Alpha Repertoire Research Articles

T-cell receptor-α repertoire of CD8+ T cells following allogeneic stem cell transplantation using next-generation sequencing.

Alloreactivity or opportunistic infections following allogeneic stem cell transplantation are difficult to predict and contribute to post-transplantation mortality. How these immune reactions result in changes to the T-cell receptor repertoire remains largely unknown. Using next-generation sequencing, the T-cell receptor alpha (TRα) repertoire of naïve and memory CD8+ T cells from 25 patients who had received different forms of allogeneic transplantation was analyzed. In parallel, reconstitution of the CD8+/CD4+ T-cell subsets was mapped using flow cytometry. When comparing the influence of anti-T-cell therapy, a delay in the reconstitution of the naïve CD8+ T-cell repertoire was observed in patients who received in vivo T-cell depletion using antithymocyte globulin or post-transplantation cyclophosphamide in case of haploidentical transplantation. Sequencing of the TRα identified a repertoire consisting of more dominant clonotypes (>1% of reads) in these patients at 6 and 18 months post transplantation. When comparing donor and recipient, approximately 50% and approximately 80% of the donors’ memory repertoire were later retrieved in the naïve and memory CD8+ T-cell receptor repertoire of the recipients, respectively. Although there was a remarkable expansion of single clones observed in the recipients’ memory CD8+ TRα repertoire, no clear association between graft-versus-host disease or cytomegalovirus infection and T-cell receptor diversity was identified. A lower TRα diversity was observed in recipients of a cytomegalovirus-seropositive donor (P=0.014). These findings suggest that CD8+ T-cell reconstitution in transplanted patients is influenced by the use of T-cell depletion or immunosuppression and the donor repertoire.

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8+ T cell receptor alpha repertoire following allogeneic transplantation

Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αβ sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting.

Mucosa-Associated Invariant T Cells Are Systemically Depleted in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Mucosa-associated invariant T (MAIT) cells contribute to host immune protection against a wide range of potential pathogens via the recognition of bacterial metabolites presented by the major histocompatibility complex class I-related molecule MR1. Although bacterial products translocate systemically in human immunodeficiency virus (HIV)-infected individuals and simian immunodeficiency virus (SIV)-infected Asian macaques, several studies have shown that MAIT cell frequencies actually decrease in peripheral blood during the course of HIV/SIV disease. However, the mechanisms underlying this proportional decline remain unclear. In this study, we characterized the phenotype, activation status, functionality, distribution, and clonotypic structure of MAIT cell populations in the peripheral blood, liver, mesenteric lymph nodes (MLNs), jejunum, and bronchoalveolar lavage (BAL) fluid of healthy and SIV-infected rhesus macaques (RMs). Low frequencies of MAIT cells were observed in the peripheral blood, MLNs, and BAL fluid of SIV-infected RMs. These numerical changes were coupled with increased proliferation and a highly public T cell receptor alpha (TCRα) repertoire in the MAIT cell compartment without redistribution to other anatomical sites. Collectively, our data show systemically decreased frequencies of MAIT cells likely attributable to enhanced turnover in SIV-infected RMs. This process may impair protective immunity against certain opportunistic infections with progression to AIDS. The data presented in this study reveal for the first time that MAIT cells are systemically depleted in an AIDS virus infection. These findings provide a new mechanistic link with our current understanding of HIV/SIV pathogenesis and implicate MAIT cell depletion in the disease process.

Restricted TCR V alpha repertoire in the T cell response to a tolerogenic determinant of type II collagen.

Tolerization of B10.RIII mice (H-2r) with i.v.-injected type II collagen (CII) renders the animals resistant to induction of collagen-induced arthritis (CIA). The B10.RIII mouse is of particular interest, in that the T cell determinants that induce tolerance are different from those that induce arthritis. To characterize T cells that react with the tolerogenic determinant and play a role in regulation of arthritis, we have developed a panel of T cell hybridomas reactive with the tolerogenic T cell epitope, CII 607-621. None of the hybrids cross-reacted with either the arthritogenic CII 445-453 or murine CII. As determined by PCR and immunofluorescence, the T cell response to the tolerogenic determinant was oligoclonal, with evident preferential usage of V alpha. Through the analysis of a large panel of T cell hybridomas, preferential usage of V alpha2, J alpha44, J beta2.7, and D beta2.1 was observed. Characterization of T cells reactive with the immunodominant determinant, CII 607-621, responsible for the induction of tolerance should prove important in developing novel therapeutic approaches for the treatment of autoimmune diseases.

