Restriction molecules involved in the interaction of human T cell clones with antigen-presenting cells

Two of three distinct human Ia molecules detected by murine monoclonal antibodies (MoAb) have been suggested to be involved in antigen presentation for T cell responses to purified protein derivatives (PPD) and herpes simplex virus (HSV). This observation was first suggested from studies on the inhibition of proliferative responses of whole T cell populations with MoAb against human Ia molecules. To determine whether a single T cell recognizes the antigen in the context of both Ia molecules or in the context of each one of two Ia molecules, we isolated and propagated PPD-reactive T cell clones from an HLA-DR heterozygous individual. They showed four different restriction patterns: type I and type II clones each appeared to be restricted to one of two HLA-DR antigens, type III clone gave anomalous patterns of response and seemed to be restricted to non-DR antigens, and type IV clone recognized antigen when both DR antigens were presented on the same antigen-presenting cells (APC) surface. Blocking study with monoclonal anti-Ia antibodies suggested that type I, II and IV clones are restricted to DR molecules and type III clones are restricted to 1B4 molecules distinct from DR or MB1 molecules. These data imply that human T cell clones recognizing PPD in the context of each one of two Ia molecules are clonally distributed.     

Antigen-presenting human T cells and antigen-presenting B cells induce a similar cytokine profile in specific T cell clones

One of the factors that may influence the cytokine secretion profile of a T cell is the antigen-presenting cell (APC). Since activated human T cells have been described to express major histocompatibility complex (MHC) class II molecules as well as costimulatory molecules for T cell activation, like e.g. ICAM-1, LFA-3 and B7, they might play a role as APC and be involved in the regulation of T-T cell interactions. To define further the role of T cells as APC we tested their capacity to induce proliferation and cytokine production in peptide- or allospecific T cell clones and compared it with conventional APC, like B lymphoblasts (B-LCL) or HTLV-1 - transformed T cells, or with non-classical APC, like activated keratinocytes or eosinophils. CD4⁺, DP-restricted T cell clones specific for a tetanus toxin peptide (amino acids 947-967) and CD4⁺, DR-restricted allospecific Tcell clones produced interleukin (IL)-2, IL-4, tumor necrosis factor-α and interferon-γ (IFN-γ) after phorbol 12-myristate 13-acetate and ionomycin stimulation and a more restricted cytokine pattern after antigen stimulation. Dose-response curves revealed that the antigen-presenting capacity of activated, MHC class II⁺, B7⁺ T cells was comparable to the one of B-LCL. Both APC induced the same cytokine profile in the T cell clones despite a weaker proliferative response with T cells as APC. Suboptimal stimulations resulted in a lower IFN-γ/IL-4 ratio. Cytokine-treated, MHC class II⁺ keratinocytes and eosinophils differed in the expression of adhesion molecules and their capacity to restimulate T cell clones. The strongly ICAM-1-positive keratinocytes induced rather high cytokine levels. In contrast, eosinophils, which express only low densities of MHC class II and no or only low levels of adhesion molecules (B7, ICAM-1 and LFA3), provided a reduced signal resulting in a diminished IFN-γ/IL-4 ratio. We conclude that non-classical APC differ in their capacity to restimulate T cell clones, whereby the intensity of MHC class II and adhesion molecules (B7, ICAM-1) expressed seems to determine the efficacy of this presentation.     

An early antigen-presenting cell defect in HIV-1-infected patients correlates with CD4 dependency in human T-cell clones.

