T-cell Receptor Repertoire Analysis Research Articles

Intralesional selection of T cell clonotypes in the immune response to melanoma antigens occurring during vaccination.

T cells infiltrating pre- and postvaccine metastases obtained from melanoma patients vaccinated with either dinitrophenyl (DNP)-modified autologous tumor or with the MAGE-3.A1 peptide display selective T cell receptor (TCR) beta chain variable region (BV) repertoire changes at the tumor site as a consequence of vaccination. Restricted sets of BV families expand in all postvaccine lesions when compared with prevaccine specimens and often contain dominant clones. A protocol devised to obtain T cell lines highly enriched for expression of a given BV region through the use of anti-BV monoclonal antibodies was used to understand whether responses to specific antigen(s) accounted for these clonal expansions. In one of the patients vaccinated with DNP-modified tumor cells, BV-driven selection of the T lymphocytes expanded in two infiltrated postvaccine metastases resulted in T cell lines able to exert HLA class I-restricted lysis of the autologous tumor. These results indicate that TCR repertoire analysis at the tumor site facilitates the detection of T cell responses elicited by a vaccine and potentially cytotoxic for the autologous tumor.

Increase in Vdelta1+ gammadelta T cells in the peripheral blood and bone marrow as a selective feature of HIV-1 but not other virus infections.

Dysregulation of T-cell receptor (TCR) alphabeta bearing lymphocytes and an increase in Vdelta1+ gammadelta T cells are typical features of HIV-1 infection. However, the role of gammadelta T cells remains unclear. Therefore, peripheral blood mononuclear cells (PBMC) of 103 HIV-1-infected patients were investigated with respect to expression of Vdelta1. These results were compared to the Vdelta1 expression of bone marrow mononuclear cells (BMMC). In contrast to healthy controls, Vdelta1+ cells dominated among both PBMC and BMMC in HIV-1-infected patients. Analysis of the coexpression of CD25, CD8, HLA-DR and CD45RO revealed a high prevalence of Vdelta1/CD45RO and Vdelta1/HLA-DR double-positive PBMC only in HIV-1-infected patients but not in healthy donors. Furthermore, analysis of the gammadelta TCR repertoire in patients infected with hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV)-1 and HSV-2 showed that the selective enhancement of Vdelta1+ cells was restricted to HIV infection and not observed in other virus diseases. Our data provide further support for the involvement of gammadelta T cells in immunosuppression and progression of HIV infection.

WS044 Phenotypic and T-cell receptor repertoire analysis of CD8+ T-cells in psoriatic arthritis

WS044 Phenotypic and T-cell receptor repertoire analysis of CD8+ T-cells in psoriatic arthritis

Analysis of γδ T-cell receptor repertoire in the human nasal mucosa

yg T cells play important roles as the first line of defense in mucosal immunity. As a feature of the human ~/8 T-cell receptor (TCR) repertoire, highly selective V-J gene usage is observed for ~ and for 8, and preferential V-J~ and V-D-Jg pairing occurs. 1 In human beings, for example, 75% of ~,8 T cells in most adult peripheral blood cells express V-/2-J~,1.2/V82-J81, and 25% express V-y1-J-y2/Vgl-JSl? The ,/8 TCR repertoire analysis of normal skin indicated the presence of V82-J81 rearrangements with minor VS1-JS1 products, a -/8 Intraepithelial lymphocytes in the small intestine and colon express predominantly V81-JS1. x Like these, certain receptors predominate at given anatomic locations. Only a few studies have been done on intraepithelial lymphocytes in the nose. To elucidate the ~/8 TCR repertoire in the nose, we performed reverse-transcription polymerase chain reaction (RT-PCR) analysis by using nasal epithelial samples obtained from normal subjects. We determined which V~/and V8 families are used and which J8 family is rearranged to VS families.

Evidence for a superantigen in the pathogenesis of tuberculosis.

