Vβ Gene Research Articles

T cell receptor β-chain repertoire analysis to reveal potential predictive biomarker for the use of immune checkpoint blockade in patients with advanced solid tumors.

e14152 Background: Immune checkpoint blockade (ICB) targeted to CTLA-4, PD-1, and PD-L1 can promote antitumor T cell immunity and clinical responses. Tracking the dynamic changes of the T Cell Receptor (TCR) Immune Repertoire 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: We sequenced the TCRβ genes from T cells contained in peripheral blood mononuclear cells (PBMCs) from 24 advanced solid tumors patients (PD/7, PR/4, SD/13) treated with immune checkpoint blockade. Peripheral blood samples were collected from baseline, 8-week, and 16-week after treatment for each patient. bioinformatic analysis was used to detect the diversity and clonality changes of TCRβ-CDR3 sequence before and after treatment with ICB in patients, and provide auxiliary technology for further guiding clinical tumor immunotherapy. Results: The analysis result of Chao index showed that the TCR-CDR3 diversity in the PR group was significantly increased after treatment than SD or PD group. (pvalue = 0.001), and the number of CDR3 aa clonotypes also increased significantly (pvalue = 0.012). A total of 61 Vβ gene fragments and 15 Jβ gene fragments were uniquely identified during immunotherapy . There was a significant difference in the number of Vβ gene fragments between baseline samples and after treatment in PR group (pvalue = 0.042). Conclusions: In this project, PBMC samples was used to evaluate the changes of TCR repertoire between before and after treatment with immune checkpoint blockade in advanced solid tumor patients. The immune function status was significantly changed before and after treatment. Patients with PR had significantly increased diversity in TCR repertoire than patients with SD or PD. The results provide T cell receptor β-chain repertoire sequencing and analysis has potential to assess whether immune checkpoint blockade promotes global T cell expansion and/or loss of T cell clonotype diversity, and to identify predictive biomarker for the use of immune checkpoint blockade.

Diversification of the TCR β Locus Vβ Repertoire by CTCF.

The architectural protein CTCF regulates the conformation and recombination of antigen receptor loci. To study the importance of CTCF in Tcrb locus repertoire formation, we created a conditional knockout mouse line that deletes Ctcf early during thymocyte development. We observed an incomplete block in thymocyte development at the double-negative to double-positive transition, resulting in greatly lowered thymic cellularity. The Tcrb repertoire was altered with a decrease in recombination of Vβ gene segments in close proximity to a CTCF binding element (CBE), resulting in an overall repertoire that was skewed in favor of Vβ gene segments with no nearby CBE. Therefore we show that CTCF functions to diversify the Tcrb repertoire.

Hypercytokine-Producing Cells and Oligoclonal T-Cell Populations in Lymph Nodes from Castleman Disease Patients

