T-cell Receptor Beta-chain Gene Research Articles

Development of a next-generation sequencing protocol for the canine T cell receptor beta chain repertoire

Profiling the T cell receptor (TCR) repertoire using next-generation sequencing has become common in both human and translational research. Companion dogs with spontaneous tumors, including canine melanoma, share several features, e.g., natural occurrence, shared environmental exposures, natural outbred population, and immunocompetence. T cells play an important role in the adaptive immune system by recognizing specific antigens via a surface TCR. As such, understanding the canine T cell response to vaccines, cancer, immunotherapies, and infectious diseases is critically important for both dog and human health. Off-the-shelf commercial reagents, kits and services are readily available for human, non-human primate, and mouse in this context. However, these resources are limited for the canine. In this study, we present a cost-effective protocol for analysis of canine TCR beta chain genes. Workflow can be accomplished in 1–2 days starting with total RNA and resulting in libraries ready for sequencing on Illumina platforms.

Context-dependent T-cell Receptor Gene Repertoire Profiles in Proliferations of T Large Granular Lymphocytes.

T cell large granular lymphocyte (T-LGL) lymphoproliferations constitute a disease spectrum ranging from poly/oligo to monoclonal. Boundaries within this spectrum of proliferations are not well established. T-LGL lymphoproliferations co-occur with a wide variety of other diseases ranging from autoimmune disorders, solid tumors, hematological malignancies, post solid organ, and hematopoietic stem cell transplantation, and can therefore arise as a consequence of a wide variety of antigenic triggers. Persistence of a dominant malignant T-LGL clone is established through continuous STAT3 activation. Using next-generation sequencing, we profiled a cohort of 27 well-established patients with T-LGL lymphoproliferations, aiming to identify the subclonal architecture of the T-cell receptor beta (TRB) chain gene repertoire. Moreover, we searched for associations between TRB gene repertoire patterns and clinical manifestations, with the ultimate objective of discriminating between T-LGL lymphoproliferations developing in different clinical contexts and/or displaying distinct clinical presentation. Altogether, our data demonstrates that the TRB gene repertoire of patients with T-LGL lymphoproliferations is context-dependent, displaying distinct clonal architectures in different settings. Our results also highlight that there are monoclonal T-LGL cells with or without STAT3 mutations that cause symptoms such as neutropenia on one end of a spectrum and reactive oligoclonal T-LGL lymphoproliferations on the other. Longitudinal analysis revealed temporal clonal dynamics and showed that T-LGL cells might arise as an epiphenomenon when co-occurring with other malignancies, possibly reactive toward tumor antigens.

Morphologic and molecular analysis of liver injury after SARS-CoV-2 vaccination reveals distinct characteristics

Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH. Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n= 6) and from patients with an initial diagnosis of AIH (n= 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing. Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8+ effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4+ effector T cells and CD79a+ B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH. Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis. Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis.

Comparison of Gold Standard Genescan with NGS-Based TCR-Beta Clonality Analysis Using Oncomine TCR Beta-Short Read Assay

