Complementarity Determining Region 3 Length Research Articles

Characteristics of T cell premature senescence in maintenance hemodialysis patients.

Uremia-associated immunodeficiency, mainly characterized by T cell dysfunction, exists in patients on maintenance hemodialysis (MHD) and promotes systemic inflammation. However, T cell senescence, one of the causes of T cell dysfunction, has not been clearly revealed yet. In this cross-sectional research, we aimed to study the manifestation of T cell premature senescence in MHD patients and further investigate the associated clinical factors. 76 MHD patients including 33 patients with cardiovascular diseases (CVD) and 28 patients with arteriovenous fistula (AVF) event history were enrolled in this study. Complementarity determining region 3 (CDR3) of T cell receptor (TCR) was analyzed by immune repertoire sequencing (IR-Seq). CD28- T cell subsets and expression of senescence marker p16 and p21 genes were detected by multicolor flow cytometry and RT-qPCR, respectively. MHD patients had significantly decreased TCR diversity (P < 0.001), increased CDR3 clone proliferation (P = 0.001) and a left-skewed CDR3 length distribution. The proportion of CD4 + CD28- T cells increased in MHD patients (P = 0.014) and showed a negative correlation with TCR diversity (P = 0.001). p16 but not p21 expression in T cells was up-regulated in MHD patients (P = 0.039). Patients with CVD exhibited increased expression of p16 and p21 genes (P = 0.010 and 0.004, respectively), and patients with AVF events showed further TCR diversity and evenness reduction (P = 0.002 and 0.017, respectively) compared to patients without the comorbidities. Moreover, age, average convection volume, total cholesterol, high-density lipoprotein cholesterol and transferrin saturation were associated with TCR diversity or CD4 + CD28- T cell proportion (P < 0.05). MHD patients undergo T cell premature senescence characterized by significant TCR diversity reduction and repertoire skew, as well as accumulation of the CD4 + CD28- subset and up-regulation of p16 gene. Patients with CVD or AVF events show higher level of immunosenescence. Furthermore, T cell senescence in MHD patients is associated with blood cholesterol and uremic toxin retention, suggesting potential intervention strategies in the future.

DNA repair mechanisms that promote insertion-deletion events during immunoglobulin gene diversification.

Insertions and deletions (indels) are low-frequency deleterious genomic DNA alterations. Despite their rarity, indels are common, and insertions leading to long complementarity-determining region 3 (CDR3) are vital for antigen-binding functions in broadly neutralizing and polyreactive antibodies targeting viruses. Because of challenges in detecting indels, the mechanism that generates indels during immunoglobulin diversification processes remains poorly understood. We carried out ultra-deep profiling of indels and systematically dissected the underlying mechanisms using passenger-immunoglobulin mouse models. We found that activation-induced cytidine deaminase-dependent ±1-base pair (bp) indels are the most prevalent indel events, biasing deleterious outcomes, whereas longer in-frame indels, especially insertions that can extend the CDR3 length, are rare outcomes. The ±1-bp indels are channeled by base excision repair, but longer indels require additional DNA-processing factors. Ectopic expression of a DNA exonuclease or perturbation of the balance of DNA polymerases can increase the frequency of longer indels, thus paving the way for models that can generate antibodies with long CDR3. Our study reveals the mechanisms that generate beneficial and deleterious indels during the process of antibody somatic hypermutation and has implications in understanding the detrimental genomic alterations in various conditions, including tumorigenesis.

Humanized V(D)J-rearranging and TdT-expressing mouse vaccine models with physiological HIV-1 broadly neutralizing antibody precursors

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.