Introduction:Castleman disease (CD) is an uncommon lymphoproliferative disorder of unclear etiology. CD is broadly subclassified into unicentric (UCD) and multicentric (MCD) disease based on based on clinical, radiological, laboratory and histomorphological findings. UCD presents with minimal symptoms, is restricted to a single lymph node station, and excision is usually curative. Multicentric CD, in contrast, presents with systemic inflammatory symptoms, multicentric lymphadenopathy, and vital organ dysfunction. MCD is further subclassified into Human Herpesvirus-8(HHV-8)-associated MCD and HHV-8-negative/idiopathic MCD (iMCD), the etiology of which is poorly understood. Histologically, UCD frequently shows a hyaline vascular pattern with atretic follicles, thickened mantle zones and increased interfollicular vascularity with hyalinization. iMCD frequently shows increased interfollicular plasma cells and hyperplastic follicles. iMCD is also characterized by a systemic hypercytokinemia that includes IL-6, VEGF, IL-2, TNF-α, IL-10 and CXCL13. However, the cell(s) involved in initiating and amplifying the cytokine network are unclear. We combined histomorphology and cytokine in situ hybridization to identify the hypercytokine-producing cells in lymph nodes from CD patients. In addition, T and B lymphocytes play important roles in initiating and amplifying the cytokine response and their clonality has not been well-studied in CD. Here, we performed deep sequencing of the immunoglobulin heavy chain and T cell receptor gene loci.Methods:Lymph node biopsies from patients with UCD and iMCD were identified from the pathology archives of the Children's Hospital of Philadelphia. 17 UCD cases and 8 HHV8-negative iMCD cases were examined. Reactive lymph nodes (N=10) with plasmacytosis and/or other CD-like features served as controls. Cytokine expression was determined by RNA in situ hybridization (RNAscope) on formalin fixed paraffin embedded tissue. IL-6, IL-6R, VEGF, IL-10, TNF-α, IL-1β, IL-2, and IL-8 RNA expression patterns were analyzed in conjunction with histomorphological features. Expression was manually quantified with a semi-quantitative grading scale (0-4) per manufacturer recommendations and statistical analysis was performed using the Chi-square test. Fresh frozen lymph node tissue was utilized for deep sequencing of the TCR Vβ and IgH gene loci. VDJ usage, clonal frequency and CDR3 sequence was determined and compared between subtypes of CD and reactive lymph node controls.Results:Lymph nodes from patients with iMCD express significantly higher levels of VEGF compared to patients with UCD and controls (75% vs. 29% vs. 0%; p=0.014). Atretic follicles and interfollicular regions were the source of increased VEGF expression. Potential cell types responsible for the increased VEGF production in these regions are follicular dendritic cells in the atretic follicles and plasma cells in the interfollicular areas. IL-6 expression was also significantly higher in iMCD cases compared to UCD and controls (75% vs. 25% vs. 20%, p=0.026) in a subset of cells within the interfollicular regions. This cellular source of the excess IL-6 in the interfollicular region may be endothelial cells. Thus, follicular dendritic cells in the germinal centers and endothelial cells, T cells, and plasma cells in the interfollicular spaces are potential sources for increased VEGF and IL-6. IL-6R, IL-10, TNF-α, IL-1β, IL-2, and IL-8 showed no significant differences between the various subtypes of CD.Deep sequencing of the TCRα gene loci revealed mildly expanded clonal T-cell populations (5% of total sequences) in a subset of iMCD cases (2/6) and UCD cases (1/9) compared to controls (0/15). B cell populations were polyclonal in both subtypes of CD and in reactive lymph nodes.Conclusion:The findings suggest that cells in the interfollicular region and atretic follicles in the lymph nodes are a potential source of the systemic hypercytokinemia in iMCD. The locations and patterns of cytokine expression implicate follicular dendritic cells, endothelial cells, and plasma cells specifically as potential hypercytokine-producing cells. Additionally, T cells in CD show oligoclonality in some cases and may play an important role in initiating or amplifying the immune response in CD. DisclosuresFajgenbaum:Janssen Pharmaceuticals, Inc.: Research Funding.

Rearranged T Cell Receptor Sequences in the Germline Genome of Channel Catfish Are Preferentially Expressed in Response to Infection.

Rearranged V(D)J genes coding for T cell receptor α and β chains are integrated into the germline genome of channel catfish. Previous analysis of expressed TCR Vβ2 repertoires demonstrated that channel catfish express multiple public clonotypes, which were shared among all the fish, following infection with a common protozoan parasite. In each case a single DNA sequence was predominately used to code for a public clonotype. We show here that the rearranged VDJ genes coding for these expressed public Vβ2 clonotypes can be amplified by PCR from germline DNA isolated from oocytes and erythrocytes. Sequencing of the Vβ2 PCR products confirmed that these expressed public Vβ2 clonotypes are integrated into the germline. Moreover, sequencing of PCR products confirmed that all five Vβ gene families and Vα1 have rearranged V(D)J genes with diverse CDR3 sequences integrated into the germline. Germline rearranged Vβ2 and Vβ4 genes retain the intron between the leader and Vβ sequence. This suggests that the germline rearranged TCR Vβ genes arose through VDJ rearrangement in T cells, and subsequently moved into the germline through DNA transposon mediated transposition. These results reveal a new dimension to the adaptive immune system of vertebrates, namely: the expression of evolutionarily conserved, rearranged V(D)J genes from the germline.

