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

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.

Repertoire analyses reveal TCR sequence features that influence T cell fate

T cells acquire a regulatory phenotype when their T cell receptors (TCRs) experience an intermediate-high affinity interaction with a self-peptide presented on MHC. Using TCR sequences from FACS-sorted human cells, we identified TCR features that shape affinity to these self-peptide-MHC complexes, finding that 1) CDR3β hydrophobicity and 2) certain TRBV genes promote Treg fate. We developed a scoring system for TCR-intrinsic regulatory potential (TiRP) and found that within the tumor microenvironment clones exhibiting Treg-Tconv plasticity had higher TiRP than expanded clones maintaining the Tconv phenotype. To elucidate drivers of these predictive TCR features, we examined the two elements of the Treg TCR ligand separately: the self-peptide via murine Tregs, and the human MHC II molecule via human memory Tconvs. These analyses revealed that CDR3β hydrophobicity promotes reactivity to self-peptides, while TRBV gene usage shapes the TCRs general propensity for MHC II restricted activation.

Germline-encoded TCR-MHC contacts promote TCR V gene bias in umbilical cord blood T cell repertoire

T cells recognize antigens as peptides bound to major histocompatibility complex (MHC) proteins through T cell receptors (TCRs) on their surface. To recognize a wide range of pathogens, each individual possesses a substantial number of TCRs with an extremely high degree of variability. It remains controversial whether germline-encoded TCR repertoire is shaped by MHC polymorphism and, if so, what is the preference between MHC genetic variants and TCR V gene compatibility. To investigate the “net” genetic association between MHC variations and TRBV genes, we applied quantitative trait locus (QTL) mapping to test the associations between MHC polymorphism and TCR β chain V (TRBV) genes usage using umbilical cord blood (UCB) samples of 201 Chinese newborns. We found TRBV gene and MHC loci that are predisposed to interact with one another differ from previous conclusions. The majority of MHC amino acid residues associated with the TRBV gene usage show spatial proximities in known structures of TCR-pMHC complexes. These results show for the first time that MHC variants bias TRBV gene usage in UCB of Chinese ancestry and indicate that germline-encoded contacts influence TCR-MHC interactions in intact T cell repertoires.

Worldwide genetic variation of the IGHV and TRBV immune receptor gene families in humans

The immunoglobulin heavy variable (IGHV) and T cell beta variable (TRBV) loci are among the most complex and variable regions in the human genome. Generated through a process of gene duplication/deletion and diversification, these loci can vary extensively between individuals in copy number and contain genes that are highly similar, making their analysis technically challenging. Here, we present a comprehensive study of the functional gene segments in the IGHV and TRBV loci, quantifying their copy number and single-nucleotide variation in a globally diverse sample of 109 (IGHV) and 286 (TRBV) humans from over a 100 populations. We find that the IGHV and TRBV gene families exhibit starkly different patterns of variation. In addition to providing insight into the different evolutionary paths of the IGHV and TRBV loci, our results are also important to the adaptive immune repertoire sequencing community, where the lack of frequencies of common alleles and copy number variants is hampering existing analytical pipelines.

Evidence for Epitope-Specific T Cell Responses in HIV-Associated Non Neoplastic Lymphadenopathy: High-Throughput Immunogenetic Evidence

