T cell repertoire and clonal deletion of Mtv superantigen-reactive T cells in mice lacking CD4 and CD8 molecules

CD4^?CD8^? double-negative T cells constitute a lymphocyte subpopulation within the thymus and peripheral lymphatic organs that express a unique T cell receptor (TCR) repertoire and do not undergo negative selection. To test whether these cells develop as a distinct lineage or due to altered selection in the absence of CD4 and CD8 expression, we analyzed the TCR repertoire in mice lacking both CD4 and CD8 accessory molecules after homologous recombination (CD4^0/0CD8^0/0). We show that mature T cells of CD4^0/0CD8^0/0 mice express an unbiased diverse TCR Vβ repertoire comparable to wild type mice. In addition, clonal deletion of mouse mammary tumor virus superantigen-reactive T cells did occur in CD4^0/0CD8^0/0 mice. These data show that the intrinsic lack of CD4 and CD8 expression has no effect on the mature TCR repertoire and that clonal deletion of superantigen-reactive cells is independent of CD4 and CD8 co-receptors.     

Influence of antigenic experience on BJ gene segment usage in human CD4+ T cells

HLA molecules influence the selection of naive CD4+ T cells as demonstrated by HLA-DR-dependent differences in BV8-BJ frequencies. The repertoire of mature peripheral T cells utilized in antigen responses is shaped by additional factors such as antigens encountered in the environment. To identify mechanisms underlying the formation of the memory repertoire, differences in the BV8-BJ repertoire of CD45RO- and CD45RO+ CD4+ T cells were examined in 21 normal donors. The naive and memory CD4+ compartments displayed unique BV8-BJ repertoires in all individuals, demonstrating that the recruitment of CD4+ T cells into the memory population is a non-random process. The frequencies of selected BV8-BJ combinations were increased among CD45RO+ T cells. Size fractionation of such expanded BV8-BJ populations demonstrated that most of them were polyclonal in nature. Twenty-five percent of the expanded BV-BJ combinations included a dominant TCR sequence, indicating monoclonal proliferation. Selection of BV8-BJ combinations for preferential use among memory T cells was HLA dependent. HLA-DR1/4+ individuals were characterized by an increased usage of BV8-BJ2S7+ TCR, and decreased usage of BV8-BJ2S1 + and BV8-BJ2S2+ TCR, whereas HLA- DR3/7+ individuals preferentially recruited BV8-BJ2S5+ T cells, and disfavored BV8-BJ2S3+ and BV8-BJ2S7+ T cells. HLA-imposed effects on the naive and memory repertoire were distinct. The BV-BJ frequencies of CD45RO+ T cells could not be predicted from the pattern of TCR found in naive CD4+ T cells, suggesting that the HLA-DR polymorphisms influence thymic selection processes differently than peripheral selection forces.      

TCRalphabeta repertoire diversity of human naturally occurring CD4+CD25+ regulatory T cells

We examined the alphabeta T cell receptor (TCR) repertoire of naturally occurring CD4+CD25+ regulatory T (Treg) cells isolated from healthy human blood. Three-color FACS analysis demonstrated that the usage of variable region segments of TCRbeta chains by CD4+CD25+ cells did not differ from those of CD4+CD25- cells. Complementarity-determining region 3 (CDR3) size distribution analyses demonstrated that the repertoire diversity of CDR3beta was almost identical between CD4+CD25+ and CD4+CD25- T cell subsets, and that there was no skewing of the CDR3beta repertoire of CD4+CD25+ T cells. In contrast, in vitro activated CD4+CD25+ T cells by cytomegalovirus-derived antigens showed a skewed CDR3 size distribution pattern. These findings support the hypothesis that naturally occurring CD4+CD25+ T cell subset in humans is 1argely composed of a T cell lineage positively selected in the thymus as a consequence of the interaction between self-peptides and TCRs and not derived from recent activation by a limited array of antigens.     

CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells

CD4+CD25+ T cells have been shown to inhibit experimentally induced organ-specific autoimmune disease and depletion of these regulatory T cells from normal mice results in development of such conditions. Furthermore, CD4+CD25+ T cells suppress the IL-2 production and thereby the proliferation of polyclonally activated CD4+CD25- T cells in vitro. The suppression in vitro is independent of secreted factors but requires interactions between CD4+CD25- and CD4+CD25+ T cells and antigen-presenting cells (APC). We have now further investigated the function of CD4+CD25+ T cells in vitro and have focused on their interactions with APC. We found that CD4+CD25+ T cells down-regulated the expression of the co-stimulatory molecules CD80 and CD86 on dendritic cells. The steady-state level of CD80 mRNA was also decreased, while the steady-state level of CD86 mRNA was not, suggesting that distinct mechanisms regulate the expression of these molecules. The down-regulation occurred even in the presence of stimuli that would normally increase the expression of CD80 and CD86 molecules. Thus, down-regulation of co-stimulatory molecules may be an additional effector function of these regulatory T cells.     