TCR repertoire profiling of tumors, adjacent normal tissues, and peripheral blood predicts survival in nasopharyngeal carcinoma

The T-cell immune responses in nasopharyngeal carcinoma patients have been extensively investigated recently for designing adoptive immunotherapy or immune checkpoint blockade therapy. However, the distribution characteristics of T cells associated with NPC pathogenesis are largely unknown. We performed deep sequencing for TCR repertoire profiling on matched tumor/adjacent normal tissue from 15 NPC patients and peripheral blood from 39 NPC patients, 39 patients with other nasopharyngeal diseases, and 33 healthy controls. We found that a lower diversity of TCR repertoire in tumors than paired tissues or a low similarity between the paired tissues was associated with a poor prognosis in NPC. A more diverse TCR repertoire was identified in the peripheral blood of NPC patients relative to the controls; this was related to a significant decrease in the proportion of high-frequency TCR clones in NPC. Higher diversity in peripheral blood of NPC patients was associated with a worse prognosis. Due to the peculiarity of the Vβ gene usage patterns in the peripheral blood of NPC patients, 15 Vβ genes were selected to distinguish NPC patients from controls by the least absolute shrinkage and selection operator analysis. We identified 11 clonotypes shared by tumors and peripheral blood samples from different NPC patients, defined as "NPC-associated" that might have value in adoptive immunotherapy. In conclusion, we here report the systematic and overall characteristics of the TCR repertoire in tumors, adjacent normal tissues, and peripheral blood of NPC patients. The data obtained may be relevant to future clinical studies in the setting of immunotherapy for NPC patients.

TCR Vβ Usage of Peripheral Blood and Liver Infiltrating Lymphocytes in Patients with Chronic Hepatitis B

Introduction. Chronic hepatitis B (CHB) is still a public health problem and its mechanism remains unclear. In this study, we detect the skewness of T cell receptor beta chain variable gene (TCR Vβ) in peripheral blood lymphocytes (PBL) and the liver infiltrating lymphocytes (LIL) of patients with CHB; and hope to provide information for further research on the pathogenic mechanism of CHB.Material and methods. Fifteen patients with CHB, ten healthy volunteers and three patients with liver cysts were recruited as the subjects. The usage of TCR Vβ of PBL and LIL were measured and compared; the associations of the TCR Vβ usage of PBL with some hematological indices, including human leukocyte antigen (HLA) alleles, percents of CD4+ and CD8+ T cells, sera levels of HBV-DNA and IFN-γ, were analyzed.Results. In PBL, Vβ12 and Vβ13.1 were the highest predominant usage genes which usage frequencies were all 46.7%; Vβ23 was the key limited usage gene (40.0%). In LIL, the mainly predominant and limited usage gene was Vβ13.1 (73.3%) and Vβ23 (46.7%), respectively. About half of the patients with CHB with HLA-DR9 or HLA-DR12 showed the predominant usage of Vβ5.2 or Vβ13.2. In patients with CHB, the percentage of CD4+ T cells was 33.41 ± 5.39 %, that of CD8+ T cells was 28.67 ± 6.77 %; the concentration of IFN-γ was 182.52 ± 44.16 pg/mL. Compared to the healthy controls, there were significant differences for these data (P < 0.05). Neither ALT nor HBV-DNA was relative to the usage of TCR Vβ.Conclusions. PBL and LIL share the common sknewness of TCR Vβ genes which probably relates to some hematological indices. However, the roles of such similarities and associations in the development of CHB need further study.

Assessment of T-cell receptor repertoire and clonal expansion in peripheral T-cell lymphoma using RNA-seq data

T-cell clonality of peripheral T-cell lymphoma (PTCL) is routinely evaluated with a PCR-based method using genomic DNA. However, there are limitations with this approach. The purpose of this study was to determine the utility of RNA-seq for assessing T-cell clonality and T-cell antigen receptor (TCR) repertoire of the neoplastic T-cells in 108 PTCL samples. TCR transcripts, including complementarity-determining region 3 (CDR3) sequences, were assessed. In normal T cells, the CDR3 sequences were extremely diverse, without any clonotype representing more than 2% of the overall TCR population. Dominant clones could be identified in 65 out of 76 PTCL cases (86%) with adequate TCR transcript expression. In monoclonal cases, the dominant clone varied between 11% and 99% of TCRβ transcripts. No unique Vα or Vβ usage was observed. Small T-cell clones were often observed in T- and NK-cell tumors in a percentage higher than observed in reactive conditions. γ chain expression was very low in tumors expressing TCRαβ, but its expression level was high and clonality was detected in a TCRγδ expressing tumor. NK cell lymphoma (NKCL) did not express significant levels of TCR Vβ or Vγ genes. RNA-seq is a useful tool for detecting and characterizing clonal TCR rearrangements in PTCL.