Non random usage of T cell receptor alpha gene expression in atopy using anchored PCR.

The T cell receptor (TCR) alpha beta heterodimer recognises antigenic peptide fragments presented by Class II MHC. This interaction initiates T cell activation and cytokine release with subsequent recruitment of inflammatory cells. Previous work from our group suggests a qualitative difference in variable alpha gene expression in atopy as compared to non atopic controls. In this study we examine TCR alpha repertoire using anchored PCR to provide a quantitative assessment of the V alpha and J alpha repertoire. One atopic (DRB1*0701,DRB1*15: DRB4*0101, DRB5*01: DQB1* 0303, DQB1*601/2) and one non-atopic (DRB1*0701,DRB1*03011/2: DRB4*01, DRB3*0x: DQB1* 0303, DQB1*0201/2) control were studied. Variable gene usage was markedly limited in the atopic individual. V alpha 1, 3, 8 accounted for 60% and J alpha 12, 31 30% of the gene usage. There was evidence of preferential V alpha-J alpha gene pairing and clonal expansion. We conclude that there is a marked non random TCR alpha gene distribution in atopy using both V alpha family and anchored PCR. This may be due in part to antigen driven clonal expansion.

Do HLA genes play a prominent role in determining T cell receptor V alpha segment usage in humans?

Previous studies in humans have demonstrated that HLA genes can profoundly influence the TCR V beta repertoire. To similarly assess the influence of HLA genes on the TCR V alpha segment repertoire, the V alpha repertoires of 12 individuals from three unrelated families were determined by quantitative PCR. Each family contained at least one pair of HLA-identical and -nonidentical siblings. Repertoire analysis was performed on purified CD4+ and CD8+ cells by using V alpha-specific primers. We were unable to demonstrate more similar V alpha repertoires between HLA-identical siblings than between HLA-nonidentical siblings. In contrast, when a similar analysis was performed on the same individuals for the V beta repertoire, HLA-identical siblings were found to have significantly more similar repertoires than HLA-nonidentical siblings. Furthermore, both the V alpha and V beta repertoires of monozygotic twins showed striking similarity. Despite our inability to show an influence of HLA genes on the V alpha repertoire, we did observe a very strong skewing in terms of preferential expression on CD4+ or CD8+ cells of several V alpha segments, notably TCRAV1, -2, -5, -6, -7, -11, -12, and -13. These studies suggest that HLA genes play less of a role in determining V alpha segment usage than V beta. Nevertheless, the pronounced skewing of V alpha segment expression in the CD4+ or CD8+ populations suggests some role for HLA genes in determining the V alpha TCR repertoire. Furthermore, the striking similarity of V alpha repertoires of identical twins suggests a major role for non-HLA genes in determining the V alpha repertoire.

Restricted heterogeneity of T cell receptor variable alpha chain transcripts in hearts of Chagas’disease cardiomyopathy patients

The role of autoimmunity in the pathogenesis and progression of heart lesions in the chronic phase of Chagas' disease is controversial. In the absence of parasites in situ, the T cell infiltrate seen in heart lesions may be the primary determinant of tissue damage ultimately leading to heart failure and death. We used the polymerase chain reaction to amplify each known T cell receptor (TCR) V alpha and V beta subfamily-specific sequence in transcripts derived from heart samples obtained from Chagas' cardiomyopathy patients. The average number of TCR V alpha subfamilies (7.1 per tissue sample) was significantly lower than that for TCR V beta subfamilies (15.1 per sample). The average percentage of tissue samples positive per TCR V alpha and V beta subfamily was respectively 39.6% vs. 73.5%. These data suggest that, in Chagas' heart lesions, the detectable TCR V alpha repertoire is significantly narrower than TCR V beta repertoire. On the other hand, in normal heart tissue, diversity of V alpha and V beta TCR is similar among the scarce circulating T cell population. Such evidence of restricted TCR V region repertoire has been described in experimental and human autoimmune diseases. Our results are consistent with the possibility that T cells responsible for heart damage in chronic Chagas' cardiomyopathy may be recognizing a few heart-specific antigenic targets.