We have used a defined panel of nine HIV peptide-specific T-cell clones (TLC) generated from a healthy volunteer to evaluate the antigen-presenting cell (APC) function of human immunodeficiency virus-1 (HIV- 1)-infected patients. Peripheral blood mononuclear cells (PBMC) from HLA-matched seropositive and uninfected volunteers were compared for their capacity to present peptide to TLC specific for the V3 loop of HIV- 1 envelope glycoprotein gp120, influenza haemagglutinin or the mycobacterial 19,000 MW antigen APC from uninfected volunteers (HIV- APC) invariably presented peptides to all TLC with comparable efficiency. In contrast using APC from HIV- 1-infected subjects (HIV+ APC) three patterns of responsiveness were observed. The first group of TLC was not stimulated by HIV+ APC even early in infection. The second responded to all APC comparably. The third and intermediate group, responded to APC from some clinically asymptomatic, but not acquired immune deficiency syndrome (AIDS), patients. The two additional TLC, derived from other donors and with specificity for non-HIV peptides, showed similar variation in response to HIV+ APC. The different patterns of response to HIV APC did not correlate with the fine specificity or cytokine phenotypes of the TLC. Neither was the defect due to decreased levels of expression of APC molecules involved in delivering the first or second signal required for T-cell activation APC mixing experiments showed no evidence of APC-derived inhibitory factor. Furthermore, the defect was independent of T cells or their products and was equally expressed in monocytes and dendritic cells. Instead, responsiveness was inversely related to the degree of CD4 dependency suggesting that the underlying mechanism was a CD4 APC-associated gp120 interaction. The early appearance of this defect in HIV- 1 infection co-incident with the loss of recall responses is consistent with a role for APC dysfunction in pathogenesis.     

Regulatory T cells suppress antigen-driven CD4 T cell reactivity following injury

Injury initiates local and systemic host responses and is known to increase CD4 Treg activity in mice and humans. This study uses a TCR transgenic T cell adoptive transfer approach and in vivo Treg depletion to determine specifically the in vivo influence of Tregs on antigen-driven CD4 T cell reactivity following burn injury in mice. We report here that injury in the absence of recipient and donor Tregs promotes high antigen-driven CD4 T cell expansion and increases the level of CD4 T cell reactivity. In contrast, CD4 T cell expansion and reactivity were suppressed significantly in injured Treg-replete mice. In additional experiments, we found that APCs prepared from burn- or sham-injured, Treg-depleted mice displayed significantly higher antigen-presenting activity than APCs prepared from normal mice, suggesting that Tregs may suppress injury responses by controlling the intensity of APC activity. Taken together, these findings demonstrate that Tregs can actively control the in vivo expansion and reactivity of antigen-stimulated, na?ve CD4 T cells following severe injury.     

CD4~+CD25~+调节性T细胞对内皮细胞抗原提呈功能的影响及机制

目的:探讨CD4+CD25+调节性T细胞(Treg)对内皮细胞抗原提呈功能的影响及机制。方法:磁性细胞分离器(MACS)分离CD4+CD25+T细胞及CD4+CD25-T细胞。在ox-LDL作用下,HUVECs与CD4+CD25+T细胞共培养,24小时后收集HUVECs。应用流式细胞术测定HUVECs抗原提呈分子(HLA DR,CD86,CD80)的表达,Cell Counting Kit-8法(CCK-8)测定HU-VECs刺激CD4+CD25-T细胞增殖的能力,Transwell小室实验初步探讨Treg作用于HUVECs的具体机制。结果:与对照组比较,Treg可显著抑制HUVECs抗原提呈分子的表达及刺激T细胞增殖的能力。用Transwell隔离后,与or-LDL刺激组比较,HU-VECs抗原提呈分子表达及其刺激T细胞增殖的能力无明显变化。结论:Treg可显著抑制HUVECs抗原提呈能力,其作用机制可能为下调CD86的表达,且依赖细胞直接接触。     

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.     

Isolation and characterization of human peripheral blood CD4+ T cell clones expressing gamma delta T cell receptors.

