TCR Transcripts Research Articles

Identification of Enhanced Vaccine Mimotopes for the p15E Murine Cancer Antigen.

The MHC-I-restricted p15E tumor rejection epitope is expressed in multiple murine cancer lines and is used as a marker of antitumor cellular immunity, but has seen limited success as a vaccine immunogen. An in vivo screening approach based on a positional peptide microlibraries is used to identify enhanced p15E mimotopes bearing amino acid mutations that induce significantly improved functional immunogenicity relative to vaccination with the wild-type epitope.

An NK-like CAR T cell transition in CAR T cell dysfunction

Chimeric antigen receptor (CAR) Tcell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR Tcell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR Tcells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of Tcell exhaustion and discover, both invitro and in CAR Tcell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like Tcell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR Tcell exhaustion. Our findings shed light on the plasticity of human CAR Tcells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR Tcell therapy in solid tumors by preventing or delaying CAR Tcell dysfunction.

76 Spatial mapping of T cell receptors and transcriptomes in renal cell carcinoma following immune checkpoint inhibitor therapy

76 Spatial mapping of T cell receptors and transcriptomes in renal cell carcinoma following immune checkpoint inhibitor therapy

Systematic Profiling of Full-Length Ig and TCR Repertoire Diversity in Rhesus Macaque through Long Read Transcriptome Sequencing.

The diversity of Ig and TCR repertoires is a focal point of immunological studies. Rhesus macaques (Macaca mulatta) are key for modeling human immune responses, placing critical importance on the accurate annotation and quantification of their Ig and TCR repertoires. However, because of incomplete reference resources, the coverage and accuracy of the traditional targeted amplification strategies for profiling rhesus Ig and TCR repertoires are largely unknown. In this study, using long read sequencing, we sequenced four Indian-origin rhesus macaque tissues and obtained high-quality, full-length sequences for over 6000 unique Ig and TCR transcripts, without the need for sequence assembly. We constructed, to our knowledge, the first complete reference set for the constant regions of all known isotypes and chain types of rhesus Ig and TCR repertoires. We show that sequence diversity exists across the entire variable regions of rhesus Ig and TCR transcripts. Consequently, existing strategies using targeted amplification of rearranged variable regions comprised of V(D)J gene segments miss a significant fraction (27-53% and 42-49%) of rhesus Ig/TCR diversity. To overcome these limitations, we designed new rhesus-specific assays that remove the need for primers conventionally targeting variable regions and allow single cell level Ig and TCR repertoire analysis. Our improved approach will enable future studies to fully capture rhesus Ig and TCR repertoire diversity and is applicable for improving annotations in any model organism.

Innate-like CD27+CD45RBhigh γδ T Cells Require TCR Signaling for Homeostasis in Peripheral Lymphoid Organs.

TCR signaling is required for homeostasis of naive αβ T cells. However, whether such a signal is necessary for γδ T cell homeostasis in the periphery remains unknown. In this study, we present evidence that a portion of Vγ2+ γδ T cells, one of the major γδ T cell subsets in the secondary lymphoid organs, requires TCR signaling for homeostasis. To attenuate γδTCR signals, we generated mice lacking Eγ4 (Eγ4-/-), an enhancer located at the 3'-most end of the TCRγ locus. Overall, we found that in thymus, Eγ4 loss altered V-J rearrangement, chromatin accessibility, and transcription of the TCRγ locus in a distance-dependent manner. Vγ2+ γδ T cells in Eγ4-/- mice developed normally both fetal and adult mouse thymi but were relatively reduced in number in spleen and lymph nodes. Although Vγ2 TCR transcription decreased in all subpopulations of Eγ4-/- mice, the number of Vγ2+ γδ T cells decreased and TCR signaling was attenuated only in the innate-like CD27+CD45RBhigh subpopulation in peripheral lymphoid organs. Consistently, CD27+CD45RBhigh Vγ2+ γδ T cells from Eγ4-/- mice transferred into Rag2-deficient mice were not efficiently recovered, suggesting that continuous TCR signaling is required for their homeostasis. Finally, CD27+CD45RBhigh Vγ2+ γδ T cells from Eγ4-/- mice showed impaired TCR-induced activation and antitumor responses. These results suggest that normal homeostasis of innate-like CD27+CD45RBhigh Vγ2+ γδ T cells in peripheral lymphoid organs requires TCR signaling.