Non-neoplastic lymphadenopathy (NNL) associated with the human immunodeficiency virus (HIV) infection may develop concurrently with the onset of HIV viremia (acute retroviral syndrome) that can persist beyond the acute phase. Histopathological findings at this early phase mainly pertain to hyperplastic changes with large lymphoid follicles; with time, the number of lymphoid follicles diminishes, while plasma cells increase; at the extreme is a pattern characterized by sclerosis of the germinal centers in the residual follicles. HIV-specific CD8+ T cell responses have been reported and certain viral protein epitopes have been identified e.g. the p24 protein, a component of the HIV particle capsid. Overall, these findings reflect an ongoing immune response that is still incompletely characterized at the molecular level, particularly as it concerns the composition of the T cell receptor (TR) gene repertoire. In order to obtain a comprehensive view into the role of antigen selection in shaping T cell responses in HIV-associated NNL [HIV(+) NNL], we studied in-depth the TR repertoire in: (i) lymph node biopsy samples from 12 patients with HIV(+) NNL, (ii) lymph node samples from 5 non-HIV patients with reactive lymphadenopathy [HIV(-) RL]; and, (iii) peripheral blood samples from 4 healthy, HIV-seronegative individuals without lymphadenopathy [healthy controls, HIV(-) HC]. Genomic DNA was isolated from either paraffin-embedded lymph nodes (for patients with lymphadenopathy) or blood mononuclear cells (for healthy individuals). TRBV-TRBD-TRBJ gene rearrangements were amplified according to the BIOMED2 protocol. PCR products were subjected to next generation sequencing (NGS) on the MiSeq Illumina Platform. NGS data analysis, interpretation and visualization was performed by a validated, in-house bioinformatics pipeline. Overall, we obtained: (i) 1,440,305 (mean: 120,025) productive rearrangement sequences in the HIV(+) NNL group; (ii) 702,533 (mean: 140,506) productive sequences in the HIV(-) RL group; and, (iii) 539,981 (mean: 134,995) productive sequences in HIV(-) HC cases. Rearrangements with identical TRBV gene usage and CDR3 sequence were defined as clonotypes. In total, we identified 15,553 unique clonotypes in patients with HIV(+) NNL (mean: 1,296, range: 337-6,212), 53,874 in HIV(-) RL (mean: 10,774, range: 3,336-16,304) and 220,069 clonotypes in HIV(-) HC cases (mean; 55,017, range: 35,430-68,916), indicating significant repertoire restriction in the former group. Indeed, this group was characterized by an increased level of oligoclonality compared to the other two groups: the mean values of the sum of relative frequencies for the 10 most frequent clonotypes were 80%, 19.6% and 16.5%, respectively. Seven of 12 HIV(+) NNL cases carried the same dominant clonotype (TRBV29-1, SVDPSGTGGEGYT) that was also found in the remaining 5 patients of this group, albeit at lower frequencies; in contrast, it was completely absent in the HIV(-) RL and HIV(-) HC groups. Regarding the TRBV gene repertoire, the TRBV29-1 gene was overrepresented (p<0.005) in the HIV(+) NNL group, whereas the TRBV6-5 and TRBV19 genes were frequent in both groups of patients with lymphadenopathy (HIV-associated or not); finally, the TRBV5-1 was underrepresented (p<0.005) in patients with lymphadenopathy (HIV-associated or not) compared to HIV(-) HC cases. Comparison of the present TR gene sequence dataset against public databases identified 2 clonotypes with an established reactivity against the p24 protein that were present in 2 different patients with HIV(+) NNL of the present cohort. In conclusion, the TR gene repertoire of patients with HIV(+) NNL displays increased level of clonality, distinct TRBV gene repertoire as well as a widely shared, specific dominant clonotype compared to HIV(-) RL cases or HIV(-) healthy controls. These findings allude to an antigen-driven, HIV-specific immune process, a claim also supported by the detection of clonotypes with established anti-HIVp24 reactivity in at least a fraction of the analyzed patients. Disclosures Gemenetzi: Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding.