Rare T cell clones bearing both CD4 and gamma delta T cell receptors (TcR gamma delta) were obtained from human peripheral blood by cell sorting using anti-CD4 and anti-TCR delta 1 antibodies. All the clones established were reactive with anti-TcR gamma delta 1 antibody, whereas only about 20% of the clones showed reactivity with anti-delta TCS1 antibody. Unlike most CD4+ T cells bearing TcR alpha beta, all the clones tested showed lectin-dependent and anti-CD3 antibody-redirected cytolytic activity. About 60% of the clones exhibited natural killer cell-like activity. Immunoprecipitation analysis of TcR gamma delta showed that each clone expressed either a disulfide-linked or non-disulfide-linked heterodimer consisting of 37-44-kDa TcR gamma and TcR delta chains.     

HLA tetramer-based artificial antigen-presenting cells for stimulation of CD4+ T cells

Current evidence suggests that immunotherapy for cancer or infectious diseases will require the activation of CD4(+) T cells in addition to the activation of cytotoxic CD8(+) T cells. To complement and overcome some of the limitations of dendritic-cell-based vaccines and ex vivo expansion of human T cells, we sought to engineer artificial antigen-presenting cells (aAPCs) for the stimulation of antigen-specific human CD4(+) T cells. We have designed a variety of aAPCs using magnetic beads as a scaffold on which to coat HLA-peptide tetrameric complexes along with costimulatory molecules such as anti-CD28. Here, we tested various forms of conjugation of the tetramers onto beads, characterized the relative concentration of antigen available on the surface of the beads, and evaluated the ability of different types of beads to promote activation of antigen-specific CD4(+) T cells. We find that an indirect coating of HLA-peptide tetramers on beads via an anti-Class II antibody provides specific stimulation of antigen-specific CD4(+) T cells.     

Induction of immunoglobulin synthesis by CD4 T cell clones

The study of mechanisms by which CD4⁺ T cells induce Ig synthesis has been greatly enhanced by the availability of CD4⁺ T cell clones with restricted cytokine profiles. We have demonstrated with in vitro and in vivo studies that both Th1 (T helper cell 1) and Th2 clones can provide MHC restricted help and induce primary as well as secondary antibody responses under cognate antigen driven conditions. In addition, we have shown that both types of clones, utilizing distinct cytokines, can effect B cell memory and affinity maturation of the Ig response, although the precise mechanisms by which this occurs are not yet clear. Using Th1 and Th2 clones, we have also shown that the pathways for IgG1 synthesis are redundant, in that induction of IgG1 synthesis in secondary responses in which B cells have already switched from IgM to 1gG1, can occur via several pathways, one involving IL-4 and IL-5, the other involving IL-2. In contrast, IgE and IgG2a synthesis require specific cytokines for synthesis in both primary and secondary B cells. Finally, the cytokines produced by Th1 and Th2 clones can 'neutralize' each other, when both types of clones are present during the induction of primary Ig responses. As an exception however, the induction of IgA synthesis is greatly augmented by the presence of both types of clones.     

CD154-dependent priming of diabetogenic CD4(+) T cells dissociated from activation of antigen-presenting cells

We followed the fate of K(d)- or I-A(g7)-restricted beta cell-autoreactive T cells in monoclonal TCR-transgenic NOD mice expressing or lacking CD154. 8.3-NOD.RAG-2(-/-)/CD154(-/-) mice, which bear autoreactive CD8(+) T cells, developed diabetes with the same incidence and tempo as 8.3-NOD.RAG-2(-/-)/CD154(+) mice. Recruitment of CD154(-/-) 8.3-CD8(+) CTL was accelerated by CD154(+)CD4(+) T cells, by expression of a B7.1 transgene in beta cells or by treatment of the mice with CpG-DNA or an agonistic anti-CD40 antibody. In contrast, the autoreactive CD4(+) T cells maturing in 4.1-NOD.RAG-2(-/-) mice lost their diabetogenic potential if they lacked CD154, even in the presence of CD154(+)CD4(+) T cells, B7.1 molecules on beta cells, CpG-DNA treatment, or systemic CD40 ligation. These results demonstrate the existence of a novel, CD154-dependent pathway of CD4(+) T cell activation that is independent of CD40-mediated activation of APCs.     

