T-cell Receptor Variable Beta Research Articles

Abstract 1192: Diversity of TCR repertoire on peripheral CD8+PD-1+ T-cell predicting the PFS of chemoradiotherapy followed by durvalumab maintenance in unresectable locally advanced NSCLC patients

Abstract Background: Chemoradiotherapy (CRT) followed by Durvalumab consolidation has been the standard treatment for unresectable locally advanced non-small cell lung cancer (LA-NSCLC) patients. This study aimed to evaluate how CRT changes TCR repertoire on peripheral CD8+PD-1+ T-cells, and the change affects the clinical outcomes of CRT followed by durvalumab treatment. Methods: This was a prospective cohort study conducted from November 2019 to May 2021 in three institutes (National Cancer Center Hospital, Kansai Medical University Hospital, and Aichi Cancer Center Hospital) in Japan. We revealed the diversity of TCR repertoire (diversity evenness 50 [DE50]) on PD-1+CD8+ T cells, and CD8+ T cell phenotype in PBMCs before and after CRT treatment. Results: A total of 40 patients treated with CRT were included. Of these patients, 32 patients received durvalumab consolidation treatment after CRT (Durva cohort). The diversity and usage of T cell Receptor Beta Variable (TRBV) and T-cell receptor beta joining gene (TRBJ) usage were significantly positively correlated before and after CRT (TRBV usage: r=0.62, p<0.001, TRBJ usage: r=0.55, p<0.001). Regarding the association of TCR repertoire diversity with CD8+ T cell phenotype, DE50 and frequencies of CD8 naive were significantly positively correlated before CRT (p=0.049), while there was no correlation between PD-L1 expression and TCR repertoire before CRT (r=0.03, p=0.89) In Durva cohort, DE50 in patients with non-recurrence was higher than those with recurrence both before and after CRT (Before CRT: 0.0032 vs. 0.0019, p=0.13, After CRT: 0.0026 vs. 0.0014, p=0.06). The progression-free survival (PFS) of patients with DE50High before CRT was significantly greater than those with DE50Low (NR vs. 14.3 months; p=0.01). Moreover, when the patients were exploratory classified based on the PD-L1 expression status and the diversity of TCR repertoire, the PFS of the CRT followed by durvalumab apparently differed (DE50High, PD-L1 TPS≥1%, NR, and DE50Low, PD-L1 TPS<1%, 5.9 (4.6-7.3) months, p=0.001). Conclusions: TCR diversity of peripheral CD8+PD-1+ T-cell predicted the recurrence in unresectable locally advanced NSCLC patients treated with chemoradiotherapy followed by durvalumab maintenance. Additionally, the TCR diversity combined with PD-L1 expression status could more accurately predict the efficacy of the CRT followed by durvalumab. Citation Format: Masayuki Shirasawa, Tatsuya Yoshida, Yuki Takeyasu, Takaji Matsutani, Shigehiro Yagishita, Shigehisa Kitano, Hiroaki Kuroda, Toyoaki Hida, Takayasu Kurata, Yuichiro Ohe. Diversity of TCR repertoire on peripheral CD8+PD-1+ T-cell predicting the PFS of chemoradiotherapy followed by durvalumab maintenance in unresectable locally advanced NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1192.

Characterization of T cell receptor repertoire in penile cancer

Tumor-infiltrating lymphocytes (TILs) play a key role in regulating the host immune response and shaping tumor microenvironment. It has been previously shown that T cell infiltration in penile tumors was associated with clinical outcomes. However, few studies have reported the T cell receptor (TCR) repertoire in patients with penile cancer. In the present study, we evaluated the TCR repertoires in tumor and adjacent normal tissues from 22 patients with penile squamous cell carcinoma (PSCC). Analysis of the T cell receptor beta-variable (TRBV) and joining (TRBJ) genes usage and analysis of complementarity determining region 3 (CDR3) length distribution did not show significant differences between tumor and matched normal tissues. Moreover, analysis of the median Jaccard index indicated a limited overlap of TCR repertoire between these groups. Compared with normal tissues, a significantly lower diversity and higher clonality of TCR repertoire was observed in tumor samples, which was associated with clinical characteristics. Further analysis of transcriptional profiles demonstrated that tumor samples with high clonality showed increased expression of genes associated with CD8 + T cells. In addition, we analyzed the TCR repertoire of CD4 + T cells and CD8 + T cells isolated from tumor tissues. We identified that expanded clonotypes were predominantly in the CD8 + T cell compartment, which presented with an exhausted phenotype. Overall, we comprehensively compared TCR repertoire between penile tumor and normal tissues and demonstrated the presence of distinct T cell immune microenvironments in patients with PSCC.