OAB-039: Distinct immunoglobulin heavy-chain variable gene repertoire and its clinical relevance in Waldenström macroglobulinemia/lymphoplasmacytic lymphoma

<h3>Background</h3> Waldenström macroglobulinemia/lymphoplasmacytic lymphoma (WM/LPL) is a heterogeneous disease whose role of immunoglobulin heavy chain variable gene (IGHV) usage remains unknown. <h3>Methods</h3> To determine the clinical relevance of IGHV repertoire in WM/LPL treated with standard immunochemotherapy, we performed the immunoglobulin gene rearrangements and complementarity determining region 3 (CDR3) analysis on 136 patients with WM/LPL. <h3>Results</h3> The IGHV gene repertoire was remarkably biased in WM/LPL. IGHV3-23, IGHV4-34, IGHV3-30, IGHV3-7 and IGHV3-74 accounted for half of the whole cohort. Most of the cases were mutated (97.0%) using a 98% IGHV germline homology cutoff. IGHV3-30 was associated with long heavy chain CDR3, indicating the specific antigen selection in WM/LPL. Patients with IGHV3-7 were significantly more likely to harbor 6q deletion (p<0.001) and complex karyotype (p=0.002). The IGHV hypermutation rate in patients with MYD88 L265P mutation was significantly higher than wildtype patients (p=0.009). IGHV3-23 and IGHV3-74 segments were more frequently detected in MYD88 mutated WM/LPL patients (p=0.025). When we looked into the five predominant IGHV segments mentioned above, we found a trend that IGHV3-23 and IGHV3-74 segments were more frequently detected in MYD88 mutated WM/LPL patients (25.7% vs. 4.3%, p=0.025). Besides, we found IGHV3-7 and IGHV4-59 were represented more in MYD88 wildtype patients compared with MYD88 mutated patients (30.4% vs. 8.9%, p=0,005). IGHV4 segments were higher expressed in MYD88 wildtype patients compared with MYD88 L265P mutated patients (39.1% vs. 21.8%, P=0.083). Moreover, Patients with IGHV4 especially IGHV4-34 had higher level of LDH. IGHV4 was a predictive marker of shorter progression-free-survival. <h3>Conclusion</h3> WM/LPL appears to be composed of different subgroups based on the IGHV repertoire. The mutational status and the IGHV CDR3 length indicated the role for antigen selection in WM/LPL development. The presence of IGHV4 genes proved to be a potential risk factor associated with outcome which deserved further study.

Landscape of variable domain of heavy-chain-only antibody repertoire from alpaca.

Heavy-chain-only antibodies (HCAbs), which are devoid of light chains, have been found naturally occurring in various species including camelids and cartilaginous fish. Because of their high thermostability, refoldability and capacity for cell permeation, the variable regions of the heavy chain of HCAbs (VHHs) have been widely used in diagnosis, bio-imaging, food safety and therapeutics. Most immunogenetic and functional studies of HCAbs are based on case studies or a limited number of low-throughput sequencing data. A complete picture derived from more abundant high-throughput sequencing (HTS) data can help us gain deeper insights. We cloned and sequenced the full-length coding region of VHHs in Alpaca (Vicugna pacos) via HTS in this study. A new pipeline was developed to conduct an in-depth analysis of the HCAb repertoires. Various critical features, including the length distribution of complementarity-determining region 3 (CDR3), V(D)J usage, VJ pairing, germline-specific mutation rate and germline-specific scoring profiles (GSSPs), were systematically characterized. The quantitative data show that V(D)J usage and VHH recombination are highly biased. Interestingly, we found that the average CDR3 length of classical VHHs is longer than that of non-classical ones, whereas the mutation rates are similar in both kinds of VHHs. Finally, GSSPs were built to quantitatively describe and compare sequences that originate from each VJ pair. Overall, this study presents a comprehensive landscape of the HCAb repertoire, which can provide useful guidance for the modeling of somatic hypermutation and the design of novel functional VHHs or VHH repertoires via evolutionary profiles.

Profiling the pattern of human TRB/IGH-CDR3 repertoire in liver transplantation patients via high-throughput sequencing analysis.

Immune processes in liver transplantation remain poorly understood. Acute allograft rejection in liver transplantation is a kind of T cell-mediated inflammatory disease accompaniedby inflammatory cell infiltration. However, the effect of acute allograft rejection on the immunological characteristics of TCRs in peripheralbloodmononuclearcell is unknown. In this study, we characterized the pattern of the human T cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) complementarity-determining region 3 (CDR3) repertoires via high-throughput sequencing in 11 acute allograft rejection (AG) cases, 23 patients with stable allograft liver function (ST) who had liver transplantation performed and 20 healthy controls (HC). The diversity of TRB-CDR3 was significantly reduced in the AG group compared with the ST group and healthy controls (HC). The CDR3 and N-addition length distribution were not significantly different between the AG and ST groups. However, N-addition length distribution was significantly changed compared to HC. It seemed that AG used more short N-additions and healthy people used more long N-additions in TRB-CDR3 repertoire. Our findings suggested that the TRB-CDR3 region of AG had distinctive V gene use compared with that of HC. The characteristics of ST seemed to be in between those of AG and HC although the difference is not significant. Cluster analysis showed that the TRB repertoire could not effectively distinguish AG from ST. This research might give to a better understanding of the immune process of liver transplantation.