The CD2 and CD28 adhesion molecules induce long-term autocrine proliferation of CD4+ T cells

In vitro human T lymphocyte activation requires two-signal triggering delivered by lectins, phorbol esters or antibodies directed against surface molecules. Stimulation of adhesion molecules by CD2 and/or CD28 antibodies defines alternative activation pathways. Activation by CD2 + CD28 monoclonal antibodies induces high-level, long-lasting and monocyte-independent proliferation of highly purified T cells. Limiting dilution cultures showed that CD28 in association with CD2 or CD3, without addition of exogenous cytokines, induced single-cell proliferation. CD2 + CD28 stimulation induced long-term interleukin (IL)-2-dependent autocrine proliferation of CD4⁺ T cell clones. We tried to elucidate this long-term proliferation by evaluating cytokine secretion and cytokine dependency. CD28 associated to CD3 or CD2 induced high levels of IL-2, tumor necrosis factor (TNF) and IL-4 secretion for 10 days, in contrast to CD3 alone which induced only TNF secretion. Cytokines of the monocytic lineage were also secreted, such as colony-stimulating factor-1, granulocyte macrophage colony-stimulating factor or IL-1, the latter being more specific of CD2 + CD28 activation. Blocking antibodies confirmed the crucial role of IL-2 in CD2 + CD28 activation. Anti-IL-4, anti-IL-7 receptor or anti-TNF antibodies had no effect on proliferation. Stimulation with CD2 + CD28 induced long-term autocrine (at least for IL-2) proliferation for CD4⁺ T cells, with no evidence for the implication of another cytokine among those tested other than IL-2. This represents a model for long-term autocrine growth for non-leukemic cells.     

Contributions of CD4+, CD8+, and CD4+CD8+ T cells to skewing within the peripheral T cell receptor beta chain repertoire of healthy macaques.

Diversity in the peripheral T cell receptor repertoire of rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) has been studied by examining the profile of CDR3 lengths in TCR beta chains. Expressed CDR3 length distribution profiles for individual TCRBV families were obtained from total peripheral blood mononuclear cells (PBMC) and T cell subsets isolated from PBMC. These studies reveal that the T cell receptor repertoire of PBMC from healthy macaques often exhibits skewing in TCRBV family CDR3 profiles. The skewing of TCRBV family CDR3 profiles was evident as discrete expanded length(s) and was detected in up to 50% of the PBMC profiles. Analyses of separated T cell populations demonstrated that the CD8+ T cell subset was responsible for the majority of observed skewing in CDR3 length profiles. However, CD4+ T cells were also shown to contribute to the skewed peripheral PBMC repertoire in these animals. While certain TCRBV families frequently displayed skewed profiles, there was no concordance in the particular CDR3 lengths expanded among the different animals. Furthermore, an additional feature of the peripheral blood of the animals studied was the presence of an unusual population of extrathymic CD4 and CD8+ (double-positive) T cells (up to 9.6% in the PBMC of rhesus macaques). The double-positive T cells could be differentiated from CD4 single-positive and CD8 single-positive T cells by their increased surface expression of LFA-1 and decreased CD62L expression. The percentage of the double-positive T cells was higher in rhesus than pig-tailed macaques and contributed substantially to the peripheral T cell repertoire.     

Mixed-haplotype MHC class II molecules select functional CD4+ T cells

MHC class II molecules are formed from polymorphic alpha and beta chains. While pairing of chains is most efficient within class II isotypes and haplotypes, limited pairing and surface expression of mixed-haplotype and -isotype class II molecules is common. The function of such molecules in antigen presentation has been established by the unique restriction of responses in F1 mice. However, it has not been established whether mixed class II molecules are able to mediate selection of functional T cells and how the reduced avidity of the TCR/MHC interaction influences the repertoire. In this report we have addressed these issues through the production of mice expressing solely mixed-haplotype class II molecules. The mixed class II molecules promote selection of a small CD4+ T cell repertoire with modified TCR use. The selected CD4+ T cells are functional in vivo and in vitro.     