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 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...

EP08.01-009 TRBV Haplotype Profile Was Related to Immune-Induced Adverse Events in Chinese Patients With Advanced NSCLC

Immune-related adverse events (irAE) are inevitable following immunotherapy, which significantly impact the quality of life and possibly cause therapy discontinuation or interruption in cancer patients. Previous studies reported that T-cell receptor beta variable (TRBV) gene was related to irAE in Caucasian patients with non-small cell lung cancer (NSCLC). However, the relationship between TRBV gene and irAE remains to be revealed in Chinese NSCLC patients.

TIL expansion with high dose IL-2 or low dose IL-2 with anti-CD3/anti-CD28 stimulation provides different quality of TIL-expanded T cell clones

Tumor infiltrating lymphocytes (TILs) are cells that are present inside the tumor environment, of which include T cells, B cells and natural killer (NK) cells. At present, TILs are used for immunotherapy in various cancers. Knowledge on adoptive transfer of TILs in ovarian cancer is still limited, especially regarding TIL expansion methods. Therefore, the aim of our study was to compare the quality of T cell clones between two expansion methods for ovarian cancer TILs. We show that TILs stimulated with the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28) and the standard stimulation method (high dose IL-2 only) both increased total number of T cells. TCR repertoire analyses revealed different TCR repertoire patterns between TIL-expanded T cells that were stimulated with the standard stimulation method (high dose IL-2 only) and the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28). Regardless, when TILs were expanded using the standard stimulation method (high dose IL-2 only), the predominant T cell receptor beta variable (TRBV) chains that were used in both TIL-expanded clones of the CD4+ and CD8+ subpopulations were similar. In addition, there were also TIL-expanded CD4+ and CD8+ T cell clones that were dominant in only one or the other subpopulations. These results reveal the bias in TIL quality after being stimulated with different protocols. Further studies are required to understand the selection of TIL expansion, in order for a more efficacy adoptive transfer treatment.

Structural basis of T cell receptor specificity and cross-reactivity of two HLA-DQ2.5-restricted gluten epitopes in celiac disease

Celiac disease is a T cell-mediated chronic inflammatory condition often characterized by human leukocyte antigen (HLA)-DQ2.5 molecules presenting gluten epitopes derived from wheat, barley, and rye. Although some T cells exhibit cross-reactivity toward distinct gluten epitopes, the structural basis underpinning such cross-reactivity is unclear. Here, we investigated the T-cell receptor specificity and cross-reactivity of two immunodominant wheat gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). We show by surface plasmon resonance that a T-cell receptor alpha variable (TRAV) 4+-T-cell receptor beta variable (TRBV) 29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1 with similar affinity, whereas a TRAV4- (TRAV9-2+) TCR recognized HLA-DQ2.5-glia-ω1 only. We further determined the crystal structures of the TRAV4+-TRBV29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1, as well as the structure of an epitope-specific TRAV9-2+-TRBV7-3+ TCR-HLA-DQ2.5-glia-ω1 complex. We found that position 7 (p7) of the DQ2.5-glia-α1a and DQ2.5-glia-ω1 epitopes made very limited contacts with the TRAV4+ TCR, thereby explaining the TCR cross-reactivity across these two epitopes. In contrast, within the TRAV9-2+ TCR-HLA-DQ2.5-glia-ω1 ternary complex, the p7-Gln was situated in an electrostatic pocket formed by the hypervariable CDR3β loop of the TCR and Arg70β from HLA-DQ2.5, a polar network which would not be supported by the p7-Leu residue of DQ2.5-glia-α1a. In conclusion, we provide additional insights into the molecular determinants of TCR specificity and cross-reactivity to two closely-related epitopes in celiac disease.

