Are primed CD4+ T lymphocytes different from unprimed cells?

Primed and unprimed lymphocytes are usually classified as separate subsets of cells, based on phenotypic and functional distinctions. In the case of CD4⁺ T lymphocytes, primed cells are thought to proliferate more vigorously, quickly and easily, and to release a different profile of cytokines, than their naive equivalent. However, most of these data were obtained from studies in which populations of lymphocytes were compared before and after antigenic stimulation, and therefore did not distinguish between the effects resulting from the clonal expansion of specific precursor cells within such populations and those due to cell differentiation per se. We have investigated the contribution of precursor cell frequency to some of the functional changes observed in populations of CD4⁺ T cells following antigenic stimulation, using approaches in which antigen-specific precursor frequencies are high in both primary and secondary stimulations: mixed leukocyte reaction responses and cells from αβ T cell receptor transgenic mice. Our data suggest that when equivalent numbers of antigen-specific naive and previously primed CD4⁺ responder T cells are compared, there is no difference in their potency to proliferate but only the previously activated subset can generate cytokines such as interferon-γ.     

Characterization of normal human CD3+ CD5- and gamma delta T cell receptor positive T lymphocytes.

The functional and phenotypic properties of normal human CD3+CD5- T cells which have a higher frequency of cytotoxic cells than CD3+CD5+ T lymphocytes have been described. Using three- and four-colour immunofluorescence flow cytometric cell sorting, the CD3+CD5- and CD3+CD5+ populations were subdivided into alpha beta or gamma delta T cell receptor positive cells. The four subsets were examined for the in vitro cytotoxic activity and were also stimulated with mitogens in limiting-dilution assays to measure the frequencies of proliferating and interleukin-2 (IL-2) producing cells. CD3+CD5- alpha beta +, CD3+CD5- gamma delta + and CD3+CD5+ gamma delta + cells had lower frequencies of proliferating and IL-2-producing cells than did CD3+CD5+ alpha beta + cells. However, the cytotoxic activity of the different phenotypes was higher in the CD3+CD5- subsets, especially when these cells were gamma delta +. Expression of gamma delta or lack of expression of CD5 appeared to be associated with the acquisition of cytolytic potentials. CD8 was expressed on 20% of fresh CD3+ gamma delta + cells. Cultured gamma delta + cells retained the expression of gamma delta, but quickly lost that of CD8 and with time modulated the expression of CD5. The expression of CD5 was found to be higher on sorted CD3+CD5+ gamma delta - than on CD3+CD5+ gamma delta + cells. These observations indicate that gamma delta is preferentially expressed on CD5-negative or weakly positive T lymphocytes and that CD3+CD5- gamma delta + cells appear to constitute a discrete small subset of mature T lymphocytes which are cytotoxic in nature. However, the exact immunological function of these cells and their place in T cell ontogeny are yet to be elucidated.     

The HIV-1 Nef protein has a dual role in T cell receptor signaling in infected CD4+ T lymphocytes

The phenotypic changes that are induced by immune activation in CD4⁺ T lymphocytes provide an optimal environment for efficient HIV-1 replication in these cells. The pathogenic Nef protein of HIV-1 modulates the T cell receptor (TCR) signaling, but whether this has a positive or negative effect on cellular activation is a matter of debate. Here we have investigated the response to TCR stimulation of primary CD4⁺ T lymphocytes infected with wt or Nef-deficient HIV-1. Results show that, in freshly isolated quiescent T cells, Nef superinduces NFAT and IL-2 production bypassing early TCR effector molecules. Conversely, the early phosphorylation of PLC-γ1, the induction of NFAT, and the expression of IL-2 are impaired by Nef in sub-optimally activated/resting T cells. Our data indicate that Nef has a dual role in the modulation of TCR signaling aimed at favoring HIV-1 replication and spread in both quiescent and metabolically active CD4⁺ T lymphocytes.     

Circulating CD3+ CD4+ CD+ T lymphocytes in multiple sclerosis

Triple-antibody flow cytometry was used to search for distinctive populations of peripheral blood lymphocyte immunophenotypes in multiple sclerosis (MS). Using monoclonal antibodies to the cell surface markers CD3, CD4, and CD8, T cell subsets were quantified on a cohort of 31 MS patients (not treated with corticosteroids for at least 6 months), 30 healthy donors, and 14 patients with other autoimmune diseases (also corticosteroid treatment-free for at least 6 months). Untreated MS patients displayed a significantly greater population of CD3+CD4+CD8+ circulating T cells than healthy donors (P = 0.023). Patients with other autoimmune diseases displayed mean populations of CD3+CD4+CD8+ cells greater than normal donors and less than MS, but not significantly different from either. An additional 45 MS patients who had received corticosteroid therapy within the previous 6 months were phenotyped. Treatment of symptomatic MS with corticosteroids was associated with a smaller population of circulating CD3+CD4+CD8+ cells. Some MS patients have significantly greater numbers of peripheral blood T lymphocytes simultaneously expressing CD3, CD4, and CD8 surface markers than healthy donors and this population of cells may be reduced by corticosteroids treatment. This triple positive phenotype may be a manifestation of a systemic immune abnormality in MS.     

