  
Paola Neri ◽  
Ranjan Maity ◽  
Sylvia McCulloch ◽  
Peter Duggan ◽  
Victor Jimenez-Zepeda ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
Research Funding ◽  
Cell Receptor ◽  
Cytotoxic T Cell ◽  
Cdr3 Sequence ◽  
Adaptive Immune ◽  
Depth Of Response

Abstract Background: A renewed interest in immune and cellular based therapeutics in multiple myeloma was recently fueled by the development of CD38 targeting monoclonal antibodies as well as the introduction of engineered CAR-T cells. Daratumumab treatment in myeloma patients was demonstrated to expand clonal CD8+T cells and T cell clonality was correlated with the depth of response consistent with a daratumumab mediated cytotoxic T cell effect. However the mechanisms behind this adaptive immune response and the identity of the T cell receptor (TCR) interacting with MHC presented tumoral peptide (pMHC) remains elusive. The large diversity of TCR combinations generated though somatic recombination of the VDJ gene sequences (~ 1015combinations) represents a major challenge for the accurate characterization of the antigen specific TCR. The aim of this study was to define the identity of the adaptive immune repertoire of the bone marrow infiltrating T lymphocytes (single cell TCR α/β paired sequencing) in myeloma patients treated to daratumumab and IMiDs based therapies. Methods and Results: BM aspirates from patients (n=24) treated with daratumumab single agent or in combination with pomalidomide or lenalidomide (MM014, MM3008 and MMY3012 trials) were collected post initiation of therapy (cycle 3, day 1) followed by magnetic beads sorting of CD3pos T cells from Ficoll generated mononuclear cell fractions. Using the 10x Genomics Single cell VDJ solution which combines single cell droplet microfluidics with 5' molecular barcoding, T cells from each patient were partitioned into droplets containing individual cells with primers specific for the constant region of the V(D)J locus allowing the PCR amplification and enrichment of α and β TCR individual cell barcoded cDNA. Paired-end sequencing was performed on Illumina NEXTseq platform. Cell Ranger VDJ pipeline was used for sample de-multiplexing, barcode processing and grouping of T cells into clonotypes with shared TCR α/β sequences. Of note, generated sequences span the full length of V-J genes (including CDR3) allowing faithful reconstruction of TCR transcripts. Consistent with the known high TCR diverse repertoire, we identified 32322 individual clonotypes corresponding to an average of 1346 clonotypes with paired α/β TCR sequences per patient. Clonotypes proportion (> 2%) and number of individual clonotypes did correlate with the depth of response (≥VGPR vs PR vs PD). Analysis of clonotype TCRs and CDR3 sequences identified 11 clonotypes with the exact paired CDR3 αβ sequences that were shared by at least 2 patients. Of interest the CDR3 sequence of one shared clonotype is predicted (https://vdjdb.cdr3.net) to bind an epitope derived for CD317 (also know as BST2 or HM1.24) previously demonstrated to be highly expressed on myeloma cells (Jalili A et al. Blood 2005). Both patients harboring this CD317 reactive T cell clone are in sCR for more than 2 years. Of note, TCRs that recognize the same peptide-MHC complexes do not always share the exact CDR3 sequences but rather have conserved CDR3 sequence features, rendering possible to predictively model epitope specificity. Indeed, recent studies demonstrated that similarity in CDR3 sequences (CDR3 differing by up to one amino acid) or shared CDR3 motifs of 2-4 amino acids in length, define the TCR clusters that are often contact points with the antigenic peptides. Such features in CDR3 sequences facilitates T cell target antigen discovery. Therefore, we applied the GLIPH algorithm (https://github.com/immunoengineer/gliph) to cluster the sequenced TCRs based on their high probability of sharing pMHC specificity owing to both conserved CDR3 motifs and global similarity in their CDR3 sequences. GLIPH grouped the TCRs sequences identified in our study into 171 unique clusters (with a minimum of 3 clones each) that are predicted to recognize the same pMHC ligands. Furthermore, it identified 26 CDR3 motifs that are elevated at least 10-fold over expected frequency in a naïve TCR reference pool (p <0.001). Functional validation of this myeloma-targeting TCR clusters through pMHC tetramer binding is ongoing. Conclusion: Single cell TCR profiling identified unique clonotypes that are highly enriched in marrow infiltrating T cells and are predicted to be reactive with myeloma peptides. This work facilitates the future development of TCR engineered T cells targeting myeloma neoepitopes. Disclosures Neri: Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. McCulloch:Takeda: Other: Travel expenses; Celgene: Honoraria. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