Introduction All mature T-cell-based lymphoid malignancies harbor identical (clonal) rearrangements of their T cell receptor (TCR) genes (van Dongen et al. Clinica Chimica Acta. 1991, 198, 1-92). The clonality assessment of the rearranged TCR gene is particularly important for the identification, characterization and monitoring of T-cell neoplasms since histology and immunophenotyping alone is not enough to make conclusive diagnosis in all cases. Diagnostic clonality testing is currently based on the parallel analysis of the rearranged TCR-gamma and TCR-beta chain genes. This is done using a multiplex PCR developed within the European BIOMED-2/EuroClonality consortium (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis of the resultant PCR products. Although, this method is considered the "gold standard", which is established worldwide in many molecular diagnostic laboratories, the resolution of this approach is limited, especially in cases with low percentage of clonally rearranged TCRs and a high mixture of non-clonally rearranged T-cells. Next generation sequencing (NGS) is a powerful tool to provide resolution at single gene level. This also holds true for NGS assays, which are able to detect all unique TCR rearrangements in a given sample with very high resolution and sensitivity. This approach can be employed for precise assessment of the immune repertoire, minimal residue disease and T-cell clonality. Detailed insights into the clonotypes provides a great potential for early diagnosis of T-cell neoplasms, and the identification of individual clones is essential for monitoring of the disease. Methods We investigated 19 formalin-fixed paraffin-embedded (FFPE) tissue samples from celiac disease patients (n = 14) and routine diagnostic cases suspicious of lymphoma (n = 5). After DNA extraction, we performed multiplex PCRs using the BIOMED-2 TCR-beta primer sets (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis. For TCR-beta NGS, the Oncomine™ TCR Beta-SR DNA Assay (Thermo Fisher Scientific) was used according to the manufacturer's instructions and sequencing was performed on the IonTorrent S5. Identification of individual clonotypes and bioinformatics analysis of the data was done with the help of the IonReporter software (Thermofisher Scientific). It is worth noting that the 14 celiac disease samples were previously analysed by a different TCR-beta NGS approach (Ritter et al. Gut. 2018, 67, 644-653) and utilized for the comparison of the NGS-data. Results 17 out of 19 cases analysed by TCR-beta multiplex PCR (BioMed-2) followed by capillary electrophoresis and by the Oncomine™ TCR Beta-SR assay displayed a very similar length distribution of the PCR products. This holds true for samples with a clonal appearance and for samples with an oligo-/polyclonal pattern. In two discrepant cases, the Primerset B of the BioMed-2 approach showed a dominant amplification product, which was not as clear by TCR-beta NGS in which both cases displayed an oligoclonal TCR-beta gene rearrangement pattern with a few dominant T-cell populations. Both samples were from celiac disease patients, which mainly present a pronounced oligoclonal TCR-beta gene rearrangement pattern. Strikingly, the NGS data from the Oncomine™ TCR Beta-SR DNA Assay delivered highly comparable results when compared to the sequencing data of Ritter et al. 2018 despite having completely different primer sets and a different NGS platform. Conclusion Our comparison of the conventional multiplex PCR (BioMed-2) and TCR-beta NGS (Oncomine™ TCR Beta-SR DNA Assay) demonstrated a very high concordance (17/19 cases) of the molecular data. The two discordant cases can be explained by an over-interpretation of dominant species BioMed-2 Primerset B amplifications, which often show up in cases with low T-cell content or oligoclonal T-cell counts. These T-cell clonotypes are detectable by TCR-beta NGS only at a low percentage because of combination of all type of rearrangements in one assay. The robustness and reliability of NGS-based TCR-beta clonality testing was demonstrated by comparison of two completely different assays, leading to very similar results for all celiac disease patients. We are thus very confident that NGS-based clonality testing will be the "gold standard" of the future. Disclosures Looney: Thermo Fisher Scientific: Employment. Allen:Thermo Fisher Scientific: Employment.

High-Throughput Sequencing of the T-Cell Receptor Beta Chain Gene Repertoire in Chronic Lymphocytic Leukemia.

High-throughput, next-generation sequencing (NGS) offers a unique opportunity for in-depth characterization of adaptive immune receptor repertoires. Nevertheless, limitations and pitfalls exist in every step of both the experimental and the analytical procedure, leading to discrepancies in the literature and incomprehensive and/or altogether misleading results. Thus, standardization of protocols in NGS immunogenetics is urgently needed.Here, we describe the experimental protocol that we developed for T-cell receptor beta chain (TRB) gene repertoire analysis in chronic lymphocytic leukemia, aiming to provide a reproducible and biologically meaningful output. Although optimized for TRBV-TRBD-TRBJ gene rearrangements, this protocol may be customized for other adaptive immune receptor sequences, as well.

Role of high-throughput sequencing in the diagnosis of cutaneous T-cell lymphoma

AimsSubstantial clinicopathological overlap exists between cutaneous T-cell lymphoma (CTCL) and benign conditions, leading to diagnostic difficulties. We sought to delineate the utility of high-throughput sequencing (HTS) across a spectrum of...

Recent Advances in Targeting CD8 T-Cell Immunity for More Effective Cancer Immunotherapy.