Characterization of the human T cell receptor α‐chain repertoire and demonstration of a genetic influence on Vα usage

Despite considerable knowledge of the determinants and profile of the T cell receptor (TCR) beta-chain repertoire, very little is known about the repertoire of the V alpha and J alpha segments of the TCR in man or mouse. We have used the anchored polymerase chain reaction (PCR) to analyze the human TCR alpha-chain repertoire at the mRNA level in peripheral blood lymphocytes. We find marked differences in the usage of individual V alpha segments. The V alpha repertoire is determined both by differences in the use of individual V alpha gene segments at the time of gene rearrangement and by genetic influences on the selection of the expressed V alpha repertoire, presumably during thymic maturation. Analysis of the J alpha repertoire shows non-random usage of individual J alpha segments but no obvious relationship to chromosomal position. We also report three new V alpha segments, three new J alpha segments and possible allelic variation in V alpha gene segments. Two unusual TCR alpha transcripts are described which add new information on the nature of TCR alpha transcript assembly.

Skewed T cell receptor V alpha repertoire among superantigen reactive murine T cells.

Reactivity of murine T cells with viral or bacterial superantigens is clearly correlated with the expression of TCR V beta domains. Thus, T cells responding to the minor lymphocyte stimulatory locus (Mls-1a) or staphylococcal enterotoxin B (SEB) express predominantly TCR V beta 6 or V beta 8.2 respectively. We have investigated the involvement of the other major variable element of the TCR, the V alpha domain, in these superantigen responses. Using a panel of anti-TCR V alpha mAbs, it is demonstrated that the TCR V alpha repertoire among superantigen stimulated V beta 6+ or V beta 8.2+ blasts (responding to Mls-1a or SEB respectively in vitro) is altered in comparison with anti-CD3 stimulated cells expressing the same V beta domains. Furthermore, the TCR V alpha repertoire is strongly skewed in TCR V beta 8.2 transgenic mice that have undergone extensive peripheral clonal deletion after SEB injection. These data imply that the V alpha domain influences superantigen recognition by the TCR.

T cell receptor V alpha-V beta repertoire and cytokine gene expression in active multiple sclerosis lesions.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with presumed autoimmune etiology. A recent study has suggested the presence of a restricted T cell receptor (TCR) V alpha repertoire in MS lesions. The presence of such a restricted TCR repertoire at the site of inflammation would have important consequences for the pathogenesis and the ultimate treatment of MS. To further characterize the TCR V alpha and V beta repertoire in MS plaque tissue, we examined a series of 26 histologically well-characterized central nervous system (CNS) tissue specimens from six MS patients as well as samples from five normal postmortem cases and a case of subacute sclerosing panencephalitis. RNA was extracted from frozen sections and cDNAs were amplified by polymerase chain reaction using primers for TCR V alpha (V alpha 1-18) and V beta (V beta 1-19) gene families. This analysis demonstrated a broad TCR V alpha-V beta repertoire in active lesions, while fewer TCR V genes were detected in chronic plaques and control samples. Even though a large number of TCR V alpha and V beta gene segments were present in the majority of active lesions, there were clear differences in the TCR repertoire between plaques from the same case, suggesting that local events influence the TCR repertoire at the level of T cell recruitment or T cell expansion. Examination of cytokine mRNAs demonstrated that IL-1 mRNA was present in the majority of acute and subacute plaques, while IL-2 and IL-4 mRNA were detected in only a few acute lesions. These data demonstrate that the TCR repertoire in MS plaques is polyclonal. However, autoreactive alpha/beta T cells thought to be critical in the initiation of the inflammatory process probably represent a minor fraction of T cells in active MS plaques and may use a limited number of TCR V gene segments for recognition of the autoantigen.

Characterization of T-cell receptor alpha beta repertoire in synovial tissue from different temporal phases of rheumatoid arthritis.

With the aim of investigating the distribution of T cells expressing different T-cell receptors (TCR) in the inflamed synovial tissue of rheumatoid arthritis patients, we have used the polymerase chain reaction to amplify TCR V alpha and V beta transcripts from synovial biopsies obtained by arthroscopy from patients with arthritis of variable duration. From each of nine patients a single biopsy was taken. Southern hybridization analysis of amplified products revealed extensive heterogeneity of TCR V beta in most patients. On the other hand, restriction in V alpha gene expression was seen in several patients. A highly restricted V alpha repertoire was observed in all cases with arthritis of short duration. In addition, two of three samples of short duration yielded a more limited number of V beta transcripts than the others. No conformity was, however, seen in usage of individual V alpha and V beta transcripts among the investigated patients. The present data thus demonstrate variability in synovial TCR expression between rheumatoid arthritis patients, but they also indicate a development towards greater diversity with increasing disease duration, implicating the necessity for careful choice of cases, preferentially selecting for early stages of disease, when further analysing rheumatoid synovial T cells for TCR usage as well as for antigen specificity.

Use of variable human V delta genes to create functional T cell receptor alpha chain transcripts.