Haplotype Analysis of the T-Cell Receptor Beta (TCRB) Locus by Long-amplicon TCRB Repertoire Sequencing

Abstract Background: Polymorphism within the human T-cell receptor beta variable (TRBV) gene has been proposed as a risk factor for autoimmune disease and immune-related adverse events (IRAEs) during immunotherapy. Previous efforts to evaluate TRBV polymorphism by whole genome sequencing have been hampered by the repetitive nature of the T-cell receptor beta (TCRB) locus. We present a novel long-amplicon TCRB repertoire sequencing approach to enable TRBV haplotype analysis from peripheral blood. Methods: Peripheral blood leukocyte total RNA from 81 Caucasians was used for sequencing of TCRB chains via the Oncomine TCRB-LR assay (amplicon spanning CDR1, 2 and 3) and the Ion Gene Studio S5. VDJ rearrangements were annotated by comparison to the IMGT database, then mined to construct TRBV allele profiles for each individual including, where detected, novel alleles not present in the ImMunoGeneTics (IMGT) database. Finally, TRBV allele profiles were subjected to principal component analysis and k-means clustering to identify TRBV allele haplotypes. Results: Clustering analysis revealed the presence of six major sets of coincident TRBV alleles, which we term haplotype groups. Allelic diversity varied markedly across haplotype groups, with approximately one third of the cohort showing limited TRBV allelic diversity and few uncommon alleles compared to members of other groups. Analysis revealed 37 putatively novel TRBV alleles that are absent from the IMGT database. Conclusion: We demonstrate a straightforward and cost-efficient method for TRBV haplotype analysis from long-amplicon TCRB sequencing data.

The variations of TRBV genes usages in the peripheral blood of a healthy population are associated with their evolution and single nucleotide polymorphisms

T cell receptors (TCRs) are a class of T cell surface molecules that recognize the antigen-derived peptides presented by the major histocompatibility complex (MHC) and are able to trigger a series of immune responses. TCRs are important members of the adaptive immune system that arose in the jawed fish 500 million years ago. T cell receptor beta variable (TRBV) genes have been widely used to characterize TCR repertoires. Studying the evolution of TRBV may help us to better understand the adaptive immune system. To investigate TRBV evolution and its impacts on the usages of TRBV genes in human populations, we compared the TRBV genes and their homologous sequences among humans, mouse, rhesus and chimpanzee, analyzed the single-nucleotide polymorphisms (SNPs) located at TRBV loci, and sequenced TCR repertoires in the peripheral blood of 97 healthy donors. We found that functional TRBVs are more evolutionarily conserved but possess more SNPs in human populations than do nonfunctional (pseudo) TRBVs. Based on the conservation levels in the four species, we classified the functional TRBVs into 2 groups: old (conserved between mouse and humans) and new (conserved only in primates). The new TRBVs evolve faster and possess more SNPs than the old TRBVs. The variations in TRBV genes frequencies in the peripheral blood of healthy donors are negatively correlated with SNP density. These observations suggest that TRBV usages may be influenced by TCR-MHC co-evolution.

Sex bias in MHC I-associated shaping of the adaptive immune system

HLA associations, T cell receptor (TCR) repertoire bias, and sex bias have independently been shown for many diseases. While some immunological differences between the sexes have been described, they do not fully explain bias in men toward many infections/cancers, and toward women in autoimmunity. Next-generation TCR variable beta chain (TCRBV) immunosequencing of 824 individuals was evaluated in a multiparametric analysis including HLA-A -B/MHC class I background, TCRBV usage, sex, age, ethnicity, and TCRBV selection/expansion dynamics. We found that HLA-associated shaping of TCRBV usage differed between the sexes. Furthermore, certain TCRBVs were selected and expanded in unison. Correlations between these TCRBV relationships and biochemical similarities in HLA-binding positions were different in CD8 T cells of patients with autoimmune diseases (multiple sclerosis and rheumatoid arthritis) compared with healthy controls. Within patients, men showed higher TCRBV relationship Spearman's rhos in relation to HLA-binding position similarities compared with women. In line with this, CD8 T cells of men with autoimmune diseases also showed higher degrees of TCRBV perturbation compared with women. Concerted selection and expansion of CD8 T cells in patients with autoimmune diseases, but especially in men, appears to be less dependent on high HLA-binding similarity than in CD4 T cells. These findings are consistent with studies attributing autoimmunity to processes of epitope spreading and expansion of low-avidity T cell clones and may have further implications for the interpretation of pathogenic mechanisms of infectious and autoimmune diseases with known HLA associations. Reanalysis of some HLA association studies, separating the data by sex, could be informative.

