T-cell Receptor Beta Research Articles

Reduced Diversities and Clonally Expanded Sequences of T-Cell Receptors in Patients With Essential Hypertension and Subclinical Carotid Atherosclerosis.

It has long been hypothesized that the abnormal immune responses contribute to the essential hypertension (EH) and its subclinical target organ damage (STOD). However, the mechanism is unclear. This study aimed at exploring the potential association with abnormal T-cell responses and EH, STOD, and early atherosclerosis in patients with EH. This cross-sectional study included 146 patients with EH and 73 age-matched normotensive individuals. The expressed peripheral TCR (T-cell receptor) β repertoire was analyzed by high through-put sequencing. The TCRβ repertoires of the patients with EH were significantly different, with significantly elevated certain TCR beta variable (TRBV) and joint (TRBJ) gene usages, significantly reduced TCRβ diversity indexes (diversity 50s) and numbers of total TCRβ clonal types, significantly elevated percentages of the biggest TCRβ clones and numbers of clones accounting >0.1% sequences, compared with those in the normotensive controls. Decreased diversity 50s and increased biggest TCRβ clone percentages were independently correlated with carotid intima-media thickness and subclinical carotid atherosclerosis (SCA) in the patients with EH. Moreover, the diversity 50s were further significantly reduced and the biggest TCRβ clone percentages were significantly increased in the patients with EH with SCA (n=89) comparing to the patients with EH/patients without SCA (n=57), and in patients with EH/SCA with carotid plaque (n=22) comparing to patients with EH/SCA/patients without carotid plaque (n=67). Importantly, specific TCRβ clones were identified in different subgroups of the patients with EH. These results reveal that abnormal T-cell responses may play important roles in the progression of EH and its SCA, especially the formation of carotid plaque. URL: https://www.chictr.org.cn; Unique identifier: ChiCTR2100054414.

T-cell tolerant fraction as a predictor of immune-related adverse events

BackgroundImmune checkpoint inhibitor (ICI) therapies may cause unpredictable and potentially severe autoimmune toxicities termed immune-related adverse events (irAEs). Because T cells mediate ICI effects, T cell profiling may provide insight...

T-cell receptor beta variable gene polymorphism predicts immune-related adverse events during checkpoint blockade immunotherapy

BackgroundImmune checkpoint inhibitors have revolutionized cancer treatment. However, they are associated with a unique spectrum of side effects, called immune-related adverse events (irAEs), which can cause significant morbidity and quickly...

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.

Unprecedented effect of vitamin D3 on T-cell receptor beta subunit and alpha7 nicotinic acetylcholine receptor expression in a 3-nitropropionic acid induced mouse model of Huntington’s disease

Introduction3-NP induction in rodent models has been shown to induce selective neurodegeneration in the striatum followed by the cortex (Brouillet, 2014). However, it remains unclear whether, under such a neurotoxic condition, characterized by neuroinflammation and oxidative stress, the gene expression of the immune resident protein, T-cell receptor beta subunit (TCR-β), α7 nicotinic acetylcholine receptor (α7 nAChRs), the nuclear factor kappa B (NF-κB), inflammatory cytokines (TNF-α and IL-6), and antioxidants (Cat and GpX4) get modulated on Vitamin D3 (VD) supplementation in the central nervous system. MethodsIn the present study, real-time polymerase chain reaction (RT-PCR) was performed to study the expression of respective genes. Male C57BL/6 mice (8–12 weeks) were divided into four groups namely, Group I: Control (saline); Group II: 3-NP induction via i.p (HD); Group III: Vitamin D3 (VD) and Group IV: (HD + VD) (Manjari et al., 2022). ResultsOn administration of 500IU/kg/day of VD, HD mice showed a significant reduction in the gene expression of the immune receptor, TCR-β subunit, nuclear factor kappa B (NF-κB), inflammatory cytokines, and key antioxidants, followed by a decrease in the acetylcholinesterase activity. ConclusionA novel neuroprotective effect of VD in HD is demonstrated by combating the immune receptor, TCR-β gene expression, antioxidant markers, and inflammatory cytokines. In addition, HD mice on VD administration for 0–15 days showed an enhancement in cholinergic signaling with restoration in α7 nAChRs mRNA and protein expression in the striatum and cortex.