Recent advances in cancer treatment have emerged from new immunotherapies targeting T-cell inhibitory receptors, including cytotoxic T-lymphocyte associated antigen (CTLA)-4 and programmed cell death (PD)-1. In this context, anti-CTLA-4 and anti-PD-1 monoclonal antibodies have demonstrated survival benefits in numerous cancers, including melanoma and non-small-cell lung carcinoma. PD-1-expressing CD8+ T lymphocytes appear to play a major role in the response to these immune checkpoint inhibitors (ICI). Cytotoxic T lymphocytes (CTL) eliminate malignant cells through recognition by the T-cell receptor (TCR) of specific antigenic peptides presented on the surface of cancer cells by major histocompatibility complex class I/beta-2-microglobulin complexes, and through killing of target cells, mainly by releasing the content of secretory lysosomes containing perforin and granzyme B. T-cell adhesion molecules and, in particular, lymphocyte-function-associated antigen-1 and CD103 integrins, and their cognate ligands, respectively, intercellular adhesion molecule 1 and E-cadherin, on target cells, are involved in strengthening the interaction between CTL and tumor cells. Tumor-specific CTL have been isolated from tumor-infiltrating lymphocytes and peripheral blood lymphocytes (PBL) of patients with varied cancers. TCRβ-chain gene usage indicated that CTL identified in vitro selectively expanded in vivo at the tumor site compared to autologous PBL. Moreover, functional studies indicated that these CTL mediate human leukocyte antigen class I-restricted cytotoxic activity toward autologous tumor cells. Several of them recognize truly tumor-specific antigens encoded by mutated genes, also known as neoantigens, which likely play a key role in antitumor CD8 T-cell immunity. Accordingly, it has been shown that the presence of T lymphocytes directed toward tumor neoantigens is associated with patient response to immunotherapies, including ICI, adoptive cell transfer, and dendritic cell-based vaccines. These tumor-specific mutation-derived antigens open up new perspectives for development of effective second-generation therapeutic cancer vaccines.

Decreased expression levels of Ifi genes is associated to the increased resistance to spontaneous arthritis disease in mice deficiency of IL-1RA.

BackgroundThe mouse strain BALB/c deficient in IL-1 receptor antagonist protein (Il-1ra) develops spontaneous arthritis disease (SAD) while the strain DBA/1 IL1rn-/- with the same deficiency does not. Previously, we mapped a QTL on chromosome 1 for SAD and then developed a congenic mouse strain BALB.D1-1-/- that contains the QTL genomic fragment associated with resistance from DBA/1-/- on a BALB/c-/- background. The congenic strain was relatively resistant to spontaneous arthritis and had delayed onset and reduced severity of disease. We obtained whole genome expression profiles from the spleen of the congenic strain BALB.D1-1-/- and four other strains, the wild type BALB/c, DBA/1 and the deficient DBA/1 IL1rn-/- and the BALB/c IL1rn-/-. We then compared the similarities and differences between the congenic strain and the four parental strains. Here we report the selected potential causal genes based on differential expression levels as well as function of genes.ResultsThere is a considerable number of genes that are differentially expressed between the congenic strain and the three parental strains, BALB/c, DBA/1, and DBA/1-/-. However there only a few differentially expressed genes were identified by comparing the congenic strain and the BALB/c-/-strain. These differentially expressed genes are mainly from T-cell receptor beta chain (Tcrb) and interferon-activatable protein (Ifi) genes. These genes are also differentially expressed between congenic strain and BALB/c strains. However, their expression levels in the congenic strain are similar to that in DBA/1 and DBA/1-/-. The expression level of Tcrb-j gene is positively associated with two genes of Ifi gene 200 cluster.ConclusionsDecreased expression levels of Ifi genes is associated to the increased resistance to spontaneous arthritis disease and with down regulation of expressions of Tcrb genes in the mouse congenic strain. Ifi genes may play an important role in the susceptibility to SAD in mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0163-y) contains supplementary material, which is available to authorized users.

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. DisclosuresStamatopoulos:Janssen Pharmaceuticals: Research Funding; Gilead Sciences: Research Funding.

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing.

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys.

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. DisclosuresNo relevant conflicts of interest to declare.