High-throughput sequencing of CD4+ T cell repertoire reveals disease-specific signatures in IgG4-related disease

BackgroundCD4+ T cells play critical roles in the pathogenesis of IgG4-related disease (IgG4-RD). The aim of this study was to investigate the TCR repertoire of peripheral blood CD4+ T cells in IgG4-RD.MethodsThe peripheral blood was collected from six healthy controls and eight IgG4-RD patients. TCR β-chain libraries of CD4+ T cells were constructed by 5′-rapid amplification of cDNA ends (5′-RACE) and sequenced by Illumina Miseq platform. The relative similarity of TCR repertoires between samples was evaluated according to the total frequencies of shared clonotypes (metric F), correlation of frequencies of shared clonotypes (metric R), and total number of shared clonotypes (metric D).ResultsThe clonal expansion and diversity of CD4+ T cell repertoire were comparable between healthy controls and IgG4-RD patients, while the proportion of expanded and coding degenerated clones, as an indicator of antigen-driven clonal expansion, was significantly higher in IgG4-RD patients. There was no significant difference in TRBV and TRBJ gene usage between healthy controls and IgG4-RD patients. The complementarity determining region 3 (CDR3) length distribution was skewed towards longer fragments in IgG4-RD. Visualization of relative similarity of TCR repertoires by multi-dimensional scaling analysis showed that TCR repertoires of IgG4-RD patients were separated from that of healthy controls in F and D metrics. We identified 11 IgG4-RD-specific CDR3 amino acid sequences that were expanded in at least 2 IgG4-RD patients, while not detected in healthy controls. According to TCR clonotype networks constructed by connecting all the CDR3 sequences with a Levenshtein distance of 1, 3 IgG4-RD-specific clusters were identified. We annotated the TCR sequences with known antigen specificity according to McPAS-TCR database and found that the frequencies of TCR sequences associated with each disease or immune function were comparable between healthy controls and IgG4-RD patients.ConclusionAccording to our study of CD4+ T cells from eight IgG4-RD patients, TCR repertoires of IgG4-RD patients were different from that of healthy controls in the proportion of expanded and coding degenerated clones and CDR3 length distribution. In addition, IgG4-RD-specific TCR sequences and clusters were identified in our study.

Immunoglobulin somatic hypermutation has clinical impact in DLBCL and potential implications for immune checkpoint blockade and neoantigen-based immunotherapies

BackgroundDiffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma.MethodsTo determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens’ immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry.ResultsSHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHMhigh) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell–like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHMhigh groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8+ T cells was significantly lower in IGHV SHMhigh than in SHMlow patients with activated B-cell–like DLBCL, whereas PD-1 expression in CD4+ T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHMhigh than in SHMlow patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM.ConclusionsThese results show for the first time that IGV SHMhigh and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

High-throughput sequencing analysis of genes encoding the B-lymphocyte receptor heavy-chain CDR3 in renal and peripheral blood of IgA nephropathy.