Public Clonotypes and Convergent Recombination Characterize the Naïve CD8+ T-Cell Receptor Repertoire of Extremely Preterm Neonates.

Respiratory support improvements have aided survival of premature neonates, but infection susceptibility remains a predominant problem. We previously reported that neonatal mice have a rapidly evolving T-cell receptor (TCR) repertoire that impairs CD8+ T cell immunity. To understand the impact of prematurity on the human CD8+ TCR repertoire, we performed next-generation sequencing of the complementarity-determining region 3 (CDR3) from the rearranged TCR variable beta (Vβ) in sorted, naïve CD8+ T cells from extremely preterm neonates (23–27 weeks gestation), term neonates (37–41 weeks gestation), children (16–56 months), and adults (25–50 years old). Strikingly, preterm neonates had an increased frequency of public clonotypes shared between unrelated individuals. Public clonotypes identified in preterm infants were encoded by germline gene sequences, and some of these clonotypes persisted into adulthood. The preterm neonatal naïve CD8+ TCR repertoire exhibited convergent recombination, characterized by different nucleotide sequences encoding the same amino acid CDR3 sequence. As determined by Pielou’s evenness and iChao1 metrics, extremely preterm neonates have less clonality, and a much lower bound for the number of unique TCR within an individual preterm neonate, which indicates a less rich and diverse repertoire, as compared to term neonates, children, and adults. This suggests that T cell selection in the preterm neonate may be less stringent or different. Our analysis is the first to compare the TCR repertoire of naïve CD8+ T cells between viable preterm neonates and term neonates. We find preterm neonates have a repertoire immaturity which potentially contributes to their increased infection susceptibility. A developmentally regulated, evenly distributed repertoire in preterm neonates may lead to the inclusion of public TCR CDR3β sequences that overlap between unrelated individuals in the preterm repertoire.

Utilizing IL-7 to generate melanoma antigen T cell receptor-modified cells for adoptive transfer in the absence of activation.

e14552 Background: Metastatic melanoma remains a fatal disease as many patients fail to respond to novel therapies. Presently, we have an ongoing trial utilizing T cell receptor (TCR)-modified cells reactive to the melanoma antigen, tyrosinase, for treatment of metastatic melanoma (NCT01586403). Current protocols using viral vectors to generate cells for adoptive transfer require activation in the presence of IL-2. While IL-2 stimulates proliferation and production of mature effector cells, it also induces T cell exhaustion. We hypothesize that tyrosinase-reactive TCR-modified cells generated in the absence of activation have an increased potential for expansion, persistence, and anti-melanoma activity. In these studies, we investigate whether IL-7, a homeostatic regulator, facilitates the transfer of a tyrosinase-reactive TCR to lymphocytes without activation. Methods: Gene-modified cells were generated using a lentiviral vector including alpha and beta chains of a TCR reactive to tyrosinase and a truncated CD34 molecule as a transduction marker. Peripheral lymphocytes from normal human donors were transduced with lentivirus following no treatment, treatment with IL-7, or activation in the presence of IL-2. Transduced CD34 positive cells were isolated using magnetic activated cell sorting (MACS) and injected intravenously into NOD scid gamma (NSG) mice. Results: We found that IL-7 treatment improved transduction in non-activated cells and resulted in expression levels of CD34 and TCR variable beta chain 12 (vbeta12) comparable to cells activated in the presence of IL-2. When non-activated tyrosinase-reactive TCR-modified human T cells were adoptively transferred into NOD NSG mice, the cells were detectable in the peripheral blood of mice up to 7 days after transfer. Conclusions: These findings suggest that generating melanoma antigen TCR-modified cells in the absence of activation for adoptive transfer is feasible with IL-7 treatment. Ongoing studies in NSG mice transplanted with melanoma will help determine whether non-activated gene-modified cells will improve the efficacy and longevity of cell therapy for melanoma and other malignancies.