Familial CD3+ T large granular lymphocyte leukemia: evidence that genetic predisposition and antigen selection promote clonal cytotoxic T-cell responses

CD3+ T-large granular lymphocyte (T-LGL) proliferations often present with cytopenias and splenomegaly and are linked to autoimmunity, especially rheumatoid arthritis and Felty's syndrome. We report here the intra-family occurrence of T-LGL leukemia in a father and son, both presenting with cytopenias and splenomegaly. Both patients carried the HLA-DRB1*04 allele, strongly associated with rheumatoid arthritis and Felty's syndrome, exhibited distinctive histopathological features suggestive of immune-mediated suppression of hematopoiesis and expressed a remarkably skewed T-cell receptor beta chain gene repertoire with overtime evolution (clonal drift). Immunoinformatics analysis and comparisons with clonotype sequences from various entities revealed (quasi)identities between (i) father and son, and (ii) father or son and patients with autoimmune disorders, T-LGL leukemia or chronic idiopathic neutropenia. Altogether, our results further corroborate antigen selection in the ontogeny of T-LGL leukemia and point to the interplay between genetics and the (micro)environment in shaping the outcome of cytotoxic T cell responses.

Pairing of T‐cell receptor chains via emulsion PCR

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.

Abstract 2338: BRAF inhibition is associated with increased clonality of tumor infiltrating lymphocytes.

Abstract There have been significant advances in the past few years with regard to BRAF-targeted therapy for metastatic melanoma, however the majority of patients have disease progression within 6 to 7 months. We have previously shown that BRAF inhibition in melanoma in vitro is associated with an increase in melanoma antigens and increased reactivity to antigen specific T cells. More recently, we corroborated these findings in vivo in patients with melanoma undergoing treatment with BRAF inhibitors, demonstrating an increase in melanoma antigens and a significant increase in CD8+ T cells following treatment. However, the nature of this immune response to BRAF inhibition is poorly understood. The goal of the present studies is to better define the T cell infiltrate, so that we can develop a therapeutic strategy to leverage their presence. To do this, we performed tumor biopsies in patients with metastatic melanoma undergoing treatment with BRAF inhibitors at two time-points: pre-treatment (day 0), and on-treatment (day 10-14). Biopsies were also taken at time of progression when applicable. DNA was isolated and the CDR3 regions of rearranged TCR beta chain genes within the tumor were sequenced and analyzed using the immunoSEQ platform by Adaptive Biotechnologies. Results demonstrated a significant increase in the clonality in the tumor infiltrating lymphocytes in 7 of the 8 patients in on-treatment vs. pre-treatment samples, suggesting that treatment with a BRAF inhibitor may induce an antigen-specific T cell response. We also sought to determine if the increase in T cells associated with BRAF inhibition is related to an infiltration of new T cell clones or a proliferation of existing clones within the tumor. To study this, we analyzed the total T cell clones in these patient samples with regard to their presence or absence in the pre-treatment compared to on-treatment samples. Results demonstrate that over 80% of the individual clones detected after initiation of BRAF-targeted therapy are new clones, suggesting that this therapy is associated with an influx of TIL into the tumors. However, if attention is focused only on the most prevalent clones rather than the total clones, there is a dichotomous relationship between change in clonal populations and magnitude of tumor regression. Namely, those who have persistence of the same dominant clones had a good treatment response, whereas those who had a change in the dominant clonal populations had a poor response. Taken together, this data suggest that though BRAF inhibition in melanoma results in infiltration of new TIL, response to treatment is dependent on a pre-existing population of TIL clones. This data has important clinical implications, and may ultimately help predict who is likely to derive the greatest benefit from BRAF-targeted therapy. A better understanding of this response may also guide us in developing rational combination therapy in the treatment of melanoma. Citation Format: Zachary A. Cooper, Dennie T. Frederick, Ryan J. Sullivan, Donald P. Lawrence, Adriano Piris, Keith T. Flaherty, David A. Fisher, Jennifer A. Wargo. BRAF inhibition is associated with increased clonality of tumor infiltrating lymphocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2338. doi:10.1158/1538-7445.AM2013-2338

Editing Human Lymphocyte Specificity for Safe and Effective Adoptive Immunotherapy of Leukemia.