Aim: IgA nephropathy (IgAN) is one of the most common chronic glomerulonephritis. Its etiology and pathogenesis remain unclear. We thus explored the immune repertoire of the B-cell receptor (BCR) and the heavy-chain complementarity-determining region 3 (CDR3) in renal tissue and peripheral blood of IgAN patients. Method: Total RNAs extracted from renal tissues and peripheral blood of patients and peripheral blood of healthy controls (HCs) were analyzed via high-throughput multiplex PCR sequencing. We amplified and sequenced BCR heavy-chain CDR3 regions to explore repertoire diversity, V/J gene family distribution, CDR3 lengths, BCR heavy-chain variants, consistency between tissue and peripheral blood data, and clones ‘shared’ by these bodily compartments. Results: We identified the renal tissue and peripheral blood BCR heavy-chain CDR3 immune repertoires of 15 IgAN patients. Top1 could be more readily cloned from peripheral blood of patients than from controls (P<0.05), the average CDR3 length was significantly shorter in patients than in HCs (P<0.05), the variant frequency of the gene encoding the BCR heavy chain was higher in patients than in HCs (P<0.05), and the BCR variant frequency was highest in IgAN kidney tissue. Preliminary screening for ‘shared’ clones showed that, in at least 13 patients, the ‘ALYFHNSAY’, ‘ARWGPMYYYMDV’, ‘ARDQGALNA’, and ‘ARVDNPADF’ CDR3 sequences were evident in peripheral blood samples from patients, but not HCs. Conclusions: We found that the ‘ALYFHNSAY’, ‘ARWGPMYYYMDV’, ‘ARDQGALNA’, and ‘ARVDNPADF’ clonal sequences may be useful for noninvasive diagnosis and treatment planning in IgAN.

A pilot study to assess peripheral blood TCR β-chain CDR3 repertoire in occupational medicamentosa-like dermatitis due to trichloroethylene using high-throughput sequencing

We exploratively characterized T cell receptor (TCR) repertoires from occupational medicamentosa-like dermatitis due to trichloroethylene (OMDT) patients to better understand the underlying pathological mechanism. We used a combination of multiplex-PCR, Illumina sequencing and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR β-chain complementarity-determining region 3 (CDR3) gene in 10 OMDT cases and 10 trichloroethylene-exposed healthy tolerant controls. Compared with the tolerant controls, OMDT cases showed no significant difference in TCR repertoire diversity including repertoire breadth, highly expanded clone, and CDR3 length distribution. However, we observed several differences in TRBV/TRBJ usage and combination between the two groups, as well as some shared and unique T cell clones in the cases. The pilot study delineated some features of TCR repertoire in OMDT patients that warrant further investigation.

A comprehensive study of immunology repertoires in both preoperative stage and postoperative stage in patients with colorectal cancer.

BackgroundColorectal cancer (CRC) is the 3rd most common cancer type in the world. The correlation between immune repertoire and prognosis of CRC has been well studied in the last decades. The diversity and stability of the immune cells can be measured by hypervariable complementarity‐determining region 3 (CDR3) segments of the T‐cell receptor (TCR).MethodsIn this study, we collected five healthy controls and 19 CRC patients’ peripheral blood mononuclear cells (PBMCs) in three stages, namely 1 day preoperative, 3 days’ postoperative, and 7 days’ postoperative, respectively. Simultaneously, we have also done the comparative analysis of these two different anesthesia methods, namely TIVA and CEGA. Sequencing of the TCR segments has been performed by multiplex PCR and high‐throughput next‐generation sequencing. We also analyzed the distribution of CDR3 length, highly expansion clones (HECs), TRBV, and TRBJ gene usage.ResultsOur result showed a significant difference between TCR CDR3 length distribution and HEC distribution between CRC patients and healthy controls. We also found that TRBV11‐2, TRBV12‐1, TRBV16, TRBV3‐2, TRBV4‐2, TRBV4‐3, TRBV5‐4, TRBV6‐8, TRBV7‐8, TRBV7‐9 and RBV11‐2, TRBV12‐1, TRBV16, TRBV3‐2, TRBV4‐2, TRBV4‐3, TRBV5‐4, TRBV6‐8, TRBV7‐8, and TRBV7‐9 usages are different between CRC patients and healthy controls.ConclusionIn conclusion, CRC patients were presented with different immune repertoire in comparison with healthy controls. In this study, significant difference in TRBV and TRBJ gene usage in between case and control group could provide some potential biomarker for the diagnosis and the treatment of the patients with CRC.

Gut and Liver B Cells of Common Clonal Origin in Primary Sclerosing Cholangitis-Inflammatory Bowel Disease.