CD4 and CD8 act as co-receptors during thymic selection of the T cell repertoire

The selection of T cell receptor specificities must logically not only involve the alpha beta-TCR but, also the CD4 and CD8 molecules, as antigen recognition by the alpha beta-TCR on mature T cells is facilitated by the CD4 and CD8 co-receptors. In this review, the studies that provided key advances in our understanding of the possible role of CD4 and CD8 in T cell development will be discussed.     

Neuropilin-1+T细胞与CD4+CD25+调节性T细胞的比较

目的:分析Neuropilin-1 T细胞(Nrp-1 T细胞)与经典CD4 CD25 调节性T细胞(Treg)的关系并比较二者的免疫调节作用。方法:流式细胞术分析BALB/c小鼠脾脏T细胞上Nrp-1与CD4、CD25的表达关系并分选Nrp-1 T细胞及CD4 CD25 Treg,通过B16-F10-luc-G5黑色素肿瘤细胞体外培养实验并利用萤光成像系统,观察比较两种细胞对NK细胞杀伤B16-F10-luc-G5黑色素瘤细胞的影响。结果:CD4 CD25 Treg中表达Nrp-1的比例为(27.28±1.17)%,明显高于CD4 CD25-T细胞的(1.63±0.08)%(P<0.01);在体外实验中,Nrp-1 T细胞与CD4 CD25 Treg均能抑制NK细胞杀伤B16-F10-luc-G5黑色素瘤细胞,Nrp-1 T细胞组的肿瘤细胞数目在6、24、48、72h分别为984±15、1015±14、1261±21、1323±38,高于CD4 CD25 Treg组的931±4、983±8、1201±18、1256±18,两组肿瘤细胞数目在各时间点均有统计学意义(P<0.01)。结论:经典CD4 CD25 Treg中表达Nrp-1的细胞比例较高,Nrp-1 T细胞有负性免疫调节作用,抑制功能比CD4 CD25 Treg更强,可以作为一类新的Treg亚群。     

Molecular cloning of rat interleukin 4 cDNA and analysis of the cytokine repertoire of subsets of CD4+ T cells

Rat peripheral CD4+ T cells may be subdivided into two functionally distinct subpopulations (OX-22highCD4+ and OX-22lowCD4+) on the basis of their reactivity with the monoclonal antibody MRCOX-22, which recognizes a restricted epitope on the leukocyte common antigen (LCA, CD45). Previous studies have demonstrated the increased activity of the OX-22highCD4+ subset in assays of cell-mediated reactivity, whereas the reciprocal OX-22lowCD4+ subset provides the majority of help for B cells in secondary antibody responses. Analyses of in vivo function have subsequently shown that the autoreactive activity associated with the OX-22highCD4+ subset can be inhibited through a suppressor activity within the OX-22lowCD4+ subset, indicating a further immunoregulatory role for these cells. Since the differential production of lymphokines such as interferon-gamma (IFN-gamma) and interleukin 4 (IL4) is believed to regulate alternative effector responses to a particular antigen, we have compared the lymphokine mRNA profiles of activated OX-22highCD4+ and OX-22lowCD4+ subsets using nucleic acid probes specific for rat IL 2, IFN-gamma and IL4, the latter of which has been isolated by a polymerase chain reaction cloning technique and its sequence is described. A higher frequency of cells expressed IL 2 mRNA in the OX-22high subset, in accordance with the relative levels of IL2 protein produced. In contrast, more IFN-gamma mRNA was detected in the OX-22lowCD4+ subset 24 h after mitogenic stimulation although these cells have consistently been shown to produce less IFN-gamma protein than the OX-22highCD4+ subset. This apparent paradox was resolved by the finding that the IFN-gamma mRNA levels in the OX-22lowCD4+ subset declined rapidly after 24 h while the levels continued to rise in the OX-22highCD4+ population such that at 48 h the relative levels were reversed. We have also demonstrated a higher level of IL4 mRNA expression within the OX-22lowCD4+ subset, which is undoubtedly involved in the increased B cell helper activity mediated by this subpopulation and may be responsible, in part, for their active suppression of cell-mediated immune responses.     