Previous studies of the human T cell receptor delta genes identified five commonly used V delta segments distinct from any of the known V alpha genes. To define better the relationship between the T cell receptor delta and alpha repertoires we amplified cDNA obtained by polyclonally activated lymphocytes with a common 3' antisense C alpha-specific primer and with five different 5' sense V delta family-specific primers. Amplified products were detected in staphylococcal enterotoxin C2, staphylococcal enterotoxin E, phytohemagglutinin, concanavalin A, anti-CD3 and anti-V beta 8-activated cells, although each cell population expressed a selective pattern of V delta genes. Sequence analysis revealed that each of the known V delta genes can productively rearrange to J alpha segments to produce functional V delta-J alpha-C alpha transcripts. These results argue strongly against the notion that the human V delta and V alpha repertoires are distinct. They further suggest that the restricted delta repertoire observed in many gamma/delta clones results from selection rather than from controlled rearrangements at the T cell receptor alpha/delta locus.

A T cell receptor V alpha region selectively expressed in CD4+ cells.

The peripheral TCR V beta repertoire is strongly influenced by the processes of negative selection (deletion) and positive selection in the thymus. In order to investigate whether such selection events influence the V alpha repertoire, we have produced an anti-V alpha 11 mAb. This antibody was made by immunization with a chimeric TCR:Ig protein containing V alpha 11 in place of the VH of an IgG2a, lambda Ig. This scheme optimizes the specificity of immunization and facilitates the screening procedure. The antibody recognizes a panel of V alpha 11-expressing T cell clones. Analysis of mouse strains indicates that the antibody recognizes V alpha 11 only in mice of the C57 background. The expression of the epitope on peripheral T cells is strongly biased to the CD4+ subset, suggesting positive selection of V alpha 11 on class II MHC molecules. In some strain comparisons, the percentage of V alpha 11-expressing T cells in the CD4+ subset was elevated in I-E+ relative to I-E- strains. These data suggest that V alpha 11 can differentially influence the selection of T cells into the CD4+/CD8+ subsets.

Diversity and organization of human T cell receptor delta variable gene segments.

Previous studies of the human TCR-delta gene identified a single commonly used V delta segment, denoted V delta 1. To better understand the extent of the human TCR-delta V gene repertoire, TCR-delta transcripts and gene rearrangements were examined in a new panel of cloned human TCR-gamma/delta lymphocytes. Through this analysis we identified and determined the structures of two new V delta segments, denoted V delta 2 and V delta 3. These V delta segments are different from previously characterized V alpha segments, supporting the notion that the human V delta and V alpha repertoires are distinct. Examination of V gamma gene segment usage in these cells reveals that the V delta 2 gene segment is used in conjunction with the V gamma 2 gene segment. Blot hybridization indicates that the V delta 2 gene segment lies between V delta 1 and D delta-J delta-C delta, and within 100 kb of the latter. Analysis of genomic clones indicates that the V delta 3 gene segment lies in an inverted orientation, approximately 2 kb 3' of C delta. This implies that rearrangement of V delta 3 to D delta-J delta-C delta occurs by inversion. Together with previous mapping studies, these results indicate that human V delta segments are dispersed, rather than clustered, within the TCR-alpha/delta locus. The analysis of rearrangements in polyclonal thymocyte DNA suggests that there may be a limited number of additional V delta gene segments yet to be characterized.

Predominant variable region gene usage by gamma/delta T cell receptor-bearing cells in the adult thymus.

Previous studies have indicated that the diversity of gamma genes expressed by gamma/delta-bearing murine T cells is limited, but comparable information concerning the expressed diversity of delta genes is lacking. In this study, we have investigated the rearrangement and expression of delta and gamma genes in T cell hybridomas that express gamma/delta T cell receptors. Three productive delta chain cDNA clones were isolated (delta 7.3, delta 7.1, and delta 2.3) that encode new variable region sequences. Two of the delta cDNAs differ significantly from those observed in the V alpha repertoire. In addition, one cDNA expressed a new J delta region (J delta 2), which was localized between J delta 1 and C delta genes. Using these and other delta gene probes and gamma gene probes, we found that five independent hybridomas expressed four different V delta s and three different V gamma s. However, analysis of an enriched population of gamma/delta-expressing cells from the adult thymus suggests that only a few V delta genes and one V gamma gene are used by the majority of the cells. These results suggest that important components of receptor chain that contribute to specificity (i.e., the germline V gene sequences) are relatively nondiverse in the thymic gamma/delta population.

Variability and repertoire size of T-cell receptor V alpha gene segments.

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.