Comprehensive profiling of TCR and BCR repertoire of infiltrated lymphocytes in the mouse injured skeletal muscle

Abstract Lymphocytes infiltrating to the injured skeletal muscle have been shown to play an important role in muscle regeneration. Site-specific infiltrating/expanding lymphocytes and its molecular features are potential resources for bioengineering such as a drug delivery system. Although some previous studies showed the accumulation of lymphocytes to the injured skeletal muscle, the actual frequency of distinct lymphocyte clones in situ is still unclear. Hence, a comprehensive picture of the lymphocyte repertoire has yet to be described in detail. To this end, we conducted a comprehensive profiling of TCR and BCR repertoire of infiltrating lymphocytes in the mouse injured skeletal muscle model by next-generation sequencing. In the TCR repertoire profiling, we found that the expression level of TCR transcripts contains unique CDR3 α and distinct population of CDR3 β were increased (7.6–17% and 6.7–15%) in infiltrating T cells in both of cardiotoxin- or cryo-injured skeletal muscle, whereas no clonal expansion of specific TCR α/β transcripts observed in control splenic T cells (<1% respectively). While in the BCR repertoire profiling, various transcripts of immunoglobulin kappa chains showed expansion in infiltrated fraction, despite the proportion of immunoglobulin heavy and lambda chain were remained similar to control splenic B cells. Together these data have allowed us to identify TCR/BCR candidates targeting injured skeletal muscles. Furthermore, we aim to utilize the T cells/B cells expressing high-affinity TCR/BCR for the drug delivery system, especially for large protein complexes.

Transcriptional regulation of Treg homeostasis and functional specification.

CD4+Foxp3+ regulatory T (Treg) cells are key players in keeping excessive inflammation in check. Mounting evidence has shown that Treg cells exert much more diverse functions in both immunological and non-immunological processes. The development, maintenance and functional specification of Treg cells are regulated by multilayered factors, including antigens and TCR signaling, cytokines, epigenetic modifiers and transcription factors (TFs). In the review, we will focus on TFs by summarizing their unique and redundant roles in Treg cells under physiological and pathophysiological conditions. We will also discuss the recent advances of Treg trajectories between lymphoid organs and non-lymphoid tissues. This review will provide an updated view of the newly identified TFs and new functions of known TFs in Treg biology.

Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing.

The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts encoding human leukocyte antigen (HLA) receptors as well as B cell and T cell receptors (BCR and TCR). Determining which alleles an individual possesses for each HLA gene (high-resolution HLA typing) is essential to establish donor–recipient compatibility in organ and bone marrow transplantations. In turn, the repertoires of millions of unique BCR and TCR transcripts in each individual carry a vast amount of health-relevant information. Both short-read RNA-seq-based HLA typing and BCR/TCR repertoire sequencing (AIRR-seq) currently rely on our incomplete knowledge of the genetic diversity at HLA and BCR/TCR loci. Here, we generated over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9% using our nanopore sequencing-based Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol. We used this data set to (1) show that deep and accurate full-length cDNA sequencing can be used to provide isoform-level transcriptome analysis for more than 9000 loci, (2) generate accurate sequences of HLA alleles, and (3) extract detailed AIRR data for the analysis of the adaptive immune system. The HLA and AIRR analysis approaches we introduce here are untargeted and therefore do not require prior knowledge of the composition or genetic diversity of HLA and BCR/TCR loci.

Mechanisms and Regulation of Nonsense-Mediated mRNA Decay and Nonsense-Associated Altered Splicing in Lymphocytes

The presence of premature termination codons (PTCs) in transcripts is dangerous for the cell as they encode potentially deleterious truncated proteins that can act with dominant-negative or gain-of-function effects. To avoid the synthesis of these shortened polypeptides, several RNA surveillance systems can be activated to decrease the level of PTC-containing mRNAs. Nonsense-mediated mRNA decay (NMD) ensures an accelerated degradation of mRNAs harboring PTCs by using several key NMD factors such as up-frameshift (UPF) proteins. Another pathway called nonsense-associated altered splicing (NAS) upregulates transcripts that have skipped disturbing PTCs by alternative splicing. Thus, these RNA quality control processes eliminate abnormal PTC-containing mRNAs from the cells by using positive and negative responses. In this review, we describe the general mechanisms of NMD and NAS and their respective involvement in the decay of aberrant immunoglobulin and TCR transcripts in lymphocytes.

Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells

Recent data suggest that porcine γδ T cells exhibit a similar degree of functional plasticity as human and murine γδ T cells. Due to the high frequency of TCR-γδ+ cells in blood and secondary lymphatic organs, the pig is an attractive model to study these cells, especially their combined features of the innate and the adaptive immune system. Using a 5’ RACE-like approach, we translated a human/murine NGS library preparation strategy to capture full-length V-(D)-J TRG and TRD clonotypes in swine. After oligo(dT) primed conversion of input RNA, the cDNA population was enriched for full-length V(D)J TCR transcripts with porcine-specific primers including Illumina adaptor sequences as overhangs for Illumina MiSeq analysis. After quality control and processing by FastQC and ea-utils, porcine TRG and TRD sequences were mapped against the human IMGT reference directory. Porcine blood-derived CD2+ and CD2‾ TCR-γδ+ cells exhibited two distinct clonotypes Vγ11JγP1 (74.6%) and Vγ10JγP1 (57.7%), respectively. Despite the high TCR-δ diversity among CD2+ cells (39 clonotypes), both subsets shared the same abundant Vδ1DδxJδ4 clonotype at approximately identically frequencies (CD2+: 31.2%; CD2‾: 37.0%). The flexible nature of this approach will facilitate the assessment of organ-specific phenotypes of γδ T cell subsets alongside with their respective TCR diversity at single cell resolution.

Ancient Use of Ig Variable Domains Contributes Significantly to the TCRδ Repertoire.

The loci encoding B and T cell Ag receptors are generally distinct in commonly studied mammals, with each receptor's gene segments limited to intralocus, cis chromosomal rearrangements. The nurse shark (Ginglymostoma cirratum) represents the oldest vertebrate class, the cartilaginous fish, with adaptive immunity provided via Ig and TCR lineages, and is one species among a growing number of taxa employing Ig-TCRδ rearrangements that blend these distinct lineages. Analysis of the nurse shark Ig-TCRδ repertoire found that these rearrangements possess CDR3 characteristics highly similar to canonical TCRδ rearrangements. Furthermore, the Ig-TCRδ rearrangements are expressed with TCRγ, canonically found in the TCRδ heterodimer. We also quantified BCR and TCR transcripts in the thymus for BCR (IgHV-IgHC), chimeric (IgHV-TCRδC), and canonical (TCRδV-TCRδC) transcripts, finding equivalent expression levels in both thymus and spleen. We also characterized the nurse shark TCRαδ locus with a targeted bacterial artifical chromosome sequencing approach and found that the TCRδ locus houses a complex of V segments from multiple lineages. An IgH-like V segment, nestled within the nurse shark TCRδ translocus, grouped with IgHV-like rearrangements we found expressed with TCRδ (but not IgH) rearrangements in our phylogenetic analysis. This distinct lineage of TCRδ-associated IgH-like V segments was termed "TAILVs." Our data illustrate a dynamic TCRδ repertoire employing TCRδVs, NARTCRVs, bona fide trans-rearrangements from shark IgH clusters, and a novel lineage in the TCRδ-associated Ig-like V segments.

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4+ memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4+ T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4+ memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood.

Describing the diversity of Ag specific receptors in vertebrates: Contribution of repertoire deep sequencing

During the last decades, gene and cDNA cloning identified TCR and Ig genes across vertebrates; genome sequencing of TCR and Ig loci in many species revealed the different organizations selected during evolution under the pressure of generating diverse repertoires of Ag receptors. By detecting clonotypes over a wide range of frequency, deep sequencing of Ig and TCR transcripts provides a new way to compare the structure of expressed repertoires in species of various sizes, at different stages of development, with different physiologies, and displaying multiple adaptations to the environment. In this review, we provide a short overview of the technologies currently used to produce global description of immune repertoires, describe how they have already been used in comparative immunology, and we discuss the future potential of such approaches. The development of these methodologies in new species holds promise for new discoveries concerning particular adaptations. As an example, understanding the development of adaptive immunity across metamorphosis in frogs has been made possible by such approaches. Repertoire sequencing is now widely used, not only in basic research but also in the context of immunotherapy and vaccination. Analysis of fish responses to pathogens and vaccines has already benefited from these methods. Finally, we also discuss potential advances based on repertoire sequencing of multigene families of immune sensors and effectors in invertebrates.