Evidence for a repertoire of functional untolerized CD4+ T cells specific for melanoma-associated antigens

Active vaccination against melanoma requires tolerance break as melanoma-associated antigens (MAA) used in vaccine formula are mostly self-antigens. While tolerance to MAA in the CD8(+) T cell compartment is well characterized, it is still not the case for the CD4(+) T cell compartment. Here, we analysed CD4(+) T cell tolerance to such antigens in mice genetically engineered to express ovalbumin (OVA) in melanocytes (Tyr-OVA mice). When we crossed Tyr-OVA mice with DO11.10 and OT-II mice transgenic for an OVA-specific TCR restricted by MHC class II, we observed different tolerization levels. Central tolerance was complete for high avidity DO11.10 CD4(+) T cells, but absent for low avidity OT-II CD4(+) T cells. OT-II CD4(+) T cells also ignored OVA in the periphery of Tyr-OVA mice, albeit being potently reactive to vaccination. OVA challenge in single transgenic Tyr-OVA mice confirmed the existence of OVA-reactive CD4(+) T cells with the induction of efficient T helper cells for antibody production and anti-tumour T cell response. In total, our study demonstrates the existence of low avidity MAA-specific CD4(+) T cells escaping by ignorance central and peripheral tolerance, but valuable in the context of vaccination against melanoma.     

The route of bacterial uptake by macrophages influences the repertoire of epitopes presented to CD4 T cells

We studied MHC class II (MHC-II)-restricted antigen processing of viable Streptococcus pyogenes by murine macrophages for presentation of two CD4 T cell epitopes of the surface M5 protein. We show that presentation of both epitopes was prevented if actin polymerization was inhibited by cytochalasin D, but not if clathrin-dependent receptor-mediated endocytosis was prevented, suggesting uptake of streptococci by phagocytosis or macropinocytosis was required for presentation of the surface M protein. However, treatment of macrophages with amiloride, which selectively blocks membrane ruffling and subsequent macropinocytosis, inhibited the response to one epitope (M5(308-319)), but had no effect on presentation of the other (M5(17-31)). The effect of the inhibitors on uptake of streptococci was analyzed by electron microscopy. Cytochalasin D completely blocked uptake of streptococci, while dimethyl-amiloride only inhibited uptake into spacious compartments. Neither of the inhibitors altered the cell-surface expression of MHC-II and costimulatory molecules analyzed by flow cytometry. The data suggest that distinct epitopes of a protein associated with viable bacteria may be presented optimally following different uptake mechanisms in the same antigen-presenting cells.     

Chronic lymphocytic leukaemia cells drive the global CD4+ T cell repertoire towards a regulatory phenotype and leads to the accumulation of CD4+ forkhead box P3+ T cells

Advanced chronic lymphocytic leukaemia (CLL) is associated with profound immunodeficiency, including changes in T regulatory cells (T(regs)). We determined the pattern of expression of forkhead box P3 (FoxP3), CD25, CD27 and CD127 and showed that the frequency of CD4+ FoxP3+ T cells was increased in CLL patients (12% versus 8% in controls). This increase was seen only in advanced disease, with selective expansion of FoxP3-expressing cells in the CD4+ CD25(low) population, whereas the number of CD4+ CD25(high) FoxP3+ cells was unchanged. CD4+ CD25(low) cells showed reduced expression of CD127 and increased CD27, and this regulatory phenotype was also seen on all CD4 T cells subsets in CLL patients, irrespective of CD25 or FoxP3 expression. Incubation of CD4+ T cells with primary CLL tumours led to a sixfold increase in the expression of FoxP3 in CD4+ CD25- T cells. Patients undergoing treatment with fludarabine demonstrated a transient increase in the percentage of CD4+ FoxP3+ T cells, but this reduced to normal levels post-treatment. This work demonstrates that patients with CLL exhibit a systemic T cell dysregulation leading to the accumulation of CD4+ FoxP3+ T cells. This appears to be driven by interaction with malignant cells, and increased understanding of the mechanisms that are involved could provide novel avenues for treatment.     

CD4 regulatory T cells

There is now compelling evidence that CD4(+) T cells that specialize in the suppression of immune responses play a key role in the control of immune pathology. Recently, there have been a number of reports that have provided information on the generation of CD4(+) regulatory T cells in the thymus and in the periphery. These cells have also been identified in humans, paving the way for analysis of the function of CD4(+) regulatory T cells in immune-mediated disease.     