Human bone marrow stromal cells hamper specific interactions of CD4 and CD8 T lymphocytes with antigen-presenting cells

Bone marrow stromal cells (BMSCs) may inhibit T-cell functions in vitro and thus have been proposed as immunoregulators to control in vivo graft-versus-host disease (GVHD) in haploidentical hemopoietic stem cell transplants. To better investigate this phenomenon, we used a defined experimental system in which responding T cells are antigen-specific and devoid of alloreactivity against BMSC from a different subject. Thus, we established antigen-specific human CD4 and CD8 T-cell lines as the readout system. Antigen-dependent proliferation was reduced with both T-cell subsets cultured on confluent BMSCs, and also on confluent human skin fibroblasts (HSF) inhibited T-cell proliferation with similar efficiency. Morphological observations of the cocultures showed impairment of physical interactions between T-cell and antigen-presenting cells in the presence of BMSC, with lack of formation of antigen-dependent clusters of T cells and antigen-presenting cells (APCs). In contrast, no effects were seen with BMSC-conditioned medium. Since suppression was seen only with confluent mesenchymal cells, this phenomenon may not be relevant in vivo, where BMSCs are at low frequency. In addition, if the reported suppressive effect of BMSCs on GVHD in vivo is confirmed, a different in vitro system should be envisaged to better understand and exploit the underlying mechanism.     

Comparative analysis of CD8 expressed on mature CD4+ CD8+ T cell clones cultured with IL-4 and that on CD8+ T cell clones: implication for functional significance of CD8 beta

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.     

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vbeta repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RB(low) subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen-specific stimulation in vivo. This suggests that anergic/suppressive CD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non-professional antigen-presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.     

Liver sinusoidal endothelial cells veto CD8 T cell activation by antigen-presenting dendritic cells

The liver is known to induce tolerance rather than immunity through tolerogenic antigen presentation or elimination of effector T cells. In particular, hepatic dendritic cells (DC) are known to be little immunogenic for CD8 T cells. Here, we investigated whether this peculiar phenotype resulted from interaction with resident hepatic cell populations. Contact of DC with liver sinusoidal endothelial cells (LSEC) but not hepatocytes or B cells vetoed antigen-presenting DC to fully activate naive CD8 T cells. This MHC-independent regulatory effect of LSEC on DC function was not connected to soluble mediators but required physical contact. Because interaction with third-party LSEC still allowed antigen-presenting DC to stimulate expression of initial activation markers on naive CD8 T cells and to stimulate activated CD8 T cells, we hypothesize that LSEC controlled the DC costimulatory function. Indeed, contact with LSEC led to reduced DC expression levels of CD80/86 or IL-12, but supplementation of these signals failed to rescue the ability to prime naive CD8 T cells, indicating involvement of further molecules. Taken together, our results reveal a novel principle operative in hepatic tolerance induction, in which LSEC not only tolerize T cells themselves but also suppress neighboring APC normally capable of inducing T cell immunity.     

Antibody-mediated delivery of antigen to chemokine receptors on antigen-presenting cells results in enhanced CD4+ T cell responses

Here, we have investigated if targeting of T cell epitopes to chemokine receptors results in improved CD4+ T cell responses. Mouse monoclonal antibodies (mAb) with kappaL chains were targeted to various chemokine receptors expressed on human monocytes or immature dendritic cells (DC), and proliferation of cloned human, DR4-restricted CD4+ T cells specific for mouse Ckappa(40-48) was measured. When using monocytes as antigen-presenting cells, mAb specific for CCR1, CCR2, CCR5, and CXCR4 were 100-10,000-fold more efficient at inducing T cell proliferation when compared to isotype-matched control mAb on a per molecule basis. Targeting of immature DC was less effective and was only seen with anti-CCR1 and anti-CXCR4 mAb. Anti-chemokine receptors mAb required to be processed by the conventional endosomal MHC class II presentation pathway. The mAb did not induce signaling through the chemokine receptors as they failed to induce mobilization of cytosolic Ca2+ and actin polymerization. They also failed to induce APC maturation. The results strongly suggest that chemokine receptors channel antigen into the endocytic pathway for presentation on MHC class II molecules. Targeting T cell epitopes to chemokine receptors by recombinant antibody should be a useful vaccine strategy for the induction of strong CD4+ T cell responses.     