A novel approach to T-cell receptor beta chain (TCRB) repertoire encoding using lossless string compression.

T-cell receptor beta chain (TCRB) repertoires are crucial for understanding immune responses. However, their high diversity and complexity present significant challenges in representation and analysis. The main motivation of this study is to develop a unified and compact representation of a TCRB repertoire that can efficiently capture its inherent complexity and diversity and allow for direct inference. We introduce a novel approach to TCRB repertoire encoding and analysis, leveraging the Lempel-Ziv 76 algorithm. This approach allows us to create a graph-like model, identify-specific sequence features, and produce a new encoding approach for an individual's repertoire. The proposed representation enables various applications, including generation probability inference, informative feature vector derivation, sequence generation, a new measure for diversity estimation, and a new sequence centrality measure. The approach was applied to four large-scale public TCRB sequencing datasets, demonstrating its potential for a wide range of applications in big biological sequencing data. Python package for implementation is available https://github.com/MuteJester/LZGraphs.

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.

Single-cell atlas of the human neonatal small intestine affected by necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a gastrointestinal complication of premature infants with high rates of morbidity and mortality. A comprehensive view of the cellular changes and aberrant interactions that underlie NEC is lacking. This study aimed at filling in this gap. We combine single-cell RNA sequencing (scRNAseq), T-cell receptor beta (TCRβ) analysis, bulk transcriptomics, and imaging to characterize cell identities, interactions, and zonal changes in NEC. We find an abundance of proinflammatory macrophages, fibroblasts, endothelial cells as well as T cells that exhibit increased TCRβ clonal expansion. Villus tip epithelial cells are reduced in NEC and the remaining epithelial cells up-regulate proinflammatory genes. We establish a detailed map of aberrant epithelial-mesenchymal-immune interactions that are associated with inflammation in NEC mucosa. Our analyses highlight the cellular dysregulations of NEC-associated intestinal tissue and identify potential targets for biomarker discovery and therapeutics.

Spatially resolved immune receptor repertoires from Visium transcriptomic libraries

Abstract Recent advances have enabled the integration of T cell receptor (TCR) sequencing into spatial transcriptomics. Current approaches yield critical information about in situ T cell responses directly from relevant tissues, yet are largely limited to alpha/beta TCRs, leaving the clonal dynamics of B cells and gamma/delta T cells underexplored. We therefore modified our previously reported ultra-efficient immune receptor repertoire sequencing (IRR-seq) method, Framework Region 3 AmplifiKation Sequencing (FR3AK-seq), for compatibility with the 10X Genomics Visium spatial transcriptomics platform. Here we report simultaneous acquisition of alpha/beta TCR, gamma/delta TCR, and immunoglobulin (B cell receptor, BCR) heavy/light chain sequences from a single multiplexed FR3AK-seq reaction using Visium transcriptomics-generated cDNA libraries. More specifically, we used the Visium spatial transcriptomics protocol to generate cDNA from serial sections of a lymph node with a metastatic lesion obtained from a patient with pancreatic ductal adenocarcinoma previously treated with immunotherapy. Notably, we detected sequences from all IRR chains as expected from the abundant immune infiltrates within these sections. Furthermore, we found that multiplexed FR3AK-seq yields technically reproducible repertoires, with the TCR beta chain repertoires also comparable to those obtained using a previously published method. We anticipate that this simple extension of the Visium technology will accelerate the simultaneous and comprehensive examination of in situ IRR dynamics beyond alpha/beta T cells.

Germline-enforced enrichment for charged amino acids in TCR beta chain (TCRβ) complementarity determining region 3 (CDR-B3) alters T cell development, repertoire content, and antigen recognition.

