An octapeptide analogue of HIV gp120 modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes.

Several lines of evidence support an important role for activated T lymphocytes in the perpetuation of autoimmune intraocular inflammatory disease (posterior uveitis). In this study peripheral blood lymphocytes (PBL) were examined by three-colour flow cytometry to assess the distribution of IL-2 receptors (IL-2R) among CD4+ and CD8+ T cell subsets in patients with active posterior uveitis and control subjects. Patients with uveitis (n = 70) showed a significant increase in PBL expressing the alpha-chain (Tac) of the IL-2R compared with controls (n = 28) (34.2% versus 29.6%) (P < 0.05). This increased Tac expression was present on both the CD4+ subset (25.7% versus 20.9%) (P < 0.05) and the CD8+ subset (2.5% versus 1.8%) (P < 0.05) of lymphocytes. We also examined whether the activated CD4+ PBL from uveitis patients (n = 30) showed a dominant pattern of T cell receptor (TCR) gene rearrangement, suggestive of an oligoclonal response to a small number of antigenic peptides. A significant increase in the usage of the V alpha 2.3 TCR family by activated but not by non-activated CD4+ PBL was detected in patients (3.9% versus 3.4%) (P < 0.05) compared with controls. There was evidence of oligoclonal activation of CD4+ PBL in 11/30 patients (36.7%) but in none of the controls (n = 10). However, different V alpha or V beta TCR families were selectively activated among and even within individual patients. The heterogeneity in TCR expression among patients with active intraocular inflammatory disease is discussed.     

An Uneven Expression of T Cell Receptor V Genes in the Arterial Wall and Peripheral Blood in Giant Cell Arteritis

The aim of the study was to investigate T cell receptor (TCR) usage at the time of diagnosis of giant cell arteritis (GCA) and to estimate the degree of clonality of T-cells infiltrating the lesion. Seven patients with biopsy-proven giant cell arteritis were included in the study. Immunocytochemistry in biopsies from the temporal arteries and flow cytometric analysis of peripheral blood lymphocytes (PBL) was performed using monoclonal antibodies specific for CD3, CD4 and CD8 and 13 TCR Vα and Vβ gene segment products. The CDR3 fragment length polymorphism was assessed by gel electrophoresis of PCR-amplified TCR segments. The T lymphocytes were found to be concentrated to the adventitia rather than the media or intima. Six of the seven patients with GCA had expansions of T lymphocytes, expressing selected TCR V genes in the arterial wall. None of these expansions was found in PBL. The infiltrating T-cells were poly- or oligoclonal. In conclusion, the dominating part of the inflammatory infiltrate in GCA emanates from the adventitial microvessels. There is an uneven expression of TCR V genes by T lymphocytes in the inflammatory infiltrates as compared to peripheral blood T lymphocytes at the time of diagnosis, consistent with an antigen-driven immunological reaction in the arterial wall.     

T cells in the lung of patients with hypersensitivity pneumonitis accumulate in a clonal manner

Hypersensitivity pneumonitis (HP) is characterized by an alveolitis sustained by CD8(+) T lymphocytes showing a limited expression of the T cell receptor (TCR). We previously demonstrated that a bias in T cell selection occurs in the lower respiratory tract of patients with HP, with a compartmentalization in the lung of CD8(+) T cells bearing (TCR)-beta variable (TCRBV) #2, 3, 5, 6, 8, and 13 gene segments. We herein characterized the clonal T cell populations present in the lung and in the blood of patients with HP. Heteroduplex analyses, cloning, and sequencing T cells bearing TCR indicate oligoclonal expansions of T cells expressing homologous or identical complementary-determining region 3. Furthermore, T cell clones isolated from the two compartments expressed similar, sometimes identical, junctional regions. Removal from antigenic exposure led to the disappearance of T cell clones. Our findings indicate that expansions of T lymphocytes bearing clonal TCRBV region gene segments take place in the lung of patients with HP during exposure. The evidence that identical T cell clones are present in the lung and the blood of the same patient suggests that the immune reaction occurring at lung level gives rise to a systemic reaction.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