Increased Frequency of Paroxysmal Nocturmal Hemoglobinuria Type Cells in Patients with Chronic Idiopathic Neutropenia Correlates with the Severity of the Disease and the Presence of Skewed T-Cell Expansions

Introduction: Chronic idiopathic neutropenia (CIN) of adults is an acquired disorder of granulopoiesis characterized by an unexplained, prolonged reduction in the number of peripheral blood (PB) neutrophils. We have previously shown that CIN pathophysiology is related to T-cell activation and increased apoptosis of granulocytic progenitor cells. Notably, in our cohort of patients we exclude cases with antineutrophil antibody activity, i.e. autoimmune neutropenia cases.Aim of the study: Preliminary data have shown the presence of minor populations of paroxysmal nocturmal hemoglobinuria (PNH) type cells in the PB of CIN patients. The present study aims to investigate further the presence of PNH type cells in the PB of a large cohort of patients fulfilling the diagnostic criteria of CIN and probe the possible association with the severity of the disease and the skewed T-cell profile.Patients - Methods: We have studied 91 adults with CIN and 55 hematologically healthy subjects, age- and sex-matched with the patients. We have used flow cytometry for the detection of PNH type cells according to the International Clinical Cytometry Society guidelines and for the identification of T-cell expansions based on the analysis of T-cell receptor beta variable (TRBV) gene repertoire.Results: CIN patients displayed increased proportion of PB PNH type FLAER-/CD24- neutrophils (0.05% ± 0.18%) and FLAER-/CD14- monocytes (1.33% ± 1.38%) compared to healthy individuals (0.005% ± 0.01% and 0.64% ± 0.66%, respectively; P =0.0044 and P =0.011, respectively). Similarly, the patients displayed increased proportion of both, type II CD235+/CD59dim and type III CD235+/CD59- (0.059% ± 0.29% and 0.065% ± 0.28%, respectively) PB PNH red blood cells, compared to healthy controls (0.01% ± 0.03% and 0.01% ± 0.03%, respectively; P<0.0001 and P< 0.0001, respectively). Interestingly, an inverse correlation was found between the proportion of PB PNH type neutrophils and the number of absolutely neutrophil counts in CIN patients (r=-0.2525, P =0.0157) whereas no correlation was found between the proportion of PNH red blood cells or monocytes and absolutely neutrophil counts. In accordance with our previously reported data, increased frequency of skewed T-cell expansions were identified in CIN patients (71.4%) compared to healthy subjects (29.9%; P<0.0001). PNH type neutrophils (FLAER-/CD24-) were identified at a higher frequency in patients with skewed TRBV repertoire (63.08%) compared to those with normal TRBV distribution (34.61%) (P = 0.0194) suggesting a possible pathogenetic/pathophysiologic association between T-cell activation and emergence of PNH neutrophils in CIN.Conclusion: CIN patients display minor populations of PNH type PB neutrophils, monocytes and red blood cells. The frequency of PNH type neutrophils is associated with the severity of neutropenia and it is higher among patients with skewed TRBV repertoire. These data support further the hypothesis that CIN displays common pathophysiologic features with immune-mediated bone marrow failure syndromes and should be included in this spectrum of disease entities. DisclosuresPapadaki:Alexion Pharmaceuticals: Research Funding.

Molecular profile of the T cell receptor beta variable in peripheral blood lymphocytes from chronic asymptomatic HBV carriers.

T cell receptor beta variable (TCRBV) repertoire could imply the composition and function status of T cells in subjects with HBV infection. The gene melting spectral pattern (GMSP) can be used to determine the profile of TCRBV gene family. The molecular profile of TCRBV in peripheral lymphocytes from asymptomatic HBV carriers (AsC) remains ill-defined. Peripheral blood mononuclear cells (PBMCs) were separated and sorted, and the profiles of TCRBV complementarity-determining region 3 (CDR3) in CD4(+), CD8(+) T subsets and PBMCs were assayed using GMSP. The number of skewed TCRBV in the PBMCs was significantly lower than that in the CD4(+) or CD8(+) T subsets, and the number of monoclonal TCRBV families in the CD8(+) T subset was significantly higher than that in CD4(+) T subset. Compared to healthy donors, TCRBV11, BV13.1, BV20 and BV24 were used more frequently than other TCRBV members in PBMCs from AsC subjects. Furthermore, the relatively conserved CDR3 motifs were detected in these TCRBVs. The results indicate that the T cell response in AsC subjects involves several TCRBVs, and that the CD8(+) T subset maybe more relevant to pathogenesis of AsC. Moreover, the four relative conserved TCRBVs maybe a target for personalized treatments for persistent HBV infection.