小鼠CD4~+ CD25~+ Foxp3~+调节性T细胞体外扩增

目的:本研究旨在探讨CD4+CD25+Foxp3+调节性T细胞体外扩增的方法。方法:采用磁珠分选小鼠CD4+T细胞,αCD3单克隆抗体包被24孔板,加入αCD28单克隆抗体、雷帕霉素、rhIL-2,培养3周后,流式细胞仪测定培养细胞中CD4+CD25+T细胞的含量,实时定量PCR检测CD4+CD25+T细胞Foxp3 mRNA的表达;单向混合淋巴细胞反应和增殖抑制试验测定扩增的CD4+CD25+T细胞的增殖及其抑制功能;ELISA检测培养上清中IL-10和TGF-β1的含量。结果:小鼠CD4+T细胞培养3周后,CD4+CD25+T细胞达(76.05±2.73)%,高于未加雷帕霉素组(52.17±1.36)%(P<0.001),磁珠分选的CD4+CD25+T细胞Foxp3 mRNA的表达是未加雷帕霉素组的5倍(P<0.001),增殖能力是未加雷帕霉素组的0.29倍(P<0.001),对CD4+T细胞增殖抑制能力是未加雷帕霉素组的3.6倍(P<0.001),培养上清中IL-10和TGF-β1分别是对照组的1.8倍和1.6倍(P<0.001)。结论:小鼠CD4+T细胞在含有1μg/ml的αCD28、rhIL-2 100 U/ml和终浓度为10 nmol/L雷帕霉素的培养体系中培养3周后能有效扩增CD4+CD25+Foxp3+调节性T细胞。     

CD4-dependent and -independent association of protein tyrosine kinases to the T cell receptor/CD3 complex of CD4+ mouse T lymphocytes

Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3-TcR complex in stimulated or unstimulated cells, including p56^lck, p59^fyn and ZAP-70. We have observed, in one mouse T helper CD4 T cell line, that most TcR- or CD3-associated tyrosine kinase activity comes from CD4:p56^lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolées, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4⁺ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4⁺ T lymphocytes. Our results show that, in normal CD4⁺ T lymphocytes, as in CD4⁺ T cell lines, there is a stable and readily detectable association between CD4: p56^lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4⁺ lymphocytes have detectable amounts of p56^lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59^fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56^fyn in a TcR/CD3-independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4⁺ T cells and CD4⁺ T cell lines in kinase association to the TcR/CD3 complex.     

CD4+ T helper 2 cells--microbial triggers, differentiation requirements and effector functions

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate‐like cells with the capacity to secrete large amounts of interleukin‐5 (IL‐5), IL‐13 and IL‐9 as well as IL‐4‐producing and antigen‐processing basophils have (re)‐emerged onto the type 2 scene. To what extent these new players influence αβ⁺ CD4⁺ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T‐cell receptor ligation, co‐stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned.     

Amphiregulin promotes the immunosuppressive activity of intrahepatic CD4+ regulatory T cells to impair CD8+ T-cell immunity against hepatitis B virus infection

Hepatitis B virus (HBV) infection causes liver diseases and hepatocellular carcinoma. Immunotolerance in HBV-infected patients is one of the factors that incur failure of HBV clearance and persistent HBV amplification. However, the mechanisms underlying immunotolerance after HBV infection are yet to be thoroughly understood. Using a novel HBV mouse model, we found for the first time that epidermal growth factor receptor (EGFR) is up-regulated on intrahepatic regulatory T (Treg) cells in HBV-infected mouse livers. The EGFR-positive Treg cells are more immunosuppressive than EGFR-negative Treg cells, demonstrated by higher expression of immunosuppressive cytokines and robust inhibition of CD8⁺ T-cell proliferation in vitro. Furthermore, EGFR-positive Treg cells potently restrain CD8⁺ T-cell-mediated anti-viral activity, leading to higher HBV burden in hepatocytes. Amphiregulin, a cytokine of the EGF family, is significantly up-regulated in HBV-infected livers, but the cellular sources of amphiregulin are still elusive. Amphiregulin promotes the immunosuppressive activity of EGFR-positive Treg cells in vitro, so as to profoundly inhibit production of anti-viral components in CD8⁺ T cells. Taken together, our discovery elucidated a novel mechanism contributing to immunotolerance and viral amplification after HBV infection. Our study may provide new clues for developing therapeutic strategies against HBV infection.     