CML患者CD4+和CD8+T细胞的TCR Vβ基因谱系和克隆性分析

目的：了解慢性粒细胞白血病慢性期（CML-CP）患者外周血CD4^＋和CD8^＋T细胞中TCR Vβ亚家族T细胞的基因表达和克隆性.方法：利用RT-PCR分别扩增19例CML-CP患者外周血单个核细胞（PBMCs）、CD4^＋和CD8^＋细胞TCR Vβ亚家族基因的CDR3,阳性产物进一步经基因扫描分析其克隆性.结果：CML患者外周血CD4^＋和CD8^＋细胞分别表达部分（1～21个）Vβ亚家族,均以Vβ13的表达率最高,其次为Vβ9,多数患者的一些Vβ亚家族T细胞呈克隆性增殖,主要出现于CD8^＋细胞中,分别以Vβ21（CD4^＋）和Vβ11（CD8^＋）亚家族的克隆性增殖多见.结论：CML患者外周血CD4^＋和CD8^＋细胞的TCR Vβ谱系均存在限制性分布,患者存在的克隆性增殖CD4^＋和CD8^＋T细胞可能与宿主抗CML抗原反应有关,它们可能在抗CML效应中起主要作用.     

Therapeutic potential of self-antigen-specific CD4+ CD25+ regulatory T cells selected in vitro from a polyclonal repertoire

CD4+ CD25+ regulatory T cells (Treg) play a major role in the prevention of autoimmune diseases. Converging evidence indicates that Treg specific for self-antigens expressed by target tissues have a greater therapeutic potential than polyclonal Treg. Therefore, the selective expansion of rare self-antigen-specific T(reg) naturally present in a polyclonal repertoire of Treg is of major importance. In this work, we investigated the potential of different dendritic cell (DC) subsets to expand antigen-specific Treg in mice. The in vitro selective expansion of rare islet-specific Treg from polyclonal Treg could only be achieved efficiently by stimulation with CD8+ splenic DC presenting islet antigens. These islet-specific Treg exerted potent bystander suppression on diabetogenic T cells and prevented type 1 diabetes. This approach opens new perspectives for cell therapy of autoimmune diseases.     

Differences in the ligand specificity between CD1d-restricted T cells with limited and diverse T-cell receptor repertoire

The natural killer (NK) T-lymphocyte population consists of two subsets utilizing a diverse and restricted T-cell receptor (TCR) repertoire, respectively. Both populations have been shown to include autoreactive cells. NKT cells carrying restricted Valpha14(AV14S1)Jalpha281/Vbeta8.2(BV8S2A1 ) TCR have been shown to recognize alpha-galactosylceramide (alphaGalCer) presented in the context of murine CD1d. In this study we screened a set of murine CD1d-autoreactive T-cell hybridomas with diverse TCR for their reactivity with several glycosylated variants of ceramide, including alphaGalCer. These hybridomas showed a different pattern of reactivity to CD1d-expressing antigen-presenting cells (APC) and were not reactive with any of the tested variants of ceramide. A second set of hybridomas had been selected for expression of Valpha14 and Vbeta8.2 TCR chains. These cells responded to alphaGalCer presented on CD1d, but were only weakly reactive to syngeneic splenocytes or CD1d-transfected cells. Their fine specificity in the response to glycosylation variants of ceramide demonstrated a homogenous reactivity pattern, including reactivity to alpha-galactosylsphingosine, the variant of alphaGalCer with truncated fatty acyl chain. These findings underline the differences in ligand specificity between the two subsets of CD1d-restricted NKT cells, and demonstrate a similarity in reactivity among the hybridomas using the Valpha14-Jalpha281/Vbeta8.2 TCR.     

Divergent telomerase and CD28 expression patterns in human CD4 and CD8 T cells following repeated encounters with the same antigenic stimulus

Induction of telomerase, the enzyme that extends telomeres, accompanies human T lymphocyte activation. Nevertheless, high proportions of memory T cells with shortened telomeres are present in vivo during HIV infection and aging. To elucidate the long-term telomerase dynamics in human T cells, longitudinal analyses were performed on T cells subjected to repeated encounters with an allogeneic cell line in long-term culture. Whereas CD4(+) and CD8(+) T cells showed similarly dramatic increases in telomerase activity following primary stimulation, by the fourth stimulation, telomerase activity was nearly undetectable in the CD8(+) subset, but remained high in the CD4(+) subset. In addition, we document the dependence of antigen-specific telomerase inducibility on CD28 and that the decline in telomerase activity parallels the loss of CD28 expression. These findings suggest stringent telomerase regulation in human T cells, a property that may ultimately contribute to telomere shortening, finite replicative potential, and loss of control over certain pathogens.