B cells express an antigen‐specific B‐cell receptor (BCR) and may contribute to liver inflammation by recognizing shared antigens in the gut and liver. Herein, we used high‐throughput BCR sequencing of the immunoglobulin heavy chain, specifically the complementarity‐determining region 3 (CDR3), to characterize the B‐cell repertoire of freshly‐frozen paired gut and liver tissue samples from patients with primary sclerosing cholangitis (PSC) and concurrent inflammatory bowel disease (IBD) (PSC‐IBD, n = 10) and paired formalin‐fixed paraffin‐embedded (FFPE) tumor‐adjacent normal colon and liver tissue from patients with colorectal liver metastases (controls, n = 10). We observed significantly greater numbers of B cells (P < 0.01) and unique B‐cell clonotypes (P < 0.05) in gut samples compared to liver samples of patients with PSC‐IBD, whereas BCR sequences in FFPE normal gut and liver samples were nearly absent (14 ± 5 clonotypes; mean ± SD; n = 20). In PSC‐IBD, an average of 8.3% (range, 1.6%‐18.0%) of B‐cell clonotypes were found to overlap paired gut and liver samples following the exclusion of memory clonotypes reported in the blood of healthy controls. Overlapping gut and liver clonotypes showed stronger evidence of antigen‐driven activation compared to non‐overlapping clonotypes, including shorter CDR3 lengths and higher counts of somatic hypermutation (P < 0.0001). Conclusion: A proportion of gut and liver B cells originate from a common clonal origin (i.e., likely to recognize the same antigen) in patients with PSC which suggests B‐cell antigens are shared across the gut–liver axis. (Hepatology Communications 2018; 00:000‐000)

Association of the characteristics of B- and T-cell repertoires with papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Complementarity-determining region 3 (CDR3) of B-cell receptors (BCRs) and T-cell receptors are the major site of antigen recognition, which determines a unique clone type, and are considered to be the representative of the disease. In the present study, high-throughput sequencing was used to analyze the association of characteristics of the BCR immunoglobulin heavy chain (IGH) and the T-cell receptor β chain (TRB) CDR3 genes in PTC and corresponding pericarcinous tissues from patients. A difference of CDR3 length distributions of total IGH CDR3 sequences between the two groups was revealed. IGHV3-11/IGHJ6, TRBV2/TRBJ1-2 and TRBV2/TRBJ1-1 may be biomarkers for the development of PTC. Furthermore, it was revealed that the extent of the common clonotype expressions at the amino acid level was slightly higher compared with the nucleotide level. The Shannon entropy demonstrated a diversity reduction in PTC compared with the pericarcinous group, and the highly expended clone (HEC) expression of PTC was higher compared with that of the corresponding pericarcinous group. Additionally, the highest clone frequency percentage of IGH and TRB was at 0.1–1.0% degree of expansion, as HEC expression was higher in PTC compared with the matched group. There was no shared clone of HECs in the two groups either at the amino acid level or at the nucleotide expression level. The differential expression of CDR3 sequences of PTC have been identified in the present study. Further research is required for assessing the immune repertoire size, diversity, cloning tracking and finding public clones of T-cell and B-cell populations in the development of PTC.

Composition and Variation Analysis of the T Cell Receptor β-Chain Complementarity Determining Region 3 Repertoire in Neonatal Sepsis.

T cell receptor (TCR) diversity is clearly related to protection from infection. However, the characteristics of TCR diversity in neonates are not clear. In this study, we investigated the TCR diversity of neonates with sepsis. Twenty neonates with severe sepsis and eight matched neonates without infection were enrolled in the study. For the neonates with sepsis, EDTA-anticoagulated blood was collected on day 1 after the diagnosis of sepsis and on day 7 of treatment. For the neonates without infection, blood was collected one time. DNA was extracted from peripheral blood mononuclear cells. The complementarity determining region 3 (CDR3) gene was analysed by multiplex PCR and high-throughput sequencing. The CDR3 types and lengths were similar in patients and healthy controls. There was a significant difference in VJ gene usage among the three groups. Compared to the healthy neonates, the neonates with sepsis had different VJ pairs and generated different clonotypes. Although the TCR β-chain diversity was generally lower in the neonates with sepsis, there was no significant difference in TCR β-chain diversity between the patients and the healthy controls. Our data showed the characteristics of the TCR repertoire in neonates with sepsis, which represents a potentially valuable data set. This result is useful for understanding neonatal susceptibility to infection.