Immune activation in the context of HIV infection

Our objective was to study whether CD4⁺ or CD8⁺ T cells expressing particular T cell receptors (TCR) would accumulate in the lungs of patients with allergic asthma following allergen exposure. We thus analysed the TCR Vα and Vβ gene usage of CD4⁺ and CD8⁺lung and peripheral blood lymphocytes (PBL) of eight patients with allergic asthma before and 4 days after inhalation challenge with the relevant allergen. Lung cells obtained by bronchoalveolar lavage (BAL) and paired PBL samples were analysed by flow cytometry using a panel of anti-TCR V-specific monoclonal antibodies that encompass ≈ 50% of the T cell repertoire. Lung-limited T cell expansions were recorded in both the CD4⁺and the CD8⁺subsets. In BAL CD8⁺, out of a total of 126 analyses, the number of T cell expansions increased from two to 11 after challenge, some of them dramatic. In BAL CD4⁺the frequency of expansions was moderately increased already before challenge, but remained unchanged. A few expansions that tended to persist were noted in PBL CD8⁺. When analysing the overall change in TCR V gene usage the largest changes were also recorded in the BAL CD8⁺subset. Specific interactions between T cells and antigens may lead to an increased frequency of T cells using selected TCR V gene segments. In this study we demonstrate that following allergen bronchoprovocation in allergic asthmatic subjects, T cell expansions preferentially emerge in the lung CD8⁺T cell subset.     

Microbial colonization induces oligoclonal expansions of intraepithelial CD8 T cells in the gut

Two populations of CD8(+) IEL generally express restricted, but apparently random and non-overlapping TCR repertoires. Previous studies in mice suggested that this could be explained by a dual origin of CD8(+) IEL, i.e. that CD8alphabeta(+) IEL derive from a few peripheral CD8(+) T cell lymphoblasts stimulated by microbial antigens in gut-associated lymphoid tissue, whereas CD8alphaalpha(+) IEL descend from an inefficient intestinal maturation pathway. We show here that the gut mucosa, instead, becomes seeded with surprisingly broad and generally non-overlapping CD8 IEL repertoires and that oligoclonality is induced locally after microbial colonization. In germ-free (GF) rats, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed surprisingly diverse TCR Vbeta repertoires, although beta-chain diversity tended to be somewhat restricted in the CD8alphaalpha(+) subset. CDR3 length displays in individual Vbeta-Cbeta and Vbeta-Jbeta combinations generally revealed polyclonal distributions over 6-11 different lengths, similar to CD8(+) lymph node T cells, and CDR3beta sequencing provided further documentation of repertoire diversity. By contrast, in ex-GF rats colonized with normal commensal microflora, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed oligoclonal CDR3 length distributions for most of the Vbeta genes analyzed. Our data suggest that microbial colonization induces apparently random clonal expansions of CD8alphabeta(+) and CD8alphaalpha(+) IEL locally in the gut.     

无症状HBV携带者外周血CD4~+TCR Vβ基因亚家族克隆化特征的研究

分析无症状HBV携带者(AsC)CD4+TCR Vβ基因家族克隆化特征。采用逆转录-聚合酶链反应(RT-PCR)扩增7例AsC外周血CD4+TCR Vβ基因22个亚家族的CDR3区,基因扫描技术对CD4+TCR Vβ亚家族的克隆化进行鉴定。结果基因扫描显示,7例AsC CD4+TCR Vβ基因亚家族均出现一个或一个以上单克隆或寡克隆增生;7例健康者CD4+TCR Vβ基因亚家族均呈正态分布。提示,AsC外周血CD4+TCR Vβ亚家族存在克隆性增生,这可能与AsC免疫耐受的形成有关。     

Limited heterogeneity of biased T-cell receptor V beta gene usage in lung but not blood T cells in active pulmonary sarcoidosis.