Next Generation Sequence Immunoprofiling of the T-Cell Repertoire in Chronic Lymphocytic Leukemia Supports Selection By Shared Antigenic Elements

Chronic lymphocytic leukemia (CLL) is characterized by remarkable skewing of the B-cell receptor immunoglobulin (BcR IG) repertoire, culminating in the existence of subsets of patients with stereotyped BcR IGs. This implies antigen selection in the natural history of CLL, ultimately affecting clonal behavior. Currently, limited information is available regarding the role of antigens in the selection and activation of cognate T cells, although this is relevant in light of B and T cell interactions inducing T cell tolerance. Our preliminary next-generation sequencing (NGS) studies in 11 patients assigned to stereotyped subset #4, a clinically indolent disease subgroup, indicated T-cell receptor beta chain (TRB) gene repertoire restriction and oligoclonality. Prompted by these observations, here we sought to obtain a comprehensive view of the T-cell repertoire in CLL by extending our analysis to 36 untreated CLL patients, either assigned to major stereotyped subsets [subset #4 (n=11), subset #1 (n=10), subset #2 (n=4), subset #16 (n=1)] or non-subset cases [with mutated-M (n=5) or unmutated-UM clonotypic BcR IGs (n=5)]. Starting material was PB mononuclear cells (n=27), purified CD4+ and CD8+ T-cell subpopulations (n=10), bone marrow (n=2) or lymph node (LN) tissue (n=1). Three patients were studied overtime. Multiple sample and PCR replicates, as well as 3 age-matched healthy controls were also included. TRBV-TRBD-TRBJ gene rearrangements were amplified on cDNA (BIOMED2 protocol) and subjected to paired-end NGS, designed to cover the complementarity determining region 3 (CDR3) twice/sequence. In order to further increase the accuracy of results, raw NGS reads were subjected to a purpose-built, bioinformatics algorithm, performing: (i) length and quality filtering of raw reads; (ii) merging of filtered-in paired reads via local alignment; (iii) length and quality filtering of stitched sequences. No base calls of Q-score<30 were allowed in the 75 nucleotide stretch ahead of the FGXG motif, thus further increasing the CDR3 sequencing reliability. Filtered-in sequences were submitted to IMGT/HighV-QUEST, and metadata was processed by an in-house bioinformatics pipeline designed for clonotype computation and repertoire analysis. Overall, 66 samples were analyzed, producing 23,238,779 filtered-in sequences. Only productive, in-frame TRBV-TRBD-TRBJ rearrangements were included in the analysis (85.8% of filtered-in sequences, median 354,972/sample). For repertoire analysis, clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid CDR3 sequence) were considered (median 37,550 distinct clonotypes/sample; 13,134 expanded versus 22,550 singletons). The distribution of TRBV genes was asymmetric with only 5 genes [TRBV12-3/12-4 (8.4%), TRBV19 (6.8%), TRBV29-1 (6.6%), TRBV5-1 (6.1%), TRBV6-5 (5.2%)] collectively accounting for one-third of the repertoire, indicating functional constraints. No significant TRBV gene repertoire difference was identified across the different immunogenetic CLL subgroups analyzed. In contrast, significant (p<0.001) differences were seen between CD8+ versus CD4+ cells regarding certain genes (e.g. over-representation of TRBV7-2 in CD4+ and under-representation of TRBV5-1 in CD8+ cells), alluding to distinct immune selective processes. Notably, the TRB repertoire was significantly more oligoclonal in CLL compared to the healthy controls (median frequency of the predominant clonotype: 5.7% versus 1.27%, respectively, p<0.05), albeit with differences between cases with distinct BcR IG, perhaps reflecting their different immunopathogenesis. Overtime analysis showed persistence of most major clonotypes in 2/3 cases studied, likely in a context of persistent antigenic stimulation. Cluster analysis of all CLL cases identified 17,592 different clonotypes (excluding singletons) shared by different patients and not present in the healthy controls, arguing in favor of a disease-biased immunoprofile; formal proof of this claim will require analysis of a larger number of healthy controls, currently underway by our group. In conclusion, massive parallel sequencing documents the restricted nature of the TRB repertoire in CLL, supporting selection by shared antigenic elements. Whether these are the same antigens interacting with the malignant clone remains to be elucidated. Disclosures Stamatopoulos: Janssen Pharmaceuticals: Research Funding; Gilead Sciences: Research Funding.

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

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.