Converging evolution leads to near maximal junction diversity through parallel mechanisms in B and T cell receptors

T and B cell receptor (TCR and BCR) complementarity determining region 3 (CDR3) genetic diversity is produced through multiple diversification and selection stages. Potential holes in the CDR3 repertoire were argued to be linked to immunodeficiencies and diseases. In contrast with BCRs, TCRs have practically no Dβ germline genetic diversity, and the question emerges as to whether they can produce a diverse CDR3 repertoire.In order to address the genetic diversity of the adaptive immune system, appropriate quantitative measures for diversity and large-scale sequencing are required. Such a diversity method should incorporate the complex diversification mechanisms of the adaptive immune response and the BCR and TCR loci structure.We combined large-scale sequencing and diversity measures to show that TCRs have a near maximal CDR3 genetic diversity. Specifically, TCR have a larger junctional and V germline diversity, which starts more 5′ in Vβ than BCRs. Selection decreases the TCR repertoire diversity, but does not affect BCR repertoire. As a result, TCR is as diverse as BCR repertoire, with a biased CDR3 length toward short TCRs and long BCRs. These differences suggest parallel converging evolutionary tracks to reach the required diversity to avoid holes in the CDR3 repertoire.

Comprehensive assessment of peripheral blood TCRβ repertoire in infectious mononucleosis and chronic active EBV infection patients

Epstein-Barr virus (EBV) primary infection is usually asymptomatic, but it sometimes progresses to infectious mononucleosis (IM). Occasionally, some people develop chronic active EBV infection (CAEBV) with underlying immunodeficiency, which belongs to a continuous spectrum of EBV-associated lymphoproliferative disorders (EBV+ LPD) with heterogeneous clinical presentations and high mortality. It has been well established that T cell-mediated immune response plays a critical role in the disease evolution of EBV infection. Recently, high-throughput sequencing of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (T cell receptor β (TCRβ)) has emerged as a sensitive approach to assess the T cell repertoire. In this study, we fully characterized the diversity of peripheral blood TCRβ repertoire in IM (n=6) and CAEBV patients (n=5) and EBV-seropositive controls (n=5). Compared with the healthy EBV-seropositive controls, both IM and CAEBV patients demonstrate a significant decrease in peripheral blood TCRβ repertoire diversity, basically, including narrowed repertoire breadth, highly expanded clones, and skewed CDR3 length distribution. However, there is no significant difference between IM and CAEBV patients. Furthermore, we observed some disease-related preferences in TRBV/TRBJ usage and combinations, as well as lots of T cell clones shared by different groups (unique or overlapped) involved in public T cell responses, which provide more detailed insights into the divergent disease evolution.