Sarcoidosis is a multisystem disorder characterized by non-caseating granulomas and the accumulation of CD4+ T cells in involved tissues such as the lung. To evaluate the diversity of the CD4+ T-cell repertoire in this disorder, a detailed clonal analysis was performed in five individuals with active sarcoidosis who demonstrated preferential accumulation of T cells expressing the T-cell receptor variable gene family V beta 8 in either the lung or blood. In three individuals, analysis of unselected samples of nucleotide sequences derived from V beta 8+ lung T cells demonstrated degrees of clonality ranging from 11% to 46%, indicating the expansion of limited numbers of V beta 8+ T-cell clones in the lung. Analysis of the corresponding deduced amino acid sequences demonstrated common VDJ junctional amino acid residues in the dominant V beta 8+ T-cell clones derived from two oligoclonal V beta 8+ lung T-cell populations, consistent with an antigen-specific T-cell response. In contrast, analysis of V beta 8+ CD4+ T cells from the blood of an individual with a marked bias for peripheral blood V beta 8+ T cells demonstrated no evidence of oligoclonality, suggesting that the stimulus for circulating biased V beta-specific T cells in sarcoidosis may derive from a different, perhaps superantigenic, origin. Clinical improvement in the disease either in response to treatment with corticosteroids or as a result of spontaneous resolution was associated with a decrease in the proportion of V beta 8-specific T cells in the biased lung and/or blood T-cell compartments. Together, these observations are consistent with a role for this T-cell subset in the clinical manifestations of active granulomatous disease.     

Monoclonality and oligoclonality of T cell receptor beta gene in angioimmunoblastic T cell lymphoma

Angioimmunoblastic T cell lymphoma (AILT) is an aggressive T cell lymphoma with an incidence of approximately 1-2% of all non-Hodgkin lymphoma. The detection of clonal T cell receptor (TCR) gene rearrangements helps in the diagnosis of T cell malignancies such as AILT, where morphological and immunohistological investigations are not always sufficient to reach a definitive diagnosis. TCR beta (TCRB) and TCR gamma (TCRG) gene rearrangements were analysed from 17 WHO-defined cases of AILT by PCR for the presence of TCR clonality. TCRB gene rearrangements were sequenced to identify molecular signature(s) common among this patient group. Monoclonal and oligoclonal TCRB and TCRG gene rearrangements were detected in all cases. BV17S1 was slightly over-represented compared to the use of other Vbeta gene segments; however, no preferential usage of J gene segment(s) was detected. The results of this study emphasise that TCR clonality and oligoclonality is a diagnostic feature of AILT and that BV17S1 is over-represented with no other common molecular findings.     

CD4(+)CD8(+)TCR(low) thymocytes express low levels of glucocorticoid receptors while being sensitive to glucocorticoid-induced apoptosis

While signaling by either the TCR or glucocorticoid receptor (GR) can induce apoptosis in thymocytes, recent studies have shown that combining these signals results in survival of CD4(+)CD8(+) thymocytes. Although glucocorticoids (GC) in this way may directly affect T cell selection, no data are available addressing GR expression in thymocyte subsets and in individual cells within subsets. We studied GR expression by combining immunofluorescence cell surface staining for CD4, CD8 and TCR with intracellular staining of GR in four-color cytometry. Significant differences of GR expression were observed in various thymocyte subsets, although a homogeneous distribution of GR expression in individual thymocyte subsets emerged. The highest GR expression was found in CD4(-)CD8(-)TCR(-) thymocytes, and decreased during development via the CD4(-)CD8(+)TCR(-) subpopulation into the CD4(+)CD8(+)TCR(low) subset. Interestingly, the latter population, although expressing less than half the GR density of CD4(-)CD8(-)TCR(-) cells, is the most sensitive subset to GC-induced apoptosis. Up-regulation of TCR expression by the CD4(+)CD8(+)TCR(low) subset to CD4(+)CD8(+)TCR(high) cells was accompanied by a parallel increase in GR expression. The latter finding and the presence of a homogeneous distribution of GR in each thymocyte subset provides an experimental basis for the concept that GR can antagonize TCR-mediated signals at a constant rate relative to TCR expression.     