Circulating and Tissue-Resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

Background & AimsInteractions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4+ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation.MethodsWe collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13−30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn’s disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4+ T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction.ResultsCirculating and gut-resident CD4+ T cells from controls responded to bacteria at frequencies of 40−4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4+ T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls.ConclusionsIn an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens.

T-cell infiltration and clonality correlate with programmed cell death protein 1 and programmed death-ligand 1 expression in patients with soft tissue sarcomas.

BACKGROUNDPatients with metastatic sarcomas have poor outcomes and although the disease may be amenable to immunotherapies, information regarding the immunologic profiles of soft tissue sarcoma (STS) subtypes is limited.METHODSThe authors identified patients with the common STS subtypes: leiomyosarcoma, undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS), well‐differentiated/dedifferentiated liposarcoma, and myxoid/round cell liposarcoma. Gene expression, immunohistochemistry for programmed cell death protein (PD‐1) and programmed death‐ligand 1 (PD‐L1), and T‐cell receptor Vβ gene sequencing were performed on formalin‐fixed, paraffin‐embedded tumors from 81 patients. Differences in liposarcoma subsets also were evaluated.RESULTSUPS and leiomyosarcoma had high expression levels of genes related to antigen presentation and T‐cell infiltration. UPS were found to have higher levels of PD‐L1 (P≤.001) and PD‐1 (P≤.05) on immunohistochemistry and had the highest T‐cell infiltration based on T‐cell receptor sequencing, significantly more than SS, which had the lowest (P≤.05). T‐cell infiltrates in UPS also were more oligoclonal compared with SS and liposarcoma (P≤.05). A model adjusted for STS histologic subtype found that for all sarcomas, T‐cell infiltration and clonality were highly correlated with PD‐1 and PD‐L1 expression levels (P≤.01).CONCLUSIONSIn the current study, the authors provide the most detailed overview of the immune microenvironment in sarcoma subtypes to date. UPS, which is a more highly mutated STS subtype, provokes a substantial immune response, suggesting that it may be well suited to treatment with immune checkpoint inhibitors. The SS and liposarcoma subsets are less mutated but do express immunogenic self‐antigens, and therefore strategies to improve antigen presentation and T‐cell infiltration may allow for successful immunotherapy in patients with these diagnoses. Cancer 2017;123:3291‐304. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Abstract IA01: Engineering T cell responses to tumors: Taking the immune system where no responses have gone before

Abstract IA01: Engineering T cell responses to tumors: Taking the immune system where no responses have gone before

CD8(+) T-cell pathogenicity in Rasmussen encephalitis elucidated by large-scale T-cell receptor sequencing.

Rasmussen encephalitis (RE) is a rare paediatric epilepsy with uni-hemispheric inflammation and progressive neurological deficits. To elucidate RE immunopathology, we applied T-cell receptor (TCR) sequencing to blood (n=23), cerebrospinal fluid (n=2) and brain biopsies (n=5) of RE patients, and paediatric controls. RE patients present with peripheral CD8+ T-cell expansion and its strength correlates with disease severity. In addition, RE is the only paediatric epilepsy with prominent T-cell expansions in the CNS. Consistently, common clones are shared between RE patients, who also share MHC-I alleles. Public RE clones share Vβ genes and length of the CDR3. Rituximab/natalizumab/basiliximab treatment does not change TCR diversity, stem cell transplantation replaces the TCR repertoire with minimal overlap between donor and recipient, as observed in individual cases. Our study supports the hypothesis of an antigen-specific attack of peripherally expanded CD8+ lymphocytes against CNS structures in RE, which might be ameliorated by restricting access to the CNS.

Rapid Evolution of the CD8+ TCR Repertoire in Neonatal Mice.

Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8(+) T cell (CTL) responses in neonates. TCRβ high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vβ family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366-374)-specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366-374)-specific CTL TCR repertoire revealed different Vβ gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257-264)-specific (OT-I) CD8(+) T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8(+) T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire

How T-cell receptors (TCRs) can be intrinsically biased toward MHC proteins while simultaneously display the structural adaptability required to engage diverse ligands remains a controversial puzzle. We addressed this by examining αβ TCR sequences and structures for evidence of physicochemical compatibility with MHC proteins. We found that human TCRs are enriched in the capacity to engage a polymorphic, positively charged "hot-spot" region that is almost exclusive to the α1-helix of the common human class I MHC protein, HLA-A*0201 (HLA-A2). TCR binding necessitates hot-spot burial, yielding high energetic penalties that must be offset via complementary electrostatic interactions. Enrichment of negative charges in TCR binding loops, particularly the germ-line loops encoded by the TCR Vα and Vβ genes, provides this capacity and is correlated with restricted positioning of TCRs over HLA-A2. Notably, this enrichment is absent from antibody genes. The data suggest a built-in TCR compatibility with HLA-A2 that biases receptors toward, but does not compel, particular binding modes. Our findings provide an instructional example for how structurally pliant MHC biases can be encoded within TCRs.

Bi-Allelic TCRα or β Recombination Enhances T Cell Development but Is Dispensable for Antigen Responses and Experimental Autoimmune Encephalomyelitis.

Dual TCRα-expressing T cells outnumber dual TCRβ-expressing cells by ~10:1. As a result, efforts to understand how dual TCR T cells impact immunity have focused on dual TCRα expression; dual TCRβ expression remains understudied. We recently demonstrated, however, that dual TCRβ expression accelerated disease in a TCR transgenic model of autoimmune arthritis through enhanced positive selection efficiency, indicating that dual TCRβ expression, though rare, can impact thymic selection. Here we generated mice hemizygous for TCRα, TCRβ, or both on the C57BL/6 background to investigate the impact bi-allelic TCR chain recombination has on T cell development, repertoire diversity, and autoimmunity. Lack of bi-allelic TCRα or TCRβ recombination reduced αβ thymocyte development efficiency, and the absence of bi-allelic TCRβ recombination promoted γδ T cell development. However, we observed no differences in the numbers of naïve and expanded antigen-specific T cells between TCRα+/-β+/- and wildtype mice, and TCR repertoire analysis revealed only subtle differences in Vβ gene usage. Finally, the absence of dual TCR T cells did not impact induced experimental autoimmune encephalomyelitis pathogenesis. Thus, despite more stringent allelic exclusion of TCRβ relative to TCRα, bi-allelic TCRβ expression can measurably impact thymocyte development and is necessary for maintaining normal αβ/γδ T cell proportions.

Quantification of a Selective Expansion of T Cell Receptor Vβ by Superantigen Using Real-Time PCR.

Selective expansion of T cells bearing specific T cell receptor Vβ segments is a hallmark of superantigens. Analyzing Vβ specificity of superantigens is important for characterizing newly discovered superantigens and understanding differential T cell responses to each toxin. Here, we describe a real-time PCR method using SYBR green I and primers specific to Cβ and Vβ genes for an absolute quantification. The established method was applied to quantify a selective expansion of T cell receptor Vβ expansion by superantigens and generated accurate, reproducible, and comparable results.

The Skewness of Alpha Beta T Cell Receptors in Peripheral Blood of the Patients with Type 1 Diabetes.

To detect the skewness of TCR Vα and TCR Vβ of patients with type 1 diabetes (T1D). The heparinized venous blood was collected from ten patients with T1D. The peripheral blood lymphocytes (PBL) were isolated and used to extract mRNA. Reverse amplyfication was performed for cDNA synthesis. The skewness of TCR Vα and Vβ was detected with real-time florescence quantitative polymerase chain reaction (RQ-PCR) and analyzed by DNA melting-curve analysis technique, respectively. Among the TCR Vα genes, the skewness frequency rate (SFR) of Vα22 was 30%; both of Vα5 and Vα24 were 20%; Vα28 was the only restricted-clone gene with the SFR of 10%. In all the Vβ genes, Vβ7 and Vβ17 were the the highest expression genes, and their SFRs were both 60%. Vβ11 was near them with the SFR of 40%; the restricted clonal genes were Vβ18 and Vβ20, their SFRs were 10% and 20%, respectivley. There are skewd genes in TCR Vα and TCR Vβ, which are probably relative to the onset of T1D.

Three-dimensional structure discrepancy between HLA alleles for effective prediction of aGVHD severity and optimal selection of recipient-donor pairs: a proof-of-concept study.