Distinct Immunogenetic Signatures in IgA Versus IgG Multiple Myeloma

Immunogenetic analysis of MM has proven instrumental in elucidating disease ontogeny e.g. by revealing the clonal relationship between switch variants expressed by the bone marrow plasma cells and myeloma progenitors in the marrow and blood; demonstrating the marked under-representation of the inherently autoreactive IGHV4-34 gene; and, identifying patterns of somatic hypermutation (SHM) indicative of post-germinal center derivation. Yet, limited information exists about the composition of the immunoglobulin (IG) gene repertoire in MM cases expressing different heavy chain isotype, in particular A versus G. This is relevant in light of studies showing an overall higher SHM impact in CD27+IgA+ compared to CD27+IgG+ normal memory B cells, perhaps reflecting a distinct location of the immune response, especially considering that IgA class switching mostly occurs in mucosa-associated lymphoid tissues. From a clinical perspective, it is also relevant to note that IgA patients exhibit a higher incidence of the t(4;14) translocation, shorter progression-free survival and worse median overall survival compared to IgG patients. Here, we explored potential differences in the immunoprofiles of IgA versus IgG MM focusing on IG gene repertoire and SHM characteristics. In total, 428 patients with a diagnosis of MM following the IMWG criteria from collaborating institutions in Greece, Italy and Spain (n=355) or retrieved from the LIGM-DB (n=73) were included in the study. Of these, 135 and 293 belonged to IgA and IgG MM groups, respectively. Amongst the evaluated productive IG rearrangements, IGHV3 subgroup genes predominated in both groups (IgA: 58.5%; IgG: 52.2%). However, at the individual gene level, major asymmetries were noted, since only 7 IGHV genes accounted for 41.6% of the IgA and 46.7% of the IgG cases, respectively. Of these, 3 genes were shared between IgA and IgG MM cases: IGHV3-30 (IgA: 11.9% - IgG: 13.3%), IGHV3-23 (IgA: 5.2% - IgG: 6.8%) and IGHV3-9 (IgA: 6.7% - IgG: 4.4%), whereas the remaining 4 of the 7 most frequent genes were specific for each group with significant (p<0.05) differences regarding the IGHV3-7 (5.2% in IgA versus 1.7% in IgG) and IGHV3-21 gene (0.7% in IgA 4.1% in IgG). IGHD3 predominated in both groups (IgA: 37% - IgG: 39.6%) followed by IGHD2 in IgG MM (18.4%) and IGHD6 in IgA MM (20.7%). IGHJ4 and IGHJ6 were the most frequent IGHJ genes with no significant differences in relative frequency. Searching for restricted IGHV-IGHJ combinations, we noted that the IGHV3-9 gene preferentially paired with the IGHJ6 gene in IgA MM versus the IGHJ4 gene in IgG MM (66.7% and 46.2% of all IGHV3-9 rearrangements, respectively). The median complementarity-determining region 3 (CDR3) length was identical in both IgA and IgG MM (15 amino acids, aa), yet differences were identified for specific CDR3 lengths as in the case of 19 aa, concerning 10.4% of all IgA versus 4.8% of all IgG rearrangements (p<0.05). Turning to SHM, the vast majority of rearrangements (IgA: 90.4%, IgG: 85%) were heavily mutated (IGHV germline identity (GI) <95%) with median GI of 91.8% for IgA and 92.2% for IgG. To study the topology of SHM, we compared the ratios of replacement (R) to silent (S) mutations in the framework (FR) and complementarity determining regions (CDRs) in cases expressing common frequent IGHV genes, namely IGHV3-23, IGHV3-30 and IGHV3-9 and identified distinct SHM patterns in all 3 instances: (i) IGHV3-23: the highest R/S ratios in IgA versus IgG MM were observed in FR2 (3.88) and CDR1 (3.9), respectively; (ii) IGHV3-30: overall "normal" SHM topology with higher R/S in CDRs rather than FRs, however, compared to IgG, IgA cases also showed a very high R/S in FR2 (5.3 versus 1.4); and, (iii) IGHV3-9: significantly (p<0.05) higher R/S ratios in CDR1 and CDR2 in IgG versus IgA cases (10.2 and 8.3 versus 1 and 2.9, respectively). Overall, in-depth immunogenetic analysis in the largest to-date series of IgA MM and IgG MM patients reveals differences regarding IGH gene repertoires, CDR3 characteristics and the topology of SHM. These findings suggest distinct antigen exposure histories and/or affinity maturation processes for IgA versus IgG MM, further highlighting the importance of microenvironmental stimuli in disease pathogenesis. DisclosuresTerpos:Celgene: Honoraria; Novartis: Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

Current status and recent advances of next generation sequencing techniques in immunological repertoire.

To ward off a wide variety of pathogens, the human adaptive immune system harbors a vast array of T-cell receptors (TCRs) and B-cell receptors (BCRs), collectively referred to as the immune repertoire. High-throughput sequencing (HTS) of TCR/BCR genes allows in-depth molecular analysis of T/B-cell clones, providing an unprecedented level of detail when examining the T/B-cell repertoire of individuals. It can evaluate TCR/BCR complementarity-determining region 3 (CDR3) diversity and assess the clonal composition, including the size of the repertoire; similarities between repertoires; V(D)J segment use; nucleotide insertions and deletions; CDR3 lengths; and amino acid distributions along the CDR3s at sequence-level resolution. Deep sequencing of B-cell and T-cell repertoires offers the potential for a quantitative understanding of the adaptive immune system in healthy and disease states. Recently, paired sequencing strategies have also been developed, which can provide information about the identity of immune receptor pairs encoded by individual T or B lymphocytes. HTS technology provides a previously unimaginable amount of sequence data, accompanied, however, by numerous challenges associated with error correction and interpretation that remain to be solved. The review details some of the technologies and some of the recent achievements in this field.