In Vitro Expansion of T-Cell-Receptor Vα2.3+ CD4+ T Lymphocytes in HLA-DR17(3), DQ2+ Individuals upon Stimulation with Mycobacterium tuberculosis

The T-cell receptor (TCR) Valpha/beta gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood of Mycobacterium bovis BCG-vaccinated healthy blood donors were stimulated in vitro with live or killed M. tuberculosis or with a soluble extract thereof. TCR Valpha/beta repertoire analysis of reactive CD4(+) and CD8(+) T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of Valpha2.3(+) CD4(+) T cells upon stimulation with live M. tuberculosis or its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded Valpha2.3(+) T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2(+) individuals tested. In addition, Valpha/Vbeta repertoire analysis of T lymphocytes from a DR17(3), DQ2(+) donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of Valpha2.3(+) CD4(+) T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation of M. tuberculosis soluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting Valpha2.3(+) CD4(+) T-cell expansion.     

In vitro and in vivo T cell oligoclonality following chronic stimulation with staphylococcal superantigens

Microbial superantigens (SAg), including SEB and TSST-1, polyclonally activate T cells belonging to specific TCR BV families. A pathogenic role for SAg in various human diseases has been suggested, but enthusiasm for this view has been tempered by the T cell oligoclonality in these disorders. To assess whether T cell oligoclonality can emerge following protracted SAg stimulation, human PBMC were stimulated with SEB, TSST-1, or anti-CD3 mAb and maintained in culture with exogenous IL-2. Oligoclonality was appreciated by day 14 among CD4(+) and CD8(+) T cells. In addition, mice transgenic for human DR2 and DQ8 were injected weekly with SEB, and splenic CD4(+) and CD8(+) T cells were analyzed for oligoclonality. In mice that received one or three such injections, little-to-no oligoclonality was detected. In contrast, considerable oligoclonality was detected in mice that received eight weekly SEB injections. Many of these T cell oligoclones were identical to "spontaneously" arising oligoclones detected in SEB-naive mice. Thus, T cell oligoclonality can emerge following chronic SAg stimulation. In hosts who have lost tolerance to self Ag, chronic exposure to SAg may preferentially promote expansion of autoreactive T cells and facilitate development of clinical disease.     

HgCl(2)-induced human lymphocyte activation in vitro: a superantigenic mechanism?

Mercuric chloride (HgCl(2)) has been proposed to be a mitogen for human blood lymphocytes in vitro. In our previous study, we demonstrated that HgCl(2) preferentially stimulates the CD4+ T cell subset to blast transformation and DNA synthesis and that the reaction is dependent on CD14+ accessory cells. In order to characterise the responding cells further and to elucidate the mechanism of T cell activation, the T cell receptor (TCR) Vbeta chain expression of the blast-transformed cells was analysed by monoclonal antibodies and flow cytometry. The 22 TCR-Vbeta-specific antibodies used were found to react with 55-80% of the naive CD4+ and CD8+ blood T cells from the different donors. Six to 18% of the lymphocytes, mainly CD4+ T cells, were blast transformed after addition of HgCl(2). The distribution of the lymphoblasts carrying certain TCR Vbeta chains were skewed, and 15-40% expressed the TCR Vbeta2 chain. Furthermore, if cells were pretreated for 5 days with HgCl(2), whereafter recombinant interleukin-2 in fresh medium was added, the TCR Vbeta7+ T cell subset was also stimulated to blast transformation. The superantigen staphyloccal enterotoxin B, as a control, induced blast transformation in 10-26% of the lymphocytes, mainly CD4+ T cells, which were, as expected, positive for Vbeta3, Vbeta12, Vbeta14 or Vbeta17. We conclude that HgCl(2) has characteristics of a superantigen, activating the human lymphocytes in a Vbeta-chain-selective manner in vitro.     