T cell receptor beta chain (TCRβ) diversity (Dβ) gene segments are highly conserved across evolution, with trout Dβ1 sequence identical to human and mouse Dβ1. A key conserved feature is enrichment for glycine in all three Dβ reading frames (RFs). Previously, we found that replacement of mouse Dβ1 with a typical immunoglobulin DH sequence, which unlike Dβ is enriched for tyrosine, leads to an increase in the use of tyrosine in TCRβ complementarity determining region 3 (CDR-B3) after thymic selection, altering T cell numbers, CDR-B3 diversity, and T cell function. To test whether the incorporation of charged amino acids into the Dβ sequence in place of glycine would also influence T cell biology, we targeted the TCRβ locus with a novel glycine-deficient DβDKRQ allele that replaces Dβ1 coding sequence with charged amino acids in all three reading frames. Developing T cells using DβDKRQ expressed TCR CDR-B3s depleted of tyrosine and glycine and enriched for germline-encoded lysine, arginine, and glutamine. Total thymocytes declined in number during the process of β selection that occurs during the transition from the DN3bc to DN4 stage. Conventional thymocyte and T cell numbers remained reduced at all subsequent thymic stages and in the spleen. By contrast, regulatory T cell numbers were increased in Peyer's patches and the large intestine. In terms of functional consequences, T cell reactivity to an ovalbumin immunodominant epitope was reduced. These findings buttress the view that natural selection of Dβ sequence is used to shape the pre-immune TCRβ repertoire, affecting both conventional and regulatory T cell development and influencing epitope recognition.

Abstract 895: Tumor-wide neoantigen-specific T-cells infiltrating mutant IDH1 low-grade gliomas and persisting in peripheral blood allow for personalized TCR-based immunotherapies

Abstract BACKGROUND: The low mutational burden and immunologically “cold” microenvironment of mutant IDH1 low-grade gliomas (LGG) are considerable challenges facing immunotherapy against these tumor types. However, we hypothesize that LGG-targeting T-cells may exist at low frequency and with limited regional infiltration within the tumor. Multi-region tumor sampling coupled with high-throughput T-cell receptor (TCR) profiling across the LGG landscape detected neoantigen-specific T-cells that persisted in peripheral blood. TCR-engineered T-cells transduced with these TCRs demonstrated neoantigen-specific immunogenicity. METHODS: Maximal-anatomical sampling of at least 10 distinct tumor regions were collected at the initial resection for three WHO Grade II diffuse astrocytoma patients for exome-based prediction of clonally and subclonally expressed neoantigens, RNAseq analysis of regional immune cell composition, and TCR beta deep sequencing. We used these predictions to generate a barcoded library of patient-specific peptide-HLA multimers loaded with predicted neoepitopes. With this library, neoantigen-specific CD8+ T-cells were captured and isolated from patient peripheral blood. Single cell TCR sequencing allowed us to identify the neoantigen-reactive TCR clonotypes which were transduced subsequently into Jurkat76 cell lines for functional validation. RESULTS: We screened patient-derived peripheral blood drawn two years after initial resection in 3 mutant IDH1 LGG patients and detected a total of 20 TCR clonotypes recognizing neoepitopes derived from truncal, tumor-wide mutations in CNTNAP1 (n=8), TP53 (n=3), and MRPL46 (n=2) as well as subclonal mutations in PRMT5 (n=1) and ZDHHC5 (n=6). Multi-sampling RNAseq analysis indicated varying degrees of interpatient and intratumoral immune infiltration as well as distally located populations of neoantigen-reactive T-cells within the tumor, suggesting widespread migration of neoantigen-specific T-cells across the glioma landscape. We proceeded with TCR functional analysis for one patient (P375) with 5 detected TCR clonotypes recognizing neoantigens derived from mutations in PRMT5, MRPL46, and TP53. Jurkat76 cells transduced with the mutant-PRMT5-specific TCR demonstrated a neoantigen-specific immune response when co-cultured with mutant-PRMT5 pulsed-antigen presenting cells expressing HLA-A*0201 (T2 cells). CONCLUSION: Our study demonstrates the existence and persistence of neoantigen-targeting T-cells within the blood and tumor of mutant IDH1 LGG patients. We identified a TCR clonotype that successfully recognizes and induces an immune response against mutant-PRMT5. These findings suggest a feasible methodology to develop personalized T-cell-based immunotherapies for patients with mutant IDH1 LGGs. Citation Format: Darwin W. Kwok, Michael Y. Zhang, Cliff Wang, Nicholas Stevers, Tyler Borrman, Zheng Pan, Benjamin Yuen, Songming Peng, Diana Nguyen, Michael Martin, Chibo Hong, Stephanie Hilz, Joanna Phillips, Anny Shai, Nancy Ann Oberheim Bush, Shawn Hervey-Jumper, Michael McDermott, Stefanie Mandl, Hideho Okada, Joseph Costello. Tumor-wide neoantigen-specific T-cells infiltrating mutant IDH1 low-grade gliomas and persisting in peripheral blood allow for personalized TCR-based immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 895.