Assessment of T-Cell Receptor Repertoire and Clonal Expansion in Peripheral T-Cell Lymphoma Using RNA-Seq Data

Introduction: Diversity of the T-cell receptor (TCR) repertoire reflects the initial V(D)J recombination events as shaped by selection by self and foreign antigens. Next generation sequencing is a powerful method for profiling the TCR repertoire, including sequences encoding complementarity-determining region 3 (CDR3). Peripheral T-cell lymphoma (PTCL) is a group of malignancies that originate from mature T-cells. T-cell clonality of PTCL is routinely evaluated with a PCR-based method to detect TCR gamma and less frequently beta chain rearrangements using genomic DNA. However, there are limitations with this approach, chief among which is the lack of sequence information. To date, the TCR repertoire of different subtypes of PTCL remains poorly defined.Objective: The purpose of this study was to determine the utility of RNA-seq for assessing T-cell clonality and analyzing the TCR usage in PTCL samples.Methods: We analyzed RNA-seq data from 30 angioimmunoblastic T-cell lymphoma (AITL), 23 Anaplastic large cell lymphoma (ALCL), 10 PTCL-NOS, and 17 NKCL. Data from naïve T cells, TFH cells, and T-effector cells (CD4+ CD45RA− TCRβ+ PD-1lo CXCR5lo PSGL-1hi) were obtained from publicly available resources. Referenced TCR and immunoglobulin transcripts according to the International ImMunoGeneTics Information System (IMGT) database were quantified by Kallisto software. We divided the pattern of Vβ (T-cell receptor beta variable region) into three categories: monoclonal (mono- or bi-allelic), oligoclonal (3-4 dominant clones), and polyclonal. CDR3 sequences were extracted by MiXCR program. PCR of the gamma chain using genomic DNA was utilized to validate the clonality of selected cases. Single nucleotide variants (SNVs) were called from aligned RNA-seq data using Samtools and VarScan 2 programs.Results: Analysis of RNA-seq data identified preferential usage of TCR-Vβ, Dβ (diversity region), and Jβ (joining region), length diversity of CDR3, and usage of nontemplated bases. Dominant clones could be identified by transcriptome sequencing in most cases of AITL (21/30), ALCL (14/23), and PTCL-NOS (7/10). Median CDR3 length is 42 nucleotides (nt) in normal T cells, 41 nt in ALCL, 48 nt in PTCL-NOS, and 44 nt in AITL. In 30 AITL samples, 20 showed monoclonal Vβ with a single peak, and 9 showed polyclonal Vβ. One case had two dominant clones with different CDR3, only one of which was in frame, implying biallelic rearrangements. As many as 3511 clones supported by at least four reads could be detected in polyclonal cases. In monoclonal cases, the dominant clone varied between 11.8% and 92.8% of TCR with Vβ rearrangements. TRBV 20-1, which is the most commonly used segment in normal T cells, is also frequently used in the dominant clones in AITL. The monoclonal AITL cases showed mutation of TET2, RHOA, DNMT3A or IDH2 whereas most of the polyclonal cases were negative or had low VAF mutation suggesting low or absent of tumor infiltrate in the specimen sequenced. There is no obvious correlation of any of the mutations with Vβ usage. Clonal B cell expansion was noted in some AITL samples. The occurrence of a preferential TRBV9 expansion in PTCL-NOS was striking. More than half of ALCL samples (14/23) showed expression of clonal Vβ, but 3/14 dominant clones were out-of-frame. γ chain expression was very low in cells expressing TCRαβ, but both expression levels and clonality were higher in TCRγδ expressing tumors. NKCL did not express significant levels of TCR Vβ or Vγ genes.Discussion/Interpretation: Transcriptome sequencing is a useful tool for understanding the TCR repertoire in T cell lymphoma and for detecting clonality for diagnosis. Clonal, often out-of-frame, Vβ transcripts are detectable in most ALCL cases and preferential TRBV9 usage is found in PTCL-NOS. DisclosuresNo relevant conflicts of interest to declare.