Oligoclonal CD8+ T-Cell Expansion in Patients with Chronic Hepatitis C Is Associated with Liver Pathology and Poor Response to Interferon-α Therapy

The role of CD8(+) T lymphocytes in chronic hepatitis C virus (HCV) infection and in liver injury with subsequent development of fibrosis and cirrhosis is poorly understood. To address this question, we performed a follow-up study including 27 chronically HCV-infected individuals. We determined clonality and phenotypes of circulating CD8(+) T cells employing TCRBV spectratyping. Antigen specificity was tested by rMHC-peptide tetramer staining and stimulation with recombinant HCV antigens. In addition, T-cell clonality and phenotypes were followed during the variable clinical response of interferon- (IFN) alpha treatment. We could demonstrate that CD8(+) T-cell expansions were significantly associated with liver fibrosis and cirrhosis. Likewise, increased oligoclonality of circulating CD8(+) T cells in chronic HCV infection was identified as an indicator for poor clinical response to IFN-alpha therapy. Moreover, we also found that IFN-alpha therapy enhanced the differentiation of CD8(+) T cells towards a late differentiation phenotype (CD28(-) CD57(+)). In cases of virus elimination the disappearance of expanded terminally differentiated CD8(+) cells was observed. Thus, this study identifies an association of clonal expansions of circulating CD8(+) T cells with liver pathology and provides a possible explanation for the fact that response to IFN-alpha therapy diminishes with the duration of infection.     

Immunization of volunteers with Salmonella enterica serovar Typhi strain Ty21a elicits the oligoclonal expansion of CD8+ T cells with predominant Vbeta repertoires

CD8(+) T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-gamma) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8(+) T cells in humans. To this end, we determined the TCR beta chain (Vbeta) usage of CD8(+) T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vbeta usage varied among volunteers, we identified oligoclonal Vbeta subset expansions in individual volunteers (Vbeta 2, 5.1, 8, 17, and 22 in volunteer 1; Vbeta 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vbeta 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-gamma secretion assays on Vbeta sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7(-) CD27(-) CD45RO(+) CD62L(-)) and coexpressed gut homing molecules (e.g., high levels of integrin alpha4beta7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vbeta families. Moreover, these serovar Typhi-specific CD8(+) T cells bearing defined Vbeta specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.     

The expression of Bcl-2 family proteins (Bcl-2, Bcl-x, Bax, Bak and Bim) in human lymphocytes

Bcl-2 family proteins regulate programmed cell death, and may play an important role in the selection of lymphocytes. We investigated the expression of Bcl-2, Bcl-x, Bax, Bak and Bim in human lymphocytes using flow-cytometry. Bcl-2 was down-regulated in CD4(+)8(+) (DP) thymocytes and CD19(+)38(+) tonsillar lymphocytes (GC B cells). Among DP thymocytes, cells co-expressing CD69 up-regulated Bcl-2, suggesting that the role of Bcl-2 is promoting survival of positively selected DP cells. Unexpectedly, the expression level of Bcl-x was higher in DP cells than in Single Positive (SP) cells and in CD69(+) DP thymocytes it was lower than in CD69(+) DP thymocytes. Expression of Bim was low in DP thymocytes but high in a subset of GC B cells. Bim and Bax were expressed more highly in SP than in DP thymocytes. Among peripheral blood lymphocytes (PBL), CD8(+) T cells expressed an approximately ten-fold higher level of Bcl-x than CD4(+) T cells while both subsets expressed similar levels of Bcl-2. Bak expression was low and Bim expression was absent in PBL. These results suggest that not only Bcl-2 but other members of the Bcl-2 family are involved in T cell development in the thymus and affinity maturation of B cells in the germinal center.     

An analysis of alpha/beta TCR V gene expression in the human thymus.

We have previously analysed alpha/beta T cell receptor (TCR) V gene usage in CD4+ and CD8+ subpopulations from human peripheral blood lymphocytes (PBL) and umbilical cord blood, and described a biased usage of some of the TCR V beta genes towards the CD4+ subpopulation. In this report, the TCR V gene usage in single positive (SP) CD4+ or CD8+ human thymocytes was analysed. Three previously described mAb with a biased usage in PBL and umbilical cord blood also had a skewed reactivity towards the CD4+ subpopulation in SP human thymocytes. Thus, in all 12 cases V beta 5.1 and V beta 6.7, and in 11/12 cases V beta 12 were preferentially used in the CD4+CD8-, compared to the CD4-CD8+ thymic subpopulation. Altogether, these results suggest a selection process in the thymus, supposedly through the positive influence of MHC class II determinants, to be responsible for this non-random, skewed TCR V gene usage.