Cell Receptor Beta Chain Repertoire 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.

Colonic mucosal associated invariant T cells in Crohn's disease have a diverse and non-public T cell receptor beta chain repertoire.

Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn's disease (CD). The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+ T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood. MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin α4β7 in the colon but not in the blood, where they were enriched for α4β7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts. MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin α4β7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.

1063P Profiling of peripheral T cell receptor beta chain repertoire in non-small cell lung cancer (NSCLC) patients treated with anti-PD1

PD-L1 expression is the most implemented biomarker in clinical practice for immunotherapy in NSCLC; however, not all patients benefit from this treatment and PD-L1 testing alone is not yet an adequate biomarker for patient selection. Consequently, new approach is necessary to identify effective biomarkers. The aim of the project is to characterize T cell receptor β chain (TCRβ) repertoire in a cohort of advanced NSCLC patients treated with anti-PD1.

Abstract 3189: Dynamics characteristics and prognostic significance of profiling the peripheral blood T-cell receptor repertoire during immune checkpoint blockade in cancer

Abstract Introduction: Immune checkpoint blockade (ICB) targeted to CTLA-4, PD-1, and PD-L1 can promote anti-tumor T cell immunity and clinical responses. Tracking the dynamic changes of the T Cell Repertoire (TCR) can reflect the evolution of the immune system in tumor immunotherapy patients, predict the immune response to ICB, and provide high-value information for clinical monitoring of immune function status of cancer patients to make effective clinical decisions. The purpose of this study was to explore the correlation between the peripheral blood TCR repertoire dynamic changes and the immune response of patients with anti-PD-1/PD-L1, and to accurately guide the individualized treatment of immunotherapy. Methods: To comprehensively profile the TCR repertoire, high-throughput sequencing was used to identify hypervariable rearrangements of complementarity determining region 3 (CDR3) of the TCR β chain in peripheral blood samples from 31 advanced solid tumors (most were non-small cell lung cancer) patients treated with immune checkpoint blockade. The blood samples were obtained at baseline of treatment with PD-1/PD-L1 antibody, one to two additional blood samples were obtained during treatment to assess dynamic changes. Shannon-Wiener index was used to identify the diversity of TCR receptor. D50 index was used to account for diversity from the cumulative 50% of the total CDR3s counted in the sample. Results:Shannon-Wiener index at baseline was higher in durable clinical benefit (CB) group than non-clinical benefit (NCB) group (p value = 0.0040) treated with anti-PD1/PD-L1, and significantly increased in CB group ( p value = 0.031). There is a significant correlation between D50 index and clinical benefit during treatment (p value = 0.025). The higher the D50 index, the more likely it is to have durable clinical benefit. Significant difference in progression-free survival (PFS) rate was identified between the D50 high and D50 low groups (p value = 0.037). We validated this result in an independent cohort of NSCLC patients treated with anti-PD1/PD-L1(N=12). Conclusions:This study revealed the dynamic changes of peripheral blood T cell receptor beta-chain repertoire in patients with solid tumors before and during anti-PD1/PD-L1 treatment. This study found that baseline TCR diversity was associated with clinical efficacy. Patients with durable clinical benefit had an increased overall diversity of TCR. The D50 index of the sample during treatment can reflect whether the patient could benefit from anti-PD1/PD-L1 treatment or not, and was a significant prognostic factor for progression-free (PFS). These analyses provide that the peripheral blood TCR repertoire parameters may be useful for prognosis prediction and rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors. Citation Format: Ye Li, Jiaqian Wang, Shengjie Sun, Liangliang Wu, YunXiao Zhang, Guoqing Zhang, Xiaoting Li, Shunchang Jiao. Dynamics characteristics and prognostic significance of profiling the peripheral blood T-cell receptor repertoire during immune checkpoint blockade in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3189.

Enrichment of melanoma-associated T cells in 6-thioguanine-resistant T cells from metastatic melanoma patients.

This study examines whether 6-thioguanine resistant T cells (mutant) from metastatic melanoma patients are enriched for melanoma-associated T cells compared to T cells obtained analogously without thioguanine selection (wild-type). Melanoma-associated antigen pentamer staining was performed on 5 tumour and 9 peripheral blood samples from metastatic melanoma patients. T cell receptor beta chain repertoire was examined via Sanger sequencing of mutant and wild-type in blood and tumour from metastatic melanoma patients at times of tumour progression (n = 8) and via Illumina sequencing in tumour derived T cells and in uncultured T cells (uncultured), wild-type and mutant from blood before and after immune checkpoint blockade (n = 1). Mutant from tumour (3 of 5; P < 0.001), but not blood (0 of 9), were enriched compared to wild-type for binding melanoma-associated antigen pentamers. T cell receptor beta analysis in patients with tumour progression (n = 8) detected increased melanoma associated T cells in mutant compared to wild-type from blood (Monte Carlo P = 10). Comparison of blood samples before and after immune checkpoint blockade with prior tumor from one metastatic melanoma patient detected increased T cell receptor beta sharing between tumour and mutant compared to tumour and wild-type or tumour and uncultured: 11.0% (72/656), 1.5% (206/13 639) and 1.3% (381/29 807), respectively (Monte Carlo P = 10 for mutant versus wild-type and mutant versus uncultured). These data demonstrate that mutant in metastatic melanoma patients are enriched for melanoma-associated T cells and are candidate probes to study in vivo melanoma-reactive T cells.

Another view of T cell antigen recognition: cooperative engagement of glycolipid antigens by Va14Ja18 natural T(iNKT) cell receptor [corrected

Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor beta-chain repertoire and how alpha-galactosylceramide (alpha-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-alphaGalCer- and alphaGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional alphabeta TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.

The T cell receptor V beta repertoire shows little change during treatment interruption-related viral rebound in chronic HIV infection.

In this study, changes in plasma virus load, peripheral blood CD4 T cell counts and the T cell repertoire were assessed in eight chronically HIV-infected individuals suspending antiretroviral therapy. Despite rapid increases in virus load and substantial CD4 T cell losses during treatment interruption, no marked changes in the T cell receptor beta chain repertoire were observed. The magnitude of associated T cell repertoire perturbation thus contrasts with that observed during primary HIV infection.

Modulation of the T cell receptor beta chain repertoire after heart transplantation

Background: In a previous study it was shown that pre-transplant blood transfusion was associated with a better clinical outcome after heart transplantation (HTx). In this study the effect of heart transplantation (HTx) on the T cell receptor V beta chain (TCRVβ) repertoire was investigated. Therefore, we analyzed the TCRVβ repertoire of patients after HTx to see whether a correlation with clinical outcome could be observed. Methods: Patients were analyzed at four different time points: pre-HTx, less than 1 month post-Htx, between 1 month and 2.5 month post-Htx and more than 2.5 months post-HTx. CD4 + and CD8 + T cells were purified from patient peripheral blood mononuclear cells (PBMC). TCR beta chain usage was analyzed semiquantitatively by Southern blot analysis. Results: HTx affected the TCRVβ repertoire in both the CD4 + and CD8 + T cell compartments in all patients. Changes in the TCRVβ repertoire were most pronounced within the CD8 + T cell subset. Interestingly, one patient showed modulation in TCRVβ chain usage predominantly in the CD4 + T cell compartment. Conclusions: Modulation of TCRVβ chain usage was detected in all patients analyzed. No clear-cut relation was observed between TCRVβ modulation after transplantation and clinical outcome. In some cases modulations appeared to concur with observed immunological events (clinically and/or in-vitro).

Recognition of tissue residing antigen by T cells in vasculitic lesions of giant cell arteritis.

The objective of this study was to explore the nature of the antigen-specific T cell response in giant cell arteritis by analyzing clonally expanded T cells in temporal artery specimens. In temporal artery tissue from eight patients, 10% of the T cell receptor beta chain repertoire was systematically screened for clonal T cells by reverse-transcriptase polymerase chain reaction with selected BV, BJ, and BC specific primers and by direct sequencing of the amplified product. In five additional patients tissue-derived T cell clones were characterized. All expanded clonotypes were analyzed for their presence at different sites of the inflamed artery. T cell lines were tested for their proliferation to autologous monocytes pulsed with temporal artery extracts from patients with giant cell arteritis, polymyalgia rheumatica, and unrelated diseases. Clonally expanded T cells were identified in 30% of the BV-J combinations of the sampled repertoire. A subset of these clones were encountered at different sites of the inflammation, but not in the peripheral blood. The T cell receptor beta chain sequences were diverse. The patients had between none and five such clonotypes in the sampled repertoire, suggesting that only few T cell specificities in each patient are involved in antigen recognition. One of these T cell clonotypes was shown to proliferate in response to an antigen selectively expressed in temporal artery specimens from giant cell arteritis and from polymyalgia rheumatica patients. Clonotypes with identical T cell receptor beta chain sequences can be found at distinct sites of the inflammation in giant cell arteritis, suggesting recognition of the same antigen at different locations. At least for some of these T cell clones the antigen is shared between different giant cell arteritis and polymyalgia rheumatica patients but not expressed in temporal arteries of patients with unrelated diseases. While different HLA-DR4+ patients utilize distinct T cell specificities, the actual number of responding T cells in individual patients is small and may be disease limiting.

Restrictions of T cell receptor beta chain repertoire in the peripheral blood of patients with systemic lupus erythematosus.

To investigate whether oligoclonal T cell populations occur in peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE). RNA was extracted from the lymphocytes isolated from whole peripheral blood of five female patients fulfilling ARA criteria for SLE and two healthy female controls, and synthesised into cDNA. CDR3 length spectratyping was performed using a polymerase chain reaction (PCR) run to saturation followed by a primer extension with a radioactively labelled primer. The resulting samples, one for each of the 23 V beta families, were then run on a polyacrylamide sequencing gel to examine the T cell receptor beta chain repertoire at the level of VDJ length heterogeneity. The two healthy female controls showed faint oligoclonal bands in only two and three V beta families respectively. Three of the patients showed a similar degree of oligoclonality to the controls, while the other two, who had active disease as shown by SLAM scores of 17 and 19 and in one case low C4 and raised C3dg levels, showed marked oligoclonality of their T cell beta chain repertoire affecting more than 17 of the 23 V beta families analysed. Using the technique of CDR3 length spectratyping, restriction of T cell receptor beta chain usage by peripheral blood T cells in patients with SLE has been demonstrated for the first time.

Few V gene segments dominate the T cell receptor beta-chain repertoire of the human thymus.

We undertook a large sample census of the TCR-beta repertoire from a single human thymus (300 clones from an unamplified cDNA library), to establish the statistics of the rearranged V, D, J, and C gene segments. The assortments of all germ-line segments are subject to significant biases and thus critically reduce the effective germ-line contribution to beta-chain diversity. Thirty-two V genes characterize the whole sample, but surprisingly as few as seven genes from different families encompass half of the beta-chain repertoire. Furthermore, a Spearman rank order correlation test of the thymic V beta frequencies with those inferred in other studies using RNase protection assays shows a statistically significant similarity. Thus, in the establishment of thymic V beta frequencies in man, rearrangement preferences intrinsic to the progenitors of TCR-beta expressing cells override HLA- and Ag-dependent biasing factors. By implication, a large enough pool of direct progenitor cells (maybe as high as thousands) must exist to secure V beta frequencies against large random fluctuations. Uncorrelated with the V gene bias, the representation of D beta and J beta segments is also far from even. Notably, D beta 1 and J beta 2.1 and 1.2 predominate. Using the above mentioned rank order statistic, we also find that unlike V genes, the bias in the J segment usage carries over into the frequencies of peripheral T lymphocyte populations.

Variation in human T cell receptor V beta and J beta repertoire: analysis using anchor polymerase chain reaction.

Anchor polymerase chain reaction has been applied to the study of human T cell receptor beta chain repertoire in peripheral blood. The use of this technique has demonstrated that considerable variation in V beta and J beta usage exists, both within and between individuals. Particular V beta families, including V beta 6, V beta 4 and V beta 12 are commonly utilized, while families such as V beta 10, V beta 11 and V beta 15 are rare in all individuals studied. Marked interindividual variation in V beta usage was detected for V beta 12 and V beta 4. Biased usage of J beta elements is a prominent feature of peripheral repertoire, while there is no evidence for preferential V beta-J beta recombination events. Biased J beta usage in expressed V beta-D beta-J beta-C beta transcripts, subject to selection, was the same as that in aberrant, unselected D beta-J beta-C beta transcripts, implying that bias resulted from events relating to rearrangement itself, in the absence of selection. N-region diversity showed some evidence for preferential insertion of deoxyguanosine, consistent with the action of terminal deoxytransferase. No P-nucleotide incorporation was seen in association with intact J beta elements. These data provide evidence of some of the variation in human T cell receptor beta chain repertoire and provide a basis for comparisons with sequences which may be obtained in autoimmune and superantigen-mediated diseases.