B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells

To characterize better the co-stimulatory activity of native B7-1 in the absence of other receptor/ligand interactions that might contribute to the response, B7-1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7-1 with anti-T cell receptor (TCR) mAb on cell-sized latex microspheres provided an effective stimulus for activation of both CD4⁺ and CD8⁺ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4⁺ T cell response was prolonged and resulted in efficient interleukin-2 production and clonal expansion. In contrast, CD8⁺ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7-1 mediated co-stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7-1 co-stimulation is not sufficient to sustain helper-independent CD8⁺ CTL responses. When the dose responses of CD4⁺ and CD8⁺ T cells to B7-1 were compared, CD8⁺ T cells were found to require higher densities of B7-1 to attain an equivalent level of activation, suggesting that the level of expression of B7-1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7-1 transfectants, purified B7-1 did not provide co-stimulation when presented on a surface separate from the TCR stimulus.     

Decreased 4-1BB expression on CD4+CD25 high regulatory T cells in peripheral blood of patients with multiple sclerosis

As a tumour necrosis factor receptor superfamily member, 4-1BB (CD137) is preferentially expressed in CD4+CD25+ regulatory T cells (Tregs) and has been suggested to play an important role in regulating the generation or function of Tregs. Recent studies of human Tregs have shown that blood CD4+CD25(high) T cells were much closer to Tregs in terms of their functionality. Furthermore, CD4+CD25(high) Tregs have been found to have a decreased effector function in patients with multiple sclerosis (MS). In this study, we examined the expression of 4-1BB and soluble 4-1BB (s4-1BB) protein levels in the peripheral blood of MS patients. Compared with healthy controls, MS patients had decreased 4-1BB expression in their CD4+C25(high) Tregs and increased plasma s4-1BB protein levels. Moreover, the plasma s4-1BB levels of MS patients were shown to be inversely correlated with the 4-1BB surface expression of CD4+CD25(high) Tregs. The down-regulated 4-1BB expression on CD4+CD25(high) Tregs of MS patients may be involved in the impaired immunoactivity of these Tregs. The elevated s4-1BB levels may, at least in part, function as a self-regulatory attempt to inhibit antigen-driven proliferation of Tregs or their immunosuppressive activity.     

Distinct involvement of CD45 in antigen receptor signalling in CD4+ and CD8+ primary T cells

We demonstrate that pretreatment of primary CD4⁺, but not CD8⁺ T cells with anti-CD45 inhibits activation signals induced through the T cell receptor for antigen (TCRαβ). Specifically, anti-TCRαβ-mediated tyrosine phosphorylation of phospholipase C-γ1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca²⁺ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti-CD45 in CD8⁺ T cells. Perturbation of TCRαβ signalling in CD4⁺ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti-CD45 ablates TCRαβ signalling, anti-CD3?-mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCRαβ to second messenger-generating pathways is under distinct physical and/or functional constraints in primary CD4⁺ and CD8⁺ T cells.     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

Increased hepatitis C virus (HCV)-specific CD4+CD25+ regulatory T lymphocytes and reduced HCV-specific CD4+ T cell response in HCV-infected patients with normal versus abnormal alanine aminotransferase levels

CD4+CD25+ T regulatory cells may play a role in the different clinical presentations of chronic hepatitis C virus (HCV) infection by suppressing CD4+ T cell responses. Peripheral CD4+CD25+ T cells from chronic HCV carriers with normal and abnormal alanine aminotransferase (ALT) were analysed for specificity and effect on HCV-specific CD4+ T cell reactivity by flow cytometry for intracellular cytokine production and proliferation assay. HCV-specific CD4+CD25(+high) T cells consistently produced transforming growth factor (TGF)-beta but only limited amounts of interleukin (IL)-10 and no IL-2 and interferon (IFN)-gamma. The HCV-specific TGF-beta response by CD4+CD25(+high) T cells was significantly greater in patients with normal ALT compared to patients with elevated ALT. In addition, a significant inverse correlation was found between the HCV-specific TGF-beta response by CD4+CD25(+high) T cells and liver inflammation. In peripheral blood mononuclear cells (PBMC), both HCV antigen-induced IFN-gamma production and proliferation of CD4+ T cells were greater in patients with elevated ALT compared with patients with normal ALT. Depletion of CD4+CD25+ cells from PBMC resulted in an increase of both IFN-gamma production and proliferation of HCV-specific CD4+ T cells that was significantly greater in patients with normal ALT levels compared with patients with elevated ALT. In addition, CD4+CD25+ T cells from patients with normal ALT levels proved to be significantly more potent to suppress CD4+ T cell reactivity with respect to those from patients with elevated ALT. In conclusion, these data support the hypothesis that CD4+CD25+ cells may play a role in controlling chronic inflammatory response and hepatic damage in chronic HCV carriers.     