AbstractAbstract 3764T cell receptor (TCR) gene-transfer is an attractive strategy for the adoptive immunotherapy of tumors. However, the full potential of this approach is limited by a number of technical hurdles including inefficient gene transfer, unstable transgene expression, exhaustion of gene-modified cells and most importantly, co-expression of the endogenous and exogenous tumor-specific TCR in the same cell. The co-expression of endogenous and exogenous TCR causes not only reduced cell-surface expression of the introduced tumor-specific TCR, but also the potential for T cells to acquire autoreactive specificities due to mispairing between the different TCR chains. Mispaired TCR have been shown to be autoreactive and potentially harmful in animal models. To address these limitations, we developed a novel strategy based on zinc finger nucleases (ZFNs) that allows for the first time the editing of T cell specificity at the DNA level, by combining the disruption of the endogenous TCR chain genes with the transfer of a tumor-specific TCR. We first stimulated PBL with anti-CD3 and anti-CD28 antibody-conjugated beads, and cultured these cells in low dose IL-7/IL-15 to preserve early differentiated T cells. We transiently expressed ZFNs targeting the constant region of the TCR alpha or beta chain genes in activated T lymphocytes using a chimeric Ad5/F35 vector, thus promoting the genetic disruption of the endogenous TCR. Lymphocytes targeted by each set of ZFNs abrogated expression of the CD3/TCR complex on the cell surface. CD3neg cells could be expanded in culture with IL7 and IL15 and expressed differentiation markers typical of central memory T cells (CD62L, CD127, CD27 and CD28), indistinguishably from unmodified T cells. Sorted CD3neg cells proved stable in culture and permissive to lentiviral transduction. Indeed, introduction of exogenous TCR chains on a Lentivirus restored the expression and functionality of the CD3/TCR complex and allowed selective expansion of TCR-transduced cells by stimulation via the TCR complex. As a model TCR, we selected an HLA-A2 restricted, codon-optimized cysteine-modified TCR specific for the Wilms' tumor antigen 1 (WT1), which is expressed by several solid tumors and leukemias and contributes to the uncontrolled proliferation of cancer cells. For a complete editing of T cell specificity, we established a protocol that sequentially disrupted the endogenous TCR chains followed by lentiviral transfer of the WT1-specific TCR. This procedure resulted in a population of TCR-edited lymphocytes encoding only the tumor-specific TCR that, in the absence of competition, was expressed at high and physiological levels. Accordingly, TCR-edited lymphocytes were superior to conventional TCR-transferred cells in promoting specific recognition of WT1-expressing targets, including primary leukemias, and most importantly, were devoid of residual endogenous reactivity including alloreactivity. These data demonstrate that the successful genetic re-programming of T cell specificity in primary lymphocytes results in a functionally superior target specific killing activity and thus has the potential to greatly improve the safety and therapeutic benefit of cancer immunotherapy. (Provasi and Genovese: equal contribution). Disclosures:Reik:Sangamo: Employment. Liu:Sangamo: Employment. Chu:Sangamo: Employment. Bordignon:Molmed: Employment. Holmes:Sangamo: Employment. Gregory:Sangamo: Employment. Bonini:Molmed: Consultancy.