The optimal selection of recipient-donor pair and accurate prediction of acute graft-versus-host disease (aGVHD) severity are always the two most crucial works in allogeneic hematopoietic stem cell transplantation (allo-HSCT), which currently rests mostly with HLA compatibility, the most polymorphic loci in the human genome, in clinic. Thus, there is an urgent need for a rapid and reliable quantitative system for optimal recipient-donor pairs selection and accurate prediction of aGVHD severity prior to allo-HSCT. For these reasons, we have developed a new selection/prediction system for optimal recipient-donor selection and effective prediction of aGVHD severity based on HLA three-dimensional (3D) structure modeling (HLA-TDSM) discrepancy, and applied this system in a pilot randomized clinical allo-HSCT study. The 37 patient-donor pairs in the study were typed at low- and high-resolution levels for HLA-A/-B/-DRB1/-DQB1 loci. HLA-TDSM system covering the 10000 alleles in HLA class I and II consists of the revised local and coordinate root-mean-square deviation (RMSD) values for each locus. Its accuracy and reliability were confirmed using stably transfected Hmy2.CIR-HLA-B cells, TCR Vβ gene scan, and antigen-specific alloreactive cytotoxic lymphocytes. Based on the preliminary results, we theoretically defined all HLA acceptable versus unacceptable mismatched alleles. More importantly, HLA-TDSM enabled a successful retrospective verification and prospective prediction for aGVHD severity in a pilot randomized clinical allo-HSCT study of 32 recipient-donor transplant pairs. There was a strong direct correlation between single/total revised RMSD and aGVHD severity (92% in retrospective group vs 95% in prospective group). These results seem to be closely related to the 3D structure discrepancy of mismatched HLA-alleles, but not the number or loci of mismatched HLA-alleles. Our data first provide the proof-of-concept that HLA-TDSM is essential for optimal selection of recipient-donor pairs and effective prediction of aGVHD severity before allo-HSCT.

High-throughput sequencing reveals an altered T cell repertoire in X-linked agammaglobulinemia

To examine the T cell receptor structure in the absence of B cells, the TCR β CDR3 was sequenced from DNA of 15 X-linked agammaglobulinemia (XLA) subjects and 18 male controls, using the Illumina HiSeq platform and the ImmunoSEQ analyzer. V gene usage and the V–J combinations, derived from both productive and non-productive sequences, were significantly different between XLA samples and controls. Although the CDR3 length was similar for XLA and control samples, the CDR3 region of the XLA T cell receptor contained significantly fewer deletions and insertions in V, D, and J gene segments, differences intrinsic to the V(D)J recombination process and not due to peripheral T cell selection. XLA CDR3s demonstrated fewer charged amino acid residues, more sharing of CDR3 sequences, and almost completely lacked a population of highly modified Vβ gene segments found in control DNA, suggesting both a skewed and contracted T cell repertoire in XLA.

Domain-Specific and Stage-Intrinsic Changes in Tcrb Conformation during Thymocyte Development.

Considerable cross-talk exists between mechanisms controlling genome architecture and gene expression. AgR loci are excellent models for these processes because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. Upon commitment to the double-negative stage of T cell development, Tcrb adopts a compact conformation that promotes long-range recombination between Vβ gene segments (Trbvs) and their DβJβ targets. Formation of a functional VβDβJβ join signals for robust proliferation of double-negative thymocytes and their differentiation into double-positive (DP) cells, where Trbv recombination is squelched (allelic exclusion). DP differentiation also is accompanied by decontraction of Tcrb, which has been thought to separate the entire Trbv cluster from DβJβ segments (spatial segregation-based model for allelic exclusion). However, DP cells also repress transcription of unrearranged Trbvs, which may contribute to allelic exclusion. We performed a more detailed study of developmental changes in Tcrb topology and found that only the most distal portion of the Trbv cluster separates from DβJβ segments in DP thymocytes, leaving most Trbvs spatially available for rearrangement. Preferential dissociation of distal Trbvs is independent of robust proliferation or changes in transcription, chromatin, or architectural factors, which are coordinately regulated across the entire Trbv cluster. Segregation of distal Trbvs also occurs on alleles harboring a functional VβDβJβ join, suggesting that this process is independent of rearrangement status and is DP intrinsic. Our finding that most Trbvs remain associated with DβJβ targets in DP cells revises allelic exclusion models from their current conformation-dominant to a transcription-dominant formulation.

Abstract SY31-03: Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies

Abstract SY31-03: Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies