T-cell repertoire complexity after allogeneic bone marrow transplantation

We analyzed the T-cell repertoire in patients transplanted with bone marrow from an HLA identical sibling by determining the TCR diversity through Vβ-CDR3-size spectratyping with Vβ/Cβ- and Vβ/Jβspecific primers. Using the Vβ/Cβ primers, we observed limited TCR diversity only in recipients of a T-celldepleted graft, whereas the TCR diversity of patients transplanted with an unmanipulated graft seemed to be indistinguishable from the one of a normal individual. However, with Vβ/Jβ-specific primers, increase of the resolution by approximately 10-fold also allowed the detection of imbalances in the TCR repertoire of recipients of an unmanipulated graft. This demonstrates that when high numbers of T cells are cotransfused with marrow, the TCR repertoire is more complete but still not as complete as in normal individuals, thereby emphasizing the important role of coinfused mature T cells in the restoration of the T-cell compartment after bone marrow transplantation.     

HLA-DRB1 molecules and antigenic experience shape the repertoire of CD4 T cells

Forces influencing the composition of the mature TCR repertoire have been well studied in the mouse. In particular, the contribution of MHC molecules in negative and positive selection events of T lymphocytes has been established. To understand whether the allelic polymorphism of HLA-DRB1 molecules can shape the human TCR repertoire, we compared the usage of TCR Vβ segments in two cohorts of unrelated individuals who were selected for the expression of HLA-DRB1 alleles. To investigate the potential role of antigenic experience in shaping the TCR repertoire, we compared the usage of Vβ gene elements in CD45RO⁻ CD4⁺ (naive) T cells versus CD45RO⁺ CD4⁺ (memory) T cells. A correlation between Vβ gene segment usage and HLA-DRB1 alleles could be demonstrated for the repertoire of the naive CD4⁺ T cells, suggesting a shaping force of the HLDRB1 allele on the peripheral TCR repertoire. While the HLA-DRB1 imposed profile in Vβ distribution was maintained in CD45RO⁺ CD4⁺ T cells, it was less pronounced, indicating that antigenic experience modulates the functional TCR repertoire.     

T-cell receptor variable region of the β-chain gene use in peripheral blood and multiple synovial membranes during rheumatoid arthritis

In order to look for a site-specific T-cell response in RA SM, PCR analyses using oligonucleotide primers specific for 24 TCRBV (Vβ) families were performed to compare the respective usage of each TCRBV gene by T cells present in PB and SM of 13 patients with RA. In four patients, SM cells from two or three sites of inflammation were subjected to analysis. In one patient, synovial tissue was studied at two different phases of the disease, resulting in a total number of 19 samples of SM cells, which were compared with paired samples of PB cells. The results showed that whereas all 24 TCRBV gene families could be detected in both PB and SM cells, there was some skewing of increased or decreased usage frequencies of particular TCR Vβ genes among SM cells. Three TCRBV families were often overexpressed in SM: Vβ3, Vβl7, and Vβ22. Moreover, Vβ4 was often decreased in SM (7 out of 13). This decrease was statistically significant in the RA population studied. SM from different joints of a given patient showed similar variations of T-cell repertoire compared to PB, even 6 months later in the course of the disease. These results demonstrate a biased TCRBV gene utilization in RA SM. This bias appears to be similar in different joints and at different times in the course of the disease. No correlation was found between the bias of TCR repertoire in SM and the HLA typing of these patients.     

Comparison of activation marker and TCR V beta gene product expression by CD4+ and CD8+ T cells in peripheral blood and lymph nodes from HIV-infected patients.

Since lymphoid organs constitute the site of active and progressive HIV disease, analysis of their lymphocytes may provide more accurate information on T cell abnormalities than that obtained from studying peripheral blood lymphocytes. The objective of this study was to compare the expressions of activation markers and T cell receptor (TCR) V beta gene products by CD4+ and CD8+ T cells in lymph nodes (LN) and peripheral blood (PB) from healthy individuals and asymptomatic HIV-infected patients to determine whether anomalies that could be identified at the HIV replication site could support the hypothesis of T cell activation by HIV-encoded antigens or superantigens. CD4+ and CD8+ T cells in paired LN and PB obtained from six healthy controls and five asymptomatic HIV-infected individuals were analysed by flow cytometry, using anti-CD38, anti-HLA-DR and 13 anti-V beta MoAbs that cover, approximately, 45% of the T cell repertoire. Analysis of T cell activation marker expression indicated that the percentages of CD4+ and CD8+ T cells bearing CD38 or CD38 and HLA-DR molecules were higher in patients than in controls and, in patients, higher in LN than in PB. Comparison between the V beta repertoires of CD4+ and CD8+ T cells in LN and PB showed that, in each healthy individual, a limited number of V beta families expressed by CD4+ or CD8+ T cells had different repartition in LN and PB, whereas in each HIV+ patient, more V beta families exhibited different distributions and these differences recurred among certain V beta segments, such as V beta 5.3 and V beta 21 in the CD4+ T cell population and V beta 5.2/5.3, V beta 12 and V beta 21 in the CD8+ T cell population. Taken together, these data argue for a skewed TCR repertoire in HIV infection and sustained activation of T cells by HIV-encoded antigens at the site of HIV replication, and further demonstrate that a high proportion of CD4+ T cells are in an activation state that may, indirectly, participate in their functional abnormalities.     

Analysis of the Conservation of T Cell Receptor Alpha and Beta Chain Variable Regions Gene in pp65 Peptide-Specific HLA-A＊0201-Restricted CD8^＋ T Cells

Many viral epitope specific T cell receptors （TCRs） in MHC-matched individuals have been demonstrated to involve conserved amino acid motifs in β chain complementarity-determining region 3 （CDR3）. However, it is not sure whether the conserved motifs can also be found in TCR β chain. In previous studies, we developed a modified method to enlarge the percentage of cytomegalovirus （CMV） pp65 peptide-specific CD8^＋ T cells in PBMC by continuous peptide stimulation in vitro, which provides sufficient number of specific T cells for detection. In this study, we further analyzed the restrictive usage of TCR Vα and Vβ gene families and investigated the CDR3 gene sequence of pp65 peptide-specific CD8β T cells. Analysis of CDR3 spectratypes suggested a restricted usage of TCR α chain AV8, AV12, AV21, AV31 families and TCR βchain BV3, BV14, BV21, BV23, BVll families in donor CD8^＋ T cells stimulated by pp65 peptide. The sequences of these T cells involved similar sequence （TX） G （X） A in CDR3 region of TCR α chain and L （XT） G （X） A in TCR β chain.     

Streptococcus pyogenes type 5 M protein is an antigen, not a superantigen, for human T cells

M proteins are coiled-coil dimers expressed on group A streptococcal cell surfaces. They have an importantrole in host antistreptococcal immunity and in poststreptococcal autoimmune sequelae. Controversy has arisen regardingwhether type 5 M proteins are superantigenic for human T cells. To investigate this, we have produced and tested M5 inthe form of two novel recombinant proteins. We found no evidence of superantigenicity using either recombinant whole M5protein (rM5) or recombinant pep M5 protein (rpepM5) to activate peripheral blood mononuclear cells (PBMC) from healthyadult volunteers. Short-term, rM5-specific T-cell lines from different subjects were uniformly self-APC restricted andshowed no consistent pattern of TCR Vβ usage. A synthetic peptide of M5 residues 217–237 was found tocontain epitope(s) recognized by some rM5-specific human T cells. PBMC responses to rM5 and rpepM5 in 3- and 7-dayproliferation assays were characteristic of antigenic rather than superantigenic stimulation. We conclude that type5 M protein activates human T cells as a conventional antigen.     

An alternative approach to the assessment of γδ T-cell clonality in celiac disease intestinal lesions through cDNA heteroduplex analysis of T-cell receptor VJ junctions

We have investigated the clonality of the γδ T lymphocytes infiltrating the intestinal mucosa of CD patients and control subjects by means of a simple and powerful method based on the heteroduplex analysis of the TCR VJ junctions. Each V-specific TCR chain, amplified either from fresh biopsy material or intestinal T-cell-line cDNA, is denatured and renatured to allow the random reshuffling of the various strands carrying different junctional sequences, coamplified in the same reaction. The mismatched chains (heteroduplexes) are separated from the matched ones (homoduplexes) through polyacrylamide gel electrophoresis, and whenever one or more T-cell clones are emerging over the polyclonal background, discrete bands are visible by ethidium-bromide staining. Through this method, we have estimated the diversity of the Vδ1–3 chains and a newly described V gene (Vδ8) whose homologue in mice is abundantly expressed in γδ iLs. We demonstrate that the well-documented expansion of Vγ1⁺ γδ lymphocytes in the jejunum of CD patients is polyclonal. Overall, the heteroduplex analysis on fresh intestinal and peripheral blood lymphocytes from both healthy and affected subjects shows a polyclonal pattern of all the Vδ⁺ subsets. In contrast, most intestinal T-cell lines produce oligoclonal patterns, suggesting a dramatic in vitro selection effect. The cell expansion in culture is generally not required for the TCR heteroduplex analysis, which can therefore be applied to rapidly monitor the T-cell response in a variety of physiologic and autoimmune reactions, substituting the standard approach of TCR cloning and multiple VJ sequencing. Human Immunology 40, 303–311 (1994)     

Therapeutic vaccination with a trivalent T-cell receptor (TCR) peptide vaccine restores deficient FoxP3 expression and TCR recognition in subjects with multiple sclerosis

Therapeutic vaccination using T-cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund''s adjuvant that reliably induced high frequencies of TCR-specific T cells. To evaluate induction of regulatory T-cell subtypes, immunological and clinical parameters were followed in 23 treatment-naïve subjects with relapsing-remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open-label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR-reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)-10-secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both ‘native’ CD4⁺ CD25⁺ and ‘inducible’ CD4⁺ CD25⁻ peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.     

Epitope Context and Reshaping of Activated T Helper Cell Repertoire

In recent years, a growing interest in the study of peptide antigenicity in relation to the role of flanking sequences and protein topology in processing, presentation, and recognition has been observed. However, the information available on the antigenicity of recombinant fusion proteins and their effect on the selection of antigen receptor repertoires is limited. To analyze the role of molecular topology of T epitopes in a system relevant to human pathology, we have used the bacterially expressed Schistosoma japonicum glutathione S transferase (GST) to construct recombinant antigens containing HIV-1 derived T cell determinants, and human T cell clones specific for these determinants. We found that antigenicity of a given GST—peptide combination was not the same when T cells and antigen presenting cells from different individuals were tested. Our results show that differences in processing and presentation of chimeric proteins are not dictated by the use of diverse restriction elements. We also found that the context in which an antigenic peptide is delivered affects the recruited repertoire as defined according to T cell receptor Vβ usage and fine specificities of selected T cells.     

“荧光定量PCR溶解曲线法”监测人TCRβ链CDR3谱系漂移技术的建立

目的 建立"荧光定量PCR溶解曲线分析技术"监测人外周血T细胞TCR β链CDR3谱系漂移(单/寡/多克隆增生).方法 提取4例正常人、9例大肠癌患者外周血单个核细胞(peripheral blood mononuclear cell-PBMC)中的总RNA,逆转录成cDNA,以26个人TRBV基因家族设计上游引物,共同的TRBC基因设计下游引物,荧光定量PCR(FQ-PCR)扩增26个TRBV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单/寡/多克隆增生.结果 正常人外周血T细胞TCR β链26个家族CDR3表达频率不一致,各家族PCR产物的"溶解曲线谱型图"(melting curve spectratyping)呈现溶点不同的CDR3多态性,为多克隆增生的高斯分布;9例大肠癌患者的外周血TCR β链CDR3谱系的26个家族CDR3表达频率不一致,有的患者部分家族呈缺失状态,患者各家族PCR产物的"溶解曲线谱型图"上,多数家族为多克隆增生的高斯分布,但每个患者均出现数量不等的单克隆和寡克隆增生家族.结论 "荧光定量PCR溶解曲线分析TCR CDR3谱系漂移技术",方法稳定简便,能较好的监测正常人和临床样本外周血T细胞TCR β链CDR3谱系漂移(单/寡/多克隆增生).     

T-Cell Receptor Vβ Repertoire CDR3 Length Diversity Differs within CD45RA and CD45RO T-Cell Subsets in Healthy and Human Immunodeficiency Virus-Infected Children

The T-cell receptor (TCR) CDR3 length heterogeneity is formed during recombination of individual Vβ gene families. We hypothesized that CDR3 length diversity could be used to assess the fundamental differences within the TCR repertoire of CD45RA and CD45RO T-cell subpopulations. By using PCR-based spectratyping, nested primers for all 24 human Vβ families were developed to amplify CDR3 lengths in immunomagnetically selected CD45RA and CD45RO subsets within both CD4⁺ and CD8⁺ T-cell populations. Umbilical cord blood mononuclear cells or peripheral blood mononuclear cells obtained from healthy newborns, infants, and children, as well as human immunodeficiency virus (HIV)-infected children, were analyzed. All T-cell subsets from newborn and healthy children demonstrated a Gaussian distribution of CDR3 lengths in separated T-cell subsets. In contrast, HIV-infected children had a high proportion of predominant CDR3 lengths within both CD45RA and CD45RO T-cell subpopulations, most commonly in CD8⁺ CD45RO T cells. Sharp differences in clonal dominance and size distributions were observed when cells were separated into CD45RA or CD45RO subpopulations. These differences were not apparent in unfractionated CD4⁺ or CD8⁺ T cells from HIV-infected subjects. Sequence analysis of predominant CDR3 lengths revealed oligoclonal expansion within individual Vβ families. Analysis of the CDR3 length diversity within CD45RA and CD45RO T cells provides a more accurate measure of disturbances in the TCR repertoire than analysis of unfractionated CD4 and CD8 T cells.     

Decreased oxidative burst activity of monocytes from asymptomatic HIV-infected individuals.

Although changes in function of monocytes from AIDS patients have previously been reported, the functional deficits that could occur in monocytes from asymptomatic HIV-seropositive individuals has not been extensively investigated. The goal of this study was to evaluate the oxidative burst response and cell surface marker expression of monocytes from this group. Both the oxidative burst, as measured by 2',7'-dichlorofluorescin diacetate oxidation, and cell surface markers were evaluated on CD14+ (Leu-M3) monocytes by two-color flow cytometry. A significantly lower oxidative burst capacity was observed in asymptomatic, seropositives after phorbol myristate acetate and calcium ionophore stimulation when compared to seronegative controls. However, differences in oxidative burst between the two groups were not observed after stimulation with heat-aggregated IgG. The oxidative burst of monocytes from seropositive HIV antigen+ or antigen- subjects was not significantly different. The most significant difference in monocyte surface markers between seropositive and seronegative controls was a decrease in the proportion of complement receptor 3+ (CD11b+) cells while slightly decreased numbers of CD13+ and CD33+ monocytes were observed. The decreased expression of CD11b+ cells in seropositives was found entirely within the seropositive, HIV antigen+ group. Similarly, when monocytes from seropositive HIV antigen+ and antigen- were compared, significantly lower numbers of CD4+ and HLA-DR+ cells were noted. These results indicate that significant changes in monocyte function and surface markers occur early during the course of HIV infection and are associated with the expression of serum HIV antigen.