Clonally expanded T cells from skin biopsies of patients with systemic sclerosis recognize DNA topoisomerase I and cytomegalovirus antigens (HUM3P.258)

Abstract Systemic sclerosis (SSc) is a chronic autoimmune disease, characterized by immune system dysregulation, uncontrolled deposition of collagen and microvascular damage in the skin and several internal organs. T cells have a key role in the initiation and propagation of SSc. Our laboratory has previously identified the presence of high proportions of identical α- and β-chains TCR transcripts in skin biopsies from patients with SSc of recent onset, demonstrating clonal expansion of T cells in response to one or more antigens. In order to identify these antigens, pairs of those in vivo clonally expanded α- and β-chain TCR transcripts from two patients with SSc (SSc-21 and SSc-22) were expressed in mutant TCR negative T cells, J.RT3-T3.5, generating a total of 52 T cell lines, including 10 controls. An intracellular Ca2+ mobilization assay was employed to examine whether these transduced Jurkat T cell lines recognize putative SSc antigens presented by autologous EBV-transformed B cell lines. The self-antigen, DNA topoisomerase I and CMV and parvovirus antigens were tested. Statistically significant intracellular Ca2+ mobilization was observed in response to 3 DNA topoisomerase I and 2 CMV peptides by 5 T cell lines expressing clonally expanded α- and β-chain TCR transcripts from patient SSc-21 and SSc-22. These results demonstrate that clonally expanded T cells recognizing DNA topoisomerase I or CMV antigens are present in skin biopsies of recent onset of patients with SSc.

TCR gene expression in lymphoid and mucosal tissues of Xenopus laevis (VET1P.1122)

Abstract γδ T cells are a small subset of T cells that are considered to function as a bridge between innate and adaptive immunity. Because these cells are relatively few in number in most laboratory animals, except during the fetal period and in mucosal tissues, it is difficult to clearly delineate the functions of γδ T cells. In order to begin to characterize the γδ T cell populations in Xenopus laevis, previous work from this lab established a semi-quantitative RT-PCR method of analysis of TCR γ and TCR β gene expression in X. laevis thymus and spleen. We have extended these studies to evaluation of expression of TCR γ, β, α and δ transcripts in the thymus and spleen as well as the skin, intestine and lungs of young adult X. laevis. Based on evaluation of TCR transcript levels by RT-PCR, γδ T cells are likely to be abundant in the mucosal tissues evaluated, and the levels of TCR γ are higher in the X. laevis thymus and spleen than expected for adult humans or mice. We suggest that X. laevis is a γδ T cell “high” organism, perhaps related to the position of frogs as relatively early in the evolution of the adaptive immune response. Evaluation of expression of other T cell-related genes including the RAG genes and other genes involved in T cell development and function is ongoing. These results point to the use of X. laevis as a uniquely powerful laboratory animal model for studies of the evolution, development and function of γδ T cells.

Effects of single and repeated infections with Neoparamoeba perurans on antibody levels and immune gene expression in Atlantic salmon (Salmo salar)

Amoebic gill disease (AGD) is the main health problem for the salmon industry in Tasmania, Australia and is now reported in most salmon producing countries. Antibody and gene expression responses to the pathogen, Neoparamoeba perurans, have been studied independently following primary exposure; however, the effects of sequential reinfection, which can often occur during net-pen culture of salmon, remain unclear. The association between the transcription of immunoglobulin (Ig) and their systemic and mucosal antibody levels in regards to AGD is unknown. Herein, we assessed the antibody responses as well as Ig transcription in the gills of Atlantic salmon infected only once and also sequentially with N. perurans. After four successive AGD challenges, no significant differences in plasma or skin mucus levels of IgM were observed between AGD-naïve and challenged fish. However, IgM gene expression in gill lesions of AGD-affected fish increased up to 31 d after infection, while no changes in IgT, TCR and CD8 transcription were observed. Changes at IgM transcription level did not match the lack of antibody response in mucus, which is possibly explained by weak correlations existing between protein and mRNA abundances in cells and tissues. In the second experiment, which investigated Ig responses to AGD at the transcriptional as well as antibody production level in salmon after a single infection, the levels of serum or skin mucus IgM antibody were not affected and no changes in the IgM or IgT transcription were induced.

Ex vivo transfection of trout pronephros leukocytes, a model for cell culture screening of fish DNA vaccine candidates

DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (GVHSV) – the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and GVHSV-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, GVHSV up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation.

Development of initial idiopathic nephrotic syndrome and post-transplantation recurrence: evidence of the same biological entity

Buffalo/Mna rats spontaneously develop a nephrotic syndrome (NS). We have demonstrated that this rat nephropathy recurs after renal transplantation. We studied this recurrence by kinetic analysis of graft lesions, infiltrating cells and cytokines. Kidneys from LEW.1 W rats were grafted into proteinuric Buff/Mna or healthy Wistar Furth recipients. Kidney samples were harvested before, during and after the occurrence of proteinuria and analysed for renal histology, cell populations and cytokine transcripts. Results were compared with the evolution of the initial disease studied previously. Both groups showed normal graft histology at Day 7 and an increasing podocyte swelling at Day 45 was seen only in the Buff/Mna recipients. At Day 80, glomerular atrophy with podocytosis and focal segmental glomerular sclerosis lesions, accompanied by tubular dilatation, appeared in the Buff/Mna group. At Day 122, the intensity of the tubular and glomerular lesions increased in Buff/Mna recipients but not in the control group. An analysis of desmin and Kim-1 (early markers of glomerular and tubular damage, respectively) transcripts expression showed that glomerular lesions precede tubular injury in this model. A monocyte infiltration associated with an increase in TNFα, IL1 and IL12 transcripts appeared before the recurrence. An early increase in Cbeta TCR transcripts with a predominant Th2 profile was observed, highlighting a Th2 polarization in the Buff/Mna recurrence. The comparison of profiles of recurrence and initial disease highlighted the same mediators for both events. We propose that initial Buff/Mna idiopathic nephrotic syndrome (INS) and post-transplantation recurrence represent the same entity and a valuable tool for the study of recurring INS.

Conservation of mucosal associated invariant T (MAIT) cells and the MR1 restriction element in ruminants, and abundance of MAIT cells in spleen

MHC-related protein 1 (MR1) is a highly conserved MHC class I-like molecule. Human and murine mucosal associated invariant T (MAIT) cells are restricted by MR1 and express an invariant T cell receptor. Even though MR1 protein expression on the cell surface has not been demonstrated in vivo or ex vivo, it is assumed that MR1 presents a bacterial antigen from the intestinal lumen to MAIT cells because MAIT cells are present in the lamina propria and their expansion is dependent on the presence of intestinal micro flora. The existence of bovine MAIT cells and MR1 has been demonstrated recently although ovine MAIT cells and MR1 have not yet been described. We cloned bovine and ovine MR1 transcripts, including splice variants, and identified an anti human MR1 antibody that recognizes cells transfected with the bovine homolog. Using this antibody, no MR1 staining was detected using cells freshly isolated from blood, thymus, spleen, colon, ileum, and lymph node. MAIT cells are known to be enriched in the CD4/CD8 double negative peripheral blood T cell population, but their relative abundance in different tissues is not known. Comparison of the amount of MAIT cell-specific TCR transcript to the amount of constant α chain transcript revealed that numbers of MAIT cells are low in neonates and increase by 3-weeks of age. In 3-month old animals, MAIT cells are abundant in spleen and less so in ileum, peripheral blood, lymph node, colon, and thymus.

Gamma-delta TCR+ T-cells in systemic sclerosis (135.37)

Abstract T-cell involvement may be central to the pathogenesis of systemic sclerosis (SSc). Like αβ TCR+ T-cells, γδ TCR+ T-cell diversity during T-cell development results in a repertoire of different T-cells each with a unique TCR. Therefore the probability of finding identical TCR on defined T-cell populations is negligible, except in the context of an antigen-driven T-cell response. To determine whether γδ TCR+ T-cells are clonally expanded in skin biopsies and/or peripheral blood of patients with SSc (n=7), γ-chain (VγI & Vγ9) and δ-chain (Vδ1 & Vδ2) TCR transcripts were amplified by gene-specific PCR, followed by cloning and sequencing. We report the presence of substantial proportions of identical VγI-chain transcripts (14.3%-37.2%; p<0.05), and Vγ9 transcripts (24%-83.3%; p<0.05) in skin biopsies and/or PBMC of patients with SSc. Likewise, there were multiple identical Vδ1-chain transcripts (25%-50%; p<0.05), and Vδ2-chain transcripts (18.1%-80%; p<0.05) in the samples analyzed. Extensive clonal expansions of γ- and δ-chain TCR transcripts were identified in skin biopsies and peripheral blood of patients with SSc, demonstrating the presence of oligoclonal populations of γδ TCR+ T-cells in these patients. These γδ TCR+ T-cells may have undergone activation and clonal expansion in vivo in response to as yet unidentified antigens. Future studies to identify the antigens recognized by these clonally expanded γδ TCRs will facilitate better understanding of SSc pathogenesis.