Composition and variation analysis of TCR β-chain CDR3 repertoire in the thymus and spleen of MRL/lpr mouse at different ages.

T cells play an important role in the onset and progression of systemic lupus erythematosus (SLE), and the biases in T cell receptor beta variable (TRBV) region families and complementarity determining region three (CDR3) composition in SLE patients and mouse models have been widely reported. However, the relationship between the composition and variation in the TCR β-chain CDR3 repertoire and SLE has not been established. Here, we compared and analyzed the thymic and splenic TCR β-chain CDR3 mRNA sequences by Roche 454 high-throughput sequencing from MRL/lpr mice at different ages. Our results indicate that diversity in the TCR CDR3 repertoire from the thymus and spleen from MRL/lpr mouse was significantly decreased with increased age (disease progression) and showed a bias in usage of common TRBV and TRBJ families. The N1 region insertions in the highly expressed CDR3s significantly increased with disease progression. This study provides a new perspective for studying SLE with progression of disease in clonal level of TCR, which may provide a basis for studying the mechanism of the MRL/lpr autoreactive T cells response and tailor an individualized treatment targeting these T cells.

Genomic characteristics of the T cell receptor (TRB) locus in the rabbit (Oryctolagus cuniculus) revealed by comparative and phylogenetic analyses

The present study identifies the genomic structure and the gene content of the T cell receptor beta (TRB) locus in the Oryctolagus cuniculus whole genome assembly. The rabbit locus spans less than 600Kb and the general genomic organization is highly conserved with respect to other mammalian species. A pool of 74 TRB variable (TRBV) genes distributed in 24 subgroups are located upstream of two in tandem-aligned D-J-C gene clusters, each composed of one TRBD, six TRBJ genes, and one TRBC gene, followed by a single TRBV gene with an inverted transcriptional orientation. All TRB genes (functional, ORF, pseudogenes) of this paper have been approved by the IMGT/WHO-IUIS nomenclature committee. Additionally, five potentially functional protease serine (PRSS) trypsinogen or trypsinogen-like genes were identified: two in tandem PRSS-like genes, followed by two PRSS genes with unique traits, lie downstream of the TRBV1 gene and one PRSS gene is located about 400Kb away downstream of the TRBV genes. Comparative and phylogenetic analyses revealed that multiple duplication events within a few subgroups have generated the germline repertoire of the rabbit TRBV genes, which is substantially larger than those described in humans, mice, and dogs, suggesting that a strong evolutionary pressure has selected the development of a species-specific TRBV repertoire. Hence, the genomic organization of the TRB locus in the genomes appears to be the result of a balance between the maintenance of a core-number of genes essential for the immunological performances and the requirement of newly arisen genes.

Multivariate Analysis Using High Definition Flow Cytometry Reveals Distinct T Cell Repertoires between the Fetal–Maternal Interface and the Peripheral Blood

The human T cell compartment is a complex system and while some information is known on repertoire composition and dynamics in the peripheral blood, little is known about repertoire composition at different anatomical sites. Here, we determine the T cell receptor beta variable (TRBV) repertoire at the decidua and compare it with the peripheral blood during normal pregnancy and pre-eclampsia. We found total T cell subset disparity of up to 58% between sites, including large signature TRBV expansions unique to the fetal–maternal interface. Defining the functional nature and specificity of compartment-specific T cells will be necessary if we are to understand localized immunity, tolerance, and pathogenesis.