Hemophagocytic lymphohistiocytosis associated with myelodysplastic syndromes

Hemophagocytic lymphohistiocytosis/hemophagocytic syndrome (HLH/HPS) is a reactive disorder of the mononuclear phagocytic system, characterized by generalized histiocytic proliferation with marked hemophagocytosis in the bone marrow [1]. It comprises two different categories: a primary (genetic) and a secondary (acquired) form. Malignant neoplasm-associated HPS (MAHS) is categorized as a secondary HLH. MAHS is mainly associated with lymphoma and rarely with other carcinomas [2]. To our knowledge, HLH associated with myelodysplastic syndromes (MDS) has not been described, although MDS with hemophagocytosis has been observed. Here, we describe a case of HLH associated with MDS, which presented with abundant CD8 T cells in the bone marrow and elevated plasma-soluble interleukin 2 receptor (sIL-2R). A 60-year-old Japanese man was admitted with pancytopenia, epitaxis, fever and general fatigue. A hematological examination showed a hemoglobin concentration of 6.5 g/dL, a platelet count of 14,000/lL and a leukocyte count of 3,200/lL with 42.8% atypical lymphocytes (Fig. 1a). Blood biochemistry showed increased levels of lactate dehydrogenase (LDH) (331 IU/L), ferritin (649 ng/mL) and sIL-2R (4,054 U/mL). NK cytolytic activity was measured by the standard 51-chromium (Cr) release assay. NK cytolytic activity was not reduced at both the E/T ratios of 10:1 (13.1%, normal range 8.9–29.5%) and 20:1 (22.5%, normal range 17.1–48.7%). Serological studies for hepatitis B, hepatitis C and HTLV-1 were negative. IgM antibodies to parvovirus were negative. Serological test for EBV revealed that anti-viral capsid antigen IgM was negative in the presence of anti-viral capsid antigen IgG (1609) and anti-EBV nuclear antigen antibody (409). The number of EBV DNA copies in serum was under 2.0 9 10 copies/mL (normal \200 copies/mL). Computed tomography (CT) revealed hepato-splenomegaly, ascites and bilateral pleural effusion. Bone marrow aspirate showed dyserythropoietic changes (abnormally lobulated nuclei) (Fig. 1c) and dysplastic megakaryocytes (micromegakaryocytes) (Fig. 1d). Erythrocytic precursors were 10.4% and bone marrow myeloblasts were 2.0% (Fig. 1e). Chromosome analysis showed severe complex karyotype: 54–57XY, ?1[2],?3[2],?4[4],?6[4],?8[4],add(9)(p22)[2],?11[3],add (15)(p11.2)[4],add(16)(q24)[4],add(19)(p13.1)[4],add(20) (p13)[4],?21[2],2–5mar[cp4]/46,XY[8]. A diagnosis of refractory anemia with excess blasts-1 (WHO classification) was made. In addition, bone marrow examination revealed increased numbers of atypical lymphocytes (Fig. 1b) and multiple sites of active hemophagocytosis (Fig. 2a–c). Atypical lymphocytes in bone marrow were positive for CD2, CD3 (surface, cytoplasmic), CD5, CD7, CD8, HLA-DR, TCR-ab, and negative for CD4. No monoclonal rearrangement of TCR beta, gamma and delta chain genes was detected by Southern blotting. These findings suggested that the atypical lymphocytes were reactive CD8 T cells. Combining fever, hemophagocytosis, splenomegaly, increased levels of ferritin and sIL-2R, a diagnosis of HLH was established. Although steroid therapy was administered for HLH, pancytopenia was not improved. Two months after the initial diagnosis, the levels of LDH and ferritin were further elevated (LDH 1,474 U/mL, ferritin 4,347 ng/mL). Bone marrow examination T. Tsuji (&) H. Yamasaki H. Tsuda Division of Hematology and Oncology, Kumamoto City Hospital, Kotoh 1-1-60, Kumamoto 862-8505, Japan e-mail: tsuji.takahiro@city.kumamoto.lg.jp

Extraction and characterization of the rhesus macaque T-cell receptor beta-chain genes.

Rhesus macaque models have been instrumental for the development and testing of vaccines prior to human studies and have provided fundamental insights into the determinants of immune efficacy in a variety of infectious diseases. However, the characterization of antigen-specific T cell receptor (TCR) repertoires during adaptive immune responses in these models has previously relied on human TCR gene assignments. Here, we extracted and characterized TCR β-chain (TRB) genes from the recently sequenced rhesus macaque genome that are homologous to the human TRB genes. Comparison of the rhesus macaque TRB genes with the human TRB genes revealed an average best-match similarity of 92.9%. Furthermore, we confirmed the usage of most rhesus macaque TRB genes by expressed TCRβ sequences within epitope-specific TCR repertoires. This primary description of the rhesus macaque TRB genes will provide a standardized nomenclature and enable better characterization of TCR usage in studies that utilize this species.

Dual rearrangement of immunoglobulin and T-cell receptor gene in a case of T-cell hairy-cell leukemia

We report a case of T-cell hairy-cell leukemia with a dual rearrangement of Ig- and T-cell receptor genes. The cytochemical, transmission electron microscopy, and surface antigens data (CD3+, CD8+, CD11+, HLA-DR+, CD19-, CD20-) were consistent with a T-cell hairy-cell leukemia. Molecular analysis according to Southern revealed a dual rearrangement of immunoglobulin heavy-chain (JH) and T-cell receptor beta (TcR beta) chain genes. Our findings suggest that the coexistence of JH and TcR gene rearrangements, frequently detected in acute leukemia, may also be observed in hematologic malignancies derived from more differentiated cells.