Tuberculosis (TB) is a disease that has posed a significant global health burden for over five thousand years [7]. In recent years the disease has made a world-wide resurgence due in large part to the increase in AIDS cases as well as the presence of multidrugresistant strains. This resurgence in TB has led to a renewed effort to understand this desease, including mechanisms of protection and pathogenesis. One approach towards understanding TB infection relies upon molecular and cellular techniques to characterize the immune response by the host to the pathogen. T cells have long been known to be pivotal to a resistance immune response. The first data supporting this position were derived from the inability of researchers to transfer immunity with serum from convalescent animals [35]. However, T cells transferred from an immune animal to a naive animal can provide protection to subsequent rechallenge. Perhaps the best evidence supporting a role for T cells in immunity to TB has been derived from gene knockout mice that fail to express major histocompatibility complex (MHC) class I or class II molecules [13, 25], and in humans, from the fact AIDS patients are exceptionally prone to infection from Mycobacterium tuberculosis [3, 11, 21, 39]. To identify T cell populations involved in the local immune response to M. tuberculosis, we decided to analyze T cells by examining the genes encoding the T cell receptor (TCR) in tuberculous pleuritis patients. These patients frequently have a large number of T cells in the pleural effusion that are antigen reactive and are actively secreting large amounts of interferon-"/(IFN-'7) [5, 6]. We reasoned that these T cells may be recognizing an immunodominant antigen that may be critical to the host for developing a successful immune response to the pathogen. To identify these T cells, we performed a TCR repertoire analysis on T cells present in the pleural fluid of tuberculous pleuritis patients, and compared these results to the repertoire

Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions

We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus-host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology.

The Influence of Non‐HLA Genes on the Human T‐Cell Receptor Repertoire

We previously demonstrated a central role for HLA genes in determining the T-cell receptor (TCR) repertoire. However, these studies also suggested that other genetic factors might also play a role in the development of this repertoire. In order to assess the role of non-HLA genes in the development of the TCR repertoire, we have analysed and compared the TCR repertoires of individuals in three families consisting of both monozygotic twins as well as an HLA-identical sib. TCR repertoire analysis was performed with both V-segment-specific MoAb and the polymerase chain reaction using TCRBV-segment-specific oligonucleotide primers. We observed that in every case the TCR repertoires of identical twins were more similar to each other than to their HLA-identical sib. Furthermore, in one family we were able to show by genotype analysis that most of the differences in repertoire between the identical twins and their HLA-identical sib were caused by polymorphisms in the TCR genes that influence expression levels. These studies document an important role for non-HLA genes in determining the TCR repertoire in man and raise the possibility that such TCR polymorphisms may play a significant role in determining disease susceptibility.

T-Cell Receptor Repertoire in Tumor-Infiltrating Lymphocytes. Analysis of Melanoma-Specific Long-Term Lines

Cytotoxic T-lymphocytes (CTLs) can be isolated from human melanoma biopsies that specifically lyse autologous melanoma in vitro and can be effective therapeutic agents for patients with advanced disease. Recent evidence indicates that HLA-A2-restricted, melanoma-specific tumor-infiltrating lymphocytes (TILs) recognize melanomas obtained from different HLA-A2+ patients, suggesting the presence of one or more common melanoma antigens. Furthermore, T-cell receptor (TCR) repertoire analysis by other groups of TILs from fresh melanoma biopsies suggests that there is limited TCR V gene usage in TILs. One serious limitation in analyzing the TCR repertoire in fresh tumors has been the inability to correlate TCR usage with immune function. Therefore, the TCR repertoire was determined in long-term TIL cultures that specifically lysed autologous melanoma in vitro and in many cases mediated in vivo regression of metastatic cancer in patients with advanced disease. The TCR repertoire in cultured melanoma-specific TILs was diverse, with each TIL containing an average of 9.5 +/- 5.7 of the 23 V alpha and 11.2 +/- 5.9 of the 23 V beta subfamilies. Despite the large diversity observed, several V alpha and V beta genes (V alpha 1, V alpha 2, V alpha 22, V beta 13, V beta 14, and V beta 18) are very commonly found in melanoma-specific TILs. No statistically significant associations were observed between the presence of a TCR V gene subfamily in TILs and clinical response, HLA haplotype, or age of the culture. Even though the results in this study suggest that certain TCR V gene segments may be involved in immune responses to human melanoma, we were unable to demonstrate functionally that a particular T-cell clonotype recognizes melanoma tumor-associated antigens. Only the analysis of melanoma-specific CTL clones can determine which clonotypes are important in lysis of human melanoma.

Analysis of the peripheral blood T-cell receptor (TCR) repertoire in monozygotic twins discordant for Crohn's disease.

T cell involvement in the inflammatory process of Crohn's Disease (CD) is evident by an increase in activated T cells and their cytokines in actively inflamed CD tissue. It has been suggested that CD may involve a superantigen based on the observation that a significant proportion of CD patients express elevated levels of V beta 8+ T cells in their peripheral blood compared to normal controls. In order to determine whether a superantigen might play a role in the pathogenesis of CD we have compared the TCR repertoires of four pairs of monozygotic twins discordant for CD. By using monozygotic twins, we could rule out the effects of HLA and other genes on the TCR repertoire. The TCR repertoires were analyzed by using a panel of V-segment-specific mAb and by quantitative polymerase chain reaction (qPCR) using V beta-specific oligonucleotide primers. In all cases the TCR repertoires of the affected and unaffected sibs were strikingly similar. We did not observe any TCR segment that was consistently altered in frequency or expression levels in all of the affected sibs compared to their identical twin. Furthermore, we did not see an increase in V beta 8+T cells in the peripheral blood of the CD sibs relative to their normal counterpart. These studies suggest that the presence of CD does not alter the TCR repertoire of peripheral blood in any obvious way and argue against the role of a superantigen in the etiology of pathogenesis of CD.

An alternative method for T-cell receptor repertoire analysis: Clustering of human V-beta subfamilies selected in responses to staphylococcal enterotoxins B and E

We have designed a convenient procedure for the analysis of Vβ repertoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucleotidyl transferase and submitted to one-side specificity PCR amplification with a constant region oligonucleotide primer. The amplified material is then analysed by reverse spot-test hybridization: after 32P-labelling, the amplification product is put to hybridize on a membrane where specially designed Vβ subfamily-specific probes are immobilized. The radioactivity fixed on each probe can then be easily quantified and the signal obtained is directly proportional to the initial amount of homologous RNA. We applied this technique to the study of Vβ gene selection following T-cell stimulation by staphylococcal enterotoxins B and E. We show that with these toxins two almost non-overlapping sets of T-cells are recruited and that this selection is likely to be dependent on specific amino acid residues shaping the fourth complementarity determining region of the TCR-β chain. These residues constitute two tandemly-conserved tripeptide sequences (Asp 39Pro 40Gly 41)-(Val 61)Ser 70Arg 71) and (Arg 66Phe 67Ser 68)-(Asp 88Ser 89Ala 90) in the SEB- and the SEE-responsive Vβ gene clusters respectively.

Transplantation tolerance is unrelated to superantigen-dependent deletion and anergy.

C57BL/6 (B6; I-E-, Mls-2b) nude mice, reconstituted at birth with thymic epithelium (TE) from BALB/c (BA; I-E+, Mls-2a) day 10 embryos (E10), permanently accepted BALB/c skin, when grafted as adults. T-cell receptor repertoire analyses in the periphery of these mice revealed no difference in frequencies of I-E/superantigen-reactive T-cell receptor V beta families, as compared to chimeras constructed with syngeneic B6 E10 TE. T lymphocytes bearing V beta 3, V beta 5, and V beta 11 T-cell receptors, from either allogeneic or syngeneic TE chimeras, responded equally well to in vitro receptor-dependent stimulation. Similar results were obtained with nude mice reconstituted at birth with E14 thymuses, already colonized by hemopoietic cells. These observations indicate that neither TE cells nor the progenies of hemopoietic precursors that colonize the thymus up to E14 express or functionally present the superantigens addressed here; it follows that tolerance to skin grafts and superantigen-related T-cell deletions are unrelated phenomena.