Molecular characterization of T cell receptor beta variable in the peripheral blood T cell repertoire in subjects with active tuberculosis or latent tuberculosis infection

BackgroundT cells are closely linked to the clinical manifestations of subjects with Mycobacterium tuberculosis (MTB) infection. T cell receptor beta variable (TCRBV) is a signal and indicative molecule on the membrane of T lymphocytes, reflecting the composition and specificity of T cells. The molecular profiles of TCRBV in peripheral blood mononuclear cells (PBMCs) and their subpopulations (CD4+ and CD8+ T cells) from subjects with active tuberculosis (TB) or latent TB infection (LTBI) have not been well described.MethodsIn 42 subjects with active TB or LTBI, PMBCs and their subsets were separated and sorted. The molecular profiles of the TCRBV complementarity determining region 3 (CDR3) in the three cell populations were investigated using our recently developed gene melting spectral pattern (GMSP) assay. The TCRBV members were then cloned and sequenced when their GMSP image profiles showed a single-peak.ResultsThe average number of skewed TCRBV molecules in the CD4+ cell subset was significantly higher than that in PBMCs and CD8+ T cells. TCRBV12, BV13.1, BV13.2, and BV24 were expressed more prevalently than other TCRBV gene families in the three cell populations. In addition, relatively conserved amino acid motifs were identified in TCRBV5.1 and BV20 CDR3 in PBMCs and its subsets. The monoclonal TCRBV14 and BV23 expressed were different between active TB and LTBI subjects.ConclusionsThese results indicate that the T cell immune response is complex and multi-specific in active TB and LTBI subjects. Analysis of TCRBV expression in CD4+ T cells suggest that it could be useful in assessing the composition and status of circulating T cells. Furthermore, the expression of TCRBV14, BV23 and the sequencing of CDR3 amino acid motifs of TCRBV5.1, BV20 could be used in the differential diagnosis and treatment of subjects with active TB or LTBI.

Limited T cell receptor beta variable repertoire responses to ESAT-6 and CFP-10 in subjects infected with Mycobacterium tuberculosis

Mycobacterium tuberculosis (MTB)-specific antigens, ESAT-6 or CFP-10 play a key role in diagnosis and control MTB infection. T cell receptor (TCR) reflects the status and function of T cells. However, the features of the TCR beta variable (TCRBV) repertoire used against ESAT-6 and CFP-10 from MTB subjects have not been well described. The molecular profiles of TCRBV complementarity-determining region 3 (CDR3) in PBMCs with or without ESAT-6 or CFP-10 stimulation were assayed using a gene melting spectral pattern (GMSP) assay developed in our previous study. The average number of skewed TCRBV family in PBMCs stimulated with ESAT-6 or CFP-10 was significantly higher than that in unstimulated PBMCs. TCRBV3, BV5.1, BV12, BV13.1, BV13.2, BV20 and BV24 were used more frequently than other TCRBV members in PBMCs from MTB subjects, and TCRBV3, BV5.1 in stimulated PBMCs have a preference in the usage of variable (V) and joining (J) segments and CDR3. The results indicate that the T cell immune response in MTB subjects involves a few of specific T cells. The preferred usage of certain V and J segments and CDR3s of TCRBV3 or BV5.1 may be related to ESAT-6 or CFP-10 respectively, which would help clinical differential diagnosis and treatment of MTB-infected subjects.

TRBV kinetics and its association with HLA disparity and aGVHD following allogeneic hematopoietic stem cell transplantation

The relative expression of T cell receptor (TCR) beta variable (TRBV) and TCR diversity was compared between recipients receiving human leukocyte antigen (HLA)-mismatched transplants and those receiving HLA-matched transplants, using granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells and bone marrow as grafts. The kinetics of the relative expression of TRBV family members were analyzed using real-time quantitative PCR. Additionally, the association of TRBV clonotype with acute graft-versus-host disease (aGVHD) was determined by cloning and sequence analysis of the CDR3 region. The TCR diversity in recipients receiving HLA-mismatched transplants was significantly lower than in those receiving HLA-matched transplants at 1 month and 2 months after hematopoietic stem cell transplantation (HSCT) (both P < 0.05). However, these differences disappeared 3 months after transplantation. The relative expression of TRBV27 (n = 7 recipients) at the onset of aGVHD was higher than in corresponding donors (P = 0.025), but no significant differences were observed between recipients lacking aGVHD and their donors at serial time points after HSCT. Our results suggest that HLA disparity may affect the relative expression of TRBV in the early phase after transplantation, and TRBV27 may be associated with the onset of aGVHD.