MP04-14 T CELL AND B CELL RECEPTOR SEQUENCING REVEALS ANTI-CANCER IMMUNE RESPONSE ASSOCIATED WITH IMMUNE-RELATED ADVERSE EVENTS IN ADVANCED RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT INHIBITORS

You have accessJournal of UrologyCME1 Apr 2023MP04-14 T CELL AND B CELL RECEPTOR SEQUENCING REVEALS ANTI-CANCER IMMUNE RESPONSE ASSOCIATED WITH IMMUNE-RELATED ADVERSE EVENTS IN ADVANCED RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT INHIBITORS Taigo Kato, Kazuma Kiyotani, Yu Ishizuya, Yoshiyuki Yamamoto, Koji Hatano, Atsunari Kawashima, and Norio Nonomura Taigo KatoTaigo Kato More articles by this author , Kazuma KiyotaniKazuma Kiyotani More articles by this author , Yu IshizuyaYu Ishizuya More articles by this author , Yoshiyuki YamamotoYoshiyuki Yamamoto More articles by this author , Koji HatanoKoji Hatano More articles by this author , Atsunari KawashimaAtsunari Kawashima More articles by this author , and Norio NonomuraNorio Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recently, several data are available on the relationship between immune-related adverse events (irAEs) and clinical efficacy in patients with immune checkpoint inhibitors (ICIs). In this study, we aimed to elucidate T cell receptor (TCR) and B cell receptor (BCR) repertoire in peripheral blood mononuclear cells (PBMCs) before and after ICI treatment, and at the onset of irAE for understanding of irAE-induced anti-cancer immune responses. METHODS: We collected metastatic tumor tissues and peripheral blood samples from 56 patients with advanced renal cell carcinoma before and 1 month after anti-programmed cell death protein 1 (PD-1) treatment initiation. Furthermore, we applied a next-generation sequencing approach to characterize TCR and BCR repertoires using tumor tissues and PBMCs. RESULTS: The number of irAEs was significantly higher in responder for PD-1 treatment (p<0.0001). TCR repertoire analysis revealed that diversity index (DI) for TCR alpha and beta in irAE present patients were significantly decreased at 1 month after treatment, indicating expansion of certain T cell clones even in PBMCs after the treatment (Figure 1A). We also examined morisita index to measure T cell similarity between pre- and post- treatment than irAE absent patients, indicating a larger change in the T cell clones, and inducing newly expanded T cells in post- treatment PBMC samples (Figure 1B). Importantly, irAE present patients had significantly higher numbers of peripheral T cell clones shared with metastatic tumor-infiltrating T cells than those in irAE absent patients at 1 month after treatment (p=0.038). We also confirmed that DI for BCR IgM and IgG in irAE present patients were significantly decreased at 1 month after the treatment, indicating expansion of certain B cell clones (Figure 1C). CONCLUSIONS: Our findings revealed that a certain number of irAE-induced T cell and B cell clones can circulate systemically and attack tumor cells synergistically in distant regions, leading to durable response in the patients with irAEs. Source of Funding: This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (C), grant number 18K09133 and 21K09343 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e38 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taigo Kato More articles by this author Kazuma Kiyotani More articles by this author Yu Ishizuya More articles by this author Yoshiyuki Yamamoto More articles by this author Koji Hatano More articles by this author Atsunari Kawashima More articles by this author Norio Nonomura More articles by this author Expand All Advertisement PDF downloadLoading ...

Minimal/Measurable Residual Disease Monitoring in Patients with Lymphoid Neoplasms by High-Throughput Sequencing of the T-Cell Receptor

High-throughput sequencing of the T-cell receptor beta (TRB) and gamma (TRG) loci is increasingly utilized due to its high sensitivity, specificity, and versatility in the diagnosis of various T-cell malignancies. Application of these technologies for tracking disease burden can be valuable in detecting recurrence, determining response to therapy, guiding future management of patients, and establishing endpoints for clinical trials. In this study, the performance of the commercially available LymphoTrack high-throughput sequencing assay was assessed for determining residual disease burden in patients with various T-cell malignancies. A custom bioinformatics pipeline and database was also developed to facilitate minimal/measurable residual disease analysis and clinical reporting. This assay demonstrated excellent test performance characteristics, achieving a sensitivity of 1 of 100,000 T-cell equivalents for the DNA inputs evaluated and high concordance with orthogonal testing methods. This assay was further utilized to correlate disease burden in several patients, demonstrating its potential utility for monitoring patients with T-cell malignancies.

Cross-reactivity influences changes in human influenza A virus and Epstein Barr virus specific CD8 memory T cell receptor alpha and beta repertoires between young and old.

Older people have difficulty controlling infection with common viruses such as influenza A virus (IAV), RNA virus which causes recurrent infections due to a high rate of genetic mutation, and Epstein Barr virus (EBV), DNA virus which persists in B cells for life in the 95% of people that become acutely infected. We questioned whether changes in epitope-specific memory CD8 T cell receptor (TCR) repertoires to these two common viruses could occur with increasing age and contribute to waning immunity. We compared CD8 memory TCR alpha and beta repertoires in two HLA-A2+ EBV- and IAV-immune donors, young (Y) and older (O) donors to three immunodominant epitopes known to be cross-reactive, IAV-M158-66 (IAV-M1), EBV-BMLF1280-288 (EBV-BM), and EBV-BRLF1109-117 (EBV-BR). We, therefore, also designed these studies to examine if TCR cross-reactivity could contribute to changes in repertoire with increasing age. TCR high throughput sequencing showed a significant difference in the pattern of TRBV usage between Y and O. However, there were many more differences in AV and AJ usage, between the age groups suggesting that changes in TCRα usage may play a greater role in evolution of the TCR repertoire emphasizing the importance of studying TRAV repertoires. With increasing age there was a preferential retention of TCR for all three epitopes with features in their complementarity-determining region (CDR3) that increased their ease of generation, and their cross-reactive potential. Young and older donors differed in the patterns of AV/AJ and BV/BJ pairings and usage of dominant CDR3 motifs specific to all three epitopes. Both young and older donors had cross-reactive responses between these 3 epitopes, which were unique and differed from the cognate responses having features that suggested they could interact with either ligand. There was an increased tendency for the classic IAV-M1 specific clone BV19-IRSS-JB2.7/AV27-CAGGGSQGNLIF-AJ42 to appear among the cross-reactive clones, suggesting that the dominance of this clone may relate to its cross-reactivity with EBV. These results suggest that although young and older donors retain classic TCR features for each epitope their repertoires are gradually changing with age, maintaining TCRs that are cross-reactive between these two common human viruses, one with recurrent infections and the other a persistent virus which frequently reactivates.

Characterization of anti-cancer immune response associated with immune-related adverse events in patients with kidney cancer.