scholarly journals T Cells in Chronic Lymphocytic Leukemia: A Two-Edged Sword

2021 ◽  
Vol 11 ◽  
Author(s):  
Elisavet Vlachonikola ◽  
Kostas Stamatopoulos ◽  
Anastasia Chatzidimitriou
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
T Cell Receptor ◽  
Immune Cell ◽  
Therapeutic Option ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Gene Repertoire ◽  
Disease Heterogeneity

Chronic lymphocytic leukemia (CLL) is a malignancy of mature, antigen-experienced B lymphocytes. Despite great progress recently achieved in the management of CLL, the disease remains incurable, underscoring the need for further investigation into the underlying pathophysiology. Microenvironmental crosstalk has an established role in CLL pathogenesis and progression. Indeed, the malignant CLL cells are strongly dependent on interactions with other immune and non-immune cell populations that shape a highly orchestrated network, the tumor microenvironment (TME). The composition of the TME, as well as the bidirectional interactions between the malignant clone and the microenvironmental elements have been linked to disease heterogeneity. Mounting evidence implicates T cells present in the TME in the natural history of the CLL as well as in the establishment of certain CLL hallmarks e.g. tumor evasion and immune suppression. CLL is characterized by restrictions in the T cell receptor gene repertoire, T cell oligoclonal expansions, as well as shared T cell receptor clonotypes amongst patients, strongly alluding to selection by restricted antigenic elements of as yet undisclosed identity. Further, the T cells in CLL exhibit a distinctive phenotype with features of “exhaustion” likely as a result of chronic antigenic stimulation. This might be relevant to the fact that, despite increased numbers of oligoclonal T cells in the periphery, these cells are incapable of mounting effective anti-tumor immune responses, a feature perhaps also linked with the elevated numbers of T regulatory subpopulations. Alterations of T cell gene expression profile are associated with defects in both the cytoskeleton and immune synapse formation, and are generally induced by direct contact with the malignant clone. That said, these abnormalities appear to be reversible, which is why therapies targeting the T cell compartment represent a reasonable therapeutic option in CLL. Indeed, novel strategies, including CAR T cell immunotherapy, immune checkpoint blockade and immunomodulation, have come to the spotlight in an attempt to restore the functionality of T cells and enhance targeted cytotoxic activity against the malignant clone.

scholarly journals Characterization of excised DNA intermediates associated with V(D)J recombination at the T-cell receptor delta locus.

1997 ◽  
Vol 17 (5) ◽  
pp. 2631-2641 ◽  
Author(s):  
P B Nakajima ◽  
M J Bosma
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Strand Break ◽  
Gene Repertoire ◽  
Gammadelta T Cells ◽  
Recombination Signal Sequences ◽  
V Gene

Lymphocyte development requires the assembly of antigen receptor genes through the specialized process of V(D)J recombination. This process is initiated by cleavage at the junction between coding segments (V, D, and J) and the recombination signal sequences that border these segments, resulting in generation of double-strand break intermediates. We have used a two-dimensional gel system to characterize broken molecules arising from V(D)J recombination at the T-cell receptor (TCR) delta locus and have identified linear species excised by Ddelta1-Ddelta2 and V-Ddelta2 rearrangement in thymus DNA. Relatively few (approximately 10) V-Ddelta2-excised linear species were detected in DNA from fetal thymocytes. The sizes of these species corresponded to the estimated distances between Ddelta2 and the V gene segments utilized by gammadelta T cells and indicated that both Ddelta2-proximal and -distal V gene segments are targeted for V-Ddelta2 rearrangement. Similar-sized species were observed in DNA from thymocytes of scid mice in which T-cell development is arrested prior to TCR expression. Since previous studies suggest that the TCR alpha/delta locus encodes more than 100 V gene segments, our results indicate that a few select V gene segments are predominantly targeted for rearrangement to Ddelta2, and this primarily accounts for the restricted Vdelta gene repertoire of gammadelta T cells.

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