Expression of CD8alpha identifies a distinct subset of effector memory CD4+ T lymphocytes

Circulating CD4+ CD8+ T lymphocytes have been described in the peripheral blood of humans and several animal species. However, the origin and functional properties of these cells remain poorly understood. In the present study, we evaluated the frequency, phenotype and function of peripheral CD4+ CD8+ T cells in rhesus macaques. Two distinct populations of CD4+ CD8+ T cells were identified: the dominant one was CD4hi CD8lo and expressed the CD8alphaalpha homodimer, while the minor population was CD4lo CD8hi and expressed the CD8alphabeta heterodimer. The majority of CD4hi CD8alphalo T cells exhibited an activated effector/memory phenotype (CCR5lo CD7- CD28- HLA-DR+) and expressed relatively high levels of granzyme B. Intracellular cytokine staining assays demonstrated that the frequency of cytomegalovirus-specific T cells was enriched five-fold in CD4hi CD8alphalo T cells compared to single-positive CD4+ T cells, whereas no consistent enrichment was observed for simian immunodeficiency virus (SIV)-specific T cells. Cross-sectional studies of SIV-infected animals demonstrated that the frequency of CD4hi CD8alphalo T cells was lower in wild-type SIV-infected animals compared to uninfected controls, although prospective studies of SIV-infected animals demonstrated depletion of CD4hi CD8alphalo lymphocytes only in a subset of animals. Taken together, these data suggest that CD4+ T cells expressing CD8alpha represent an effector/memory subset of CD4+ T cells and that this cell population can be depleted during the course of SIV infection.     

Anti-CD3-induced changes in protein kinase C isozymes expression in human CD4+ and CD8+ T lymphocytes

In order to determine whether there is a differential expression and activation of PKC isozymes between CD4+ and CD8+ T cells, peripheral blood mononuclear cells were stimulated with anti-CD3 monoclonal antibody (moAb) for various time intervals and the expression of calcium-dependent PKC isozymes (, , ) and calcium-independent PKC isozymes (, , ) was analyzed with dual color flow cytometry, using anti-PKC isozyme antibodies and anti-CD4 or anti-CD8 antibodies. The basal fluorescence intensity of all PKC isozymes was comparable between CD4+ T cells and CD8+ T cells. Following activation with anti-CD3 moAb a marked increase in the fluorescence intensity of all PKC isozymes in both CD4+ and CD8+ T cells, albeit to a different extent and with different kinetics was observed. Among all PKC isozymes studied, the least striking changes were observed in PKC isozyme and the most striking changes were observed in PKC- isozyme. Laser-based confocal microscopic studies confirmed that the increase in fluorescence intensity of PKC isozymes following anti-CD3 moAb stimulation, as measured by flow cytometry was accompanied by the translocation of PKC isozymes from cytosol to the plasma membrane. This study demonstrates a differential effect of anti-CD3 moAb on the expression of PKC isozymes between CD4+ and CD8+ T cells and suggests that flow cytometry can be used to study the translocation of PKC isozymes from cytosol to the plasma membrane.     

CD4+VEGFR1HIGH T cell as a novel Treg subset regulates inflammatory bowel disease in lymphopenic mice

Regulatory T cells (Tregs) are a specialized subpopulation of T cells that control the immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. Many subsets of CD4⁺ Tregs have been identified, including Foxp3⁺, Tr1, Th3, and Foxp3neg iT(R)35 cells. In this study, we identified a new subset of CD4⁺VEGFR1^high Tregs that have immunosuppressive capacity. CD4⁺VEGFR1high T cells, which constitute approximately 1.0% of CD4⁺ T cells, are hyporesponsive to T-cell antigen receptor stimulation. Surface marker and FoxP3 expression analysis revealed that CD4⁺VEGFR1^high T cells are distinct from known Tregs. CD4⁺VEGFR1^high T cells suppressed the proliferation of CD4⁺CD25⁻ T cell as efficiently as CD4⁺CD25^high natural Tregs in a contact-independent manner. Furthermore, adoptive transfer of CD4⁺VEGFR1⁺ T cells from wild type to RAG-2-deficient C57BL/6 mice inhibited effector T-cell-mediated inflammatory bowel disease. Thus, we report CD4⁺ VEGFR1^high T cells as a novel subset of Tregs that regulate the inflammatory response in the intestinal tract.