711 Background: Some reports showed promising clinical efficacy among patients who discontinued immune checkpoint inhibitors (ICIs) due to immune-related adverse events (irAEs). In this study, we aimed to characterize T cell receptor (TCR) repertoire repertoire in peripheral blood mononuclear cells (PBMCs) before and after ICI treatment, and at the onset of irAE for understanding of irAE-provoked anti-cancer immune responses. Methods: We collected metastatic tumor tissues and peripheral blood samples from 54 patients with advanced kidney cancer before anti-programmed cell death protein 1 (PD-1) treatment and 1 month after treatment initiation. Furthermore, we applied a next-generation sequencing approach to characterize TCR repertoires using tumor tissues and PBMCs. Results: The number of irAEs was significantly higher in responder for nivolumab (p &lt; 0.0001). We also confirmed that irAE present patients had better progression-free survival and overall survival when compared to irAE absent patients (p=0.0010 and p=0.011, respectively). TCR repertoire analysis revealed that diversity index for TCR alpha and beta in irAE present patients were significantly decreased at 1 month after nivolumab, indicating expansion of certain T cell clones even in PBMCs after nivolumab. We also examined morishita index to measure T cell similarity between pre- and post nivolumab samples. As a result, irAE present patients had significantly lower similarity than irAE absent patients, indicating a larger change in the T cell clones, and inducing newly expanded T cell clones in post-nivolumab PBMC samples. Importantly, irAE present patients had significantly higher numbers of peripheral T cell clones shared with metastatic tumor-infiltrating T cells than those in irAE absent patients at 1 month after nivolumab (as per the 100 most abundant T cell clonotypes in the peripheral blood; p=0.038). Conclusions: Our findings revealed that a certain number of irAE-provoked T cell clones can also circulate systemically and attack tumor cells in distant regions, leading to durable response in the patients with irAEs.

Archaic humans have contributed to large-scale variation in modern human T cell receptor genes

Human Tcell receptors (TCRs) are critical for mediating immune responses to pathogens and tumors and regulating self-antigen recognition. Yet, variations in the genes encoding TCRs remain insufficiently defined. Detailed analysis of expressed TCR alpha, beta, gamma, and delta genes in 45 donors from four human populations-African, East Asian, South Asian, and European-revealed 175 additional TCR variable and junctional alleles. Most of these contained coding changes and were present at widely differing frequencies in the populations, a finding confirmed using DNA samples from the 1000 Genomes Project. Importantly, we identified three Neanderthal-derived, introgressed TCR regions including a highly divergent TRGV4 variant, which mediated altered butyrophilin-like molecule 3 (BTNL3) ligand reactivity and was frequent in all modern Eurasian population groups. Our results demonstrate remarkable variation in TCR genes in both individuals and populations, providing a strong incentive for including allelic variation in studies of TCR function in human biology.

High-throughput Treg cell receptor sequencing reveals differential immune repertoires in rheumatoid arthritis with kidney deficiency.

Regulatory T (Treg) cells are important immune cells that are regulated by adaptive immunity in the composition of Treg-cell subsets and T-cell receptors (TCRs). Treg cells are related to most autoimmune diseases, such as rheumatoid arthritis (RA). In traditional Chinese medicine (TCM), RA is typically attributed to kidney deficiency (KD) associated with the immunosenescence that causes immune dysfunction and the impaired function of Treg cells. So far, however, no mechanism related to KD and immune repertoires has been identified in RA. Flow cytometry and high-throughput Treg-cell receptor sequencing were used to investigate the amount of different Treg-cell subsets and the diversity of TCRs between RA patients and healthy subjects, as well as between KD RA and non-KD RA patients. RT-qPCR was used to validate the high-throughput sequencing results. The data showed that the amount of naïve Treg cells in KD patients was less than in non-KD RA patients (P = 0.004) with no significant differences observed between other subsets. In the TCR of Treg cells, the length of complementarity determining region 3 (CDR3) was low and clonotypes increased in the KD group compared with the non-KD group. The diversity and abundance of Treg TCRs were low, as determined by the Hill number. In addition, several V(D)J combinations, such as T-cell receptor beta variable 7-2 (TRBV7-2), TRBV11-1, TRBV13, TRBV15, and TRBJ2-3, varied significantly between the two groups, indicating that KD causes Treg dysfunction. RT-qPCR shows that FOXP3 expression in peripheral blood Treg is lower in KD than in non-KD. The results demonstrate the close correlation between KD and immune repertoires in RA and provide a new evaluation method for RA in TCM.

Peripheral T-cell receptor repertoire dynamics in small cell lung cancer.