CD20-Positive T-Cell Large Granular Lymphocyte Leukemia: Case Report and Review of the Literature

CD20-positive T-cell malignancy is a rare disease. We report a case of CD20-positive T-cell large granular lymphocyte leukemia (T-LGLL). The leukemic cells were positive for CD20 and T cell markers, such as CD3, CD4, CD5, CD8 and CD57. A monoclonal rearrangement of the T-cell receptor (TCR) beta chain gene was detected. Twenty-three cases of well-documented CD20-positive T-cell malignancies were reviewed. Most cases were mature T-cell malignancies, especially exhibiting a cytotoxic T-cell phenotype, despite a diversity of the pathological diagnoses. Additional cases must be evaluated to clarify the implications of CD20 expression on T-cell malignancies and to elucidate whether such cases constitute a distinct biologic and clinical disease entity. The accumulation of cases will help to facilitate provision of a proper treatment for CD20-positive T-cell malignancies in the future.

LMP2-Specific TCR Gene Therapy for Hodgkin Lymphoma - Design of a Dominant TCR Construct for Immunotherapy.

Hodgkin lymphoma (HL) is the second most commonly diagnosed cancer in the 15–29 year old population. EBV-positive Hodgkin lymphoma typically demonstrates latency II antigen expression, characterised by loss of most EBV antigens except for the latent membrane protein (LMP) 1 and 2 and the EBNA-1 protein. LMP2 is expressed in Reed Sternberg cells and may serve as a target for antigen-specific immunotherapy. However, LMP2 is poorly immunogenic and it is often difficult to generate autologous LMP2-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy. T cell receptor (TCR) gene transfer using retroviral vectors containing the TCR alpha and beta chain genes can reproducibly redirect the antigen specificity of a given population of T cells. Such an approach has been used here to generate LMP2-specific CTL independent of the immuno-competence of the patient. The goal of this study was to generate a retroviral TCR construct suitable for rapid and efficient production of LMP2-specific CTL. Retrovirally introduced TCRs compete with endogenous TCRs for a limited pool of CD3 molecules required for assembly of the TCR complex. Competition for CD3 molecules may limit surface expression of the introduced TCR resulting in a transduced T cell with poor functional avidity. In an attempt to generate a ‘highly competitive' LMP2-TCR the following modifications were made to the retroviral vector construct:i.nucleotide sequences were codon optimised for efficient translation in human cells;ii.the TCR chain constant regions were altered to contain murine sequences to enhance CD3 binding; andiii.the TCR alpha and beta chain genes were linked by a self-cleaving 2A sequence from the porcine teschovirus to enhance equimolar expression of both TCR chains.The unmodified HLA-A2-restricted LMP2-specific TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. Very few CD8+Vβ13+ T cells were detectable after LMP2-TCR transduction (up to 2.5% of viable CD3+ T cells, as detected by FACs analysis using monoclonal anti-Vβ13 antibodies), which included 1.9% CD8+ T cells expressing endogenous Vβ13+ TCRs as quantified in mock-transduced control cells. Poor expression of the wild type LMP2-TCR was consistently observed in independent transduction experiments. However, transduction with the modified LMP2-TCR construct resulted in cell surface expression of the TCR in 55–65% viable CD3+ T cells. HLA-A2/LMP2 pentamer binding was demonstrated in 36–39% CD8+ CTL cells immediately post transduction. The transduced cells showed peptide-specific IFNγ and IL2 production and killed target cells displaying the LMP2 peptide. Of major importance, expression of the introduced LMP2-TCR completely suppressed the cell surface expression of almost the entire repertoire of endogenous TCR combinations, including ‘mis-paired' TCRs in the transduced primary human T cells. ‘Mis-paired' TCRs contain an introduced alpha chain paired with an endogenous beta chain and vice versa. The antigen specificity of such mispaired TCRs generated after transduction is unknown and could lead to unwanted side effects. The design of vectors containing modified TCR sequences, which produce ‘dominant' TCRs may improve the efficacy of TCR gene therapy and reduce the risk of potential auto-reactivity of endogenous and ‘mis-paired' TCR combinations. We have shown that LMP2-specific T cells can be readily generated by TCR gene transfer with minimal risk of autoreactivity.