Identifying a circulating biomarker predictive of immune checkpoint inhibitor (ICI) benefit in patients with small cell lung cancer (SCLC) remains an unmet need. Characteristics of peripheral and intratumoral T-cell receptor (TCR) repertoires have been shown to predict clinical outcomes in non-small cell lung cancer (NSCLC). Recognizing a knowledge gap, we sought to characterize circulating TCR repertoires and their relationship with clinical outcomes in SCLC. SCLC patients with limited (n=4) and extensive (n=10) stage disease were prospectively enrolled for blood collection and chart review. Targeted next-generation sequencing of TCR beta and alpha chains of peripheral blood samples was performed. Unique TCR clonotypes were defined by identical CDR3, V gene, and J gene nucleotide sequences of the beta chain and subsequently used to calculate TCR diversity indices. Patients with stable versus progressive and limited versus extensive stage disease did not demonstrate significant differences in V gene usage. Kaplan-Meier curve and log-rank analysis did not identify a statistical difference in progression-free survival (PFS) (P=0.900) or overall survival (OS) (P=0.200) between high and low on-treatment TCR diversity groups, although the high diversity group exhibited a trend toward increased OS. We report the second study investigating peripheral TCR repertoire diversity in SCLC. With a limited sample size, no statistically significant associations between peripheral TCR diversity and clinical outcomes were observed, though further study is warranted.

Anti-VEGF and Anti-EGFR Antibody Therapy on T-Cell Infiltration and TCR Variation in Metastatic Colorectal Cancer.

Chemotherapy combined with anti-EGFR or anti-VEGF monoclonal antibodies (mAb) is widely used to treat patients with metastatic colorectal cancer (mCRC). Here, we investigated the effects of these antibodies on T-cell infiltration and T-cell receptor (TCR) repertoire variation in CRC liver metastases. Ten patients with mCRC received chemotherapy in combination with anti-EGFR (n=6) or anti-VEGF (n=4) mAb. T-cell infiltration was examined for CD3 and CD8 by carrying out immunohistochemistry on biopsy or surgical specimens from liver metastases before and after treatment. TCR repertoire analysis was carried out on specimens with post-treatment CD3+ T-cell infiltration. T-cell infiltrations were approximately 83% (5/6) and 50% (2/4), following treatment with anti-EGFR or anti-VEGF mAb, respectively. TCR repertoire analysis revealed higher clonality and lower diversity of TCR alpha and beta (TRA and TRB) in the anti-VEGF mAb group than that in the anti-EGFR group mAb. Furthermore, the percentage of the common TCR clones between infiltrating T cells and T cells in peripheral blood was significantly lower in the anti-VEGF mAb group compared to that in the anti-EGFR mAb group. The population of T cells infiltrating liver metastases in the anti-VEGF mAb group differed from that in the anti-EGFR mAb group.

Targeting the melanoma-associated antigen CSPG4 with HLA-C*07:01-restricted T-cell receptors.

Chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen, is expressed in melanoma but also other tumor entities and constitutes an attractive target for immunotherapeutic approaches. While recent preclinical reports focused on anti-CSPG4 chimeric antigen receptors (CAR), we here explore T-cell receptor (TCR)-based approaches targeting CSPG4. The TCRs of two CSPG4-reactive T-cell clones (11C/73 and 2C/165) restricted by the highly prevalent HLA-C*07:01 allele were isolated and the respective αβTCR pairs were retrovirally expressed in CRISPR/Cas9-edited TCR-knockout T cells for functional testing. We also combined alpha and beta TCR chains derived from 11C/73 and 2C/165 in a cross-over fashion to assess for hemichain dominance. CSPG4+ melanoma, glioblastoma and lung cancer cell lines were identified and, if negative, retrovirally transduced with HLA-C*07:01. Functional tests confirmed specific recognition of CSPG4+HLA-C*07:01+ target cells by the αβTCR retrieved from the parental T-cell clones and in part also by the cross-over TCR construct 2Cα-11Cβ. Despite high surface expression, the 11Cα-2Cβ combination, however, was not functional. Collectively, 11C/73- and 2C/165-expressing T cells specifically and efficiently recognized CSPG4+HLA-C*07:01+ cancer cells which warrants further preclinical and clinical evaluation of these TCRs.