Establishment and characterization of Epstein-Barr virus-specific human CD4+ T lymphocyte clones.

We developed a simple method for establishing Epstein-Barr virus (EBV)-specific, human CD4+ T cell clones. The method originates from our experience that the regression of cell growth in in vitro EBV transformation of B cells occurs when round lymphoid cells appear in the culture. Peripheral blood mononuclear cells (PBMCs) were cultured with EBV, and IL-2 (20 U/ml) was added to the culture on day 17 after the virus addition. The phenotype of the growing cells was CD3+, CD4+, and CD8-. The cells were cytotoxic for autologous lymphoblastoid B cell line (LCL) and EBV-superinfected autologous LCL. The cytotoxic T lymphocytes (CTLs) were confirmed to be CD4+ T cells but not CD8+ T cells in the culture. CTL clones were established by a limiting dilution method. All the CTL clones had the phenotype of CD3+, CD4+ and CD8-, and proliferated in response to autologous LCL. They produced interferon (IFN)-gamma, interleukin 2 (IL-2) and tumour necrosis factor (TNF)-beta but not IL-4. All but one clone responded to both autologous, EBV-superinfected and non-superinfected LCLs. Proliferative and cytotoxic responses to allogenic LCLs were heterogeneous. These results suggest that this method induces heterogeneous, EBV-specific CD4+ CTL clones and is useful for analysis of CD4+ T cells in EBV infections.     

Activation requirements of circulating antigen-specific human CD8(+) memory T cells probed with insect cell-based artificial antigen-presenting cells.

We sought to define the molecular setup of an antigen-presenting cell that elicits antigen-specific T cell responses in vitro using insect cells that were infected with recombinant baculoviruses. Expression of single-chain HLA was complemented step-by-step with costimulatory molecules, including CD54 and CD80, by co-infection with the relevant viruses. Role of CD8 was assessed by introducing hybrid class I molecules where the alpha-3 domain of the HLA heavy chain molecule was replaced by its murine K(b) counterpart. Circulating T cells that respond to the EBV-derived HLA-A2-restricted peptide GLGCTLVAML were previously shown to bear hallmarks of memory cells. We found that the HLA+peptide complex alone displayed on the surface of insect cells was sufficient to elicit IFN-gamma secretion from these freshly isolated CD8(+) T cells in ELISpot assays. Binding of CD8 was absolutely required, but coexpression of costimulatory molecules resulted only in minimal increase in the number of spots. Tumor antigen-specific CTL clones also reacted in a strictly antigen-specific manner, but required CD54 for quantitative responses. The amount of IFN-gamma produced by the individual reactive T cells was evaluated as spot size, and was also influenced by the costimulatory molecules: CD54 increased also the response magnitude of cultured CTL lines, while CD80 enhanced cytokine release from freshly isolated CD8(+) T cells. Understanding the stimulatory requirements of functionally competent effector/memory T cells and their exact enumeration will be helpful for increasing the efficacy of vaccines.     

Eosinophil as antigen-presenting cell: activation of T cell clones and T cell hybridoma by eosinophils after antigen processing.

We have studied the role of murine eosinophils as antigen-presenting cells (APC). Eosinophils have several characteristics that support the hypothesis of its function as potential APC: they have phagocytic capacity, express adhesion molecules and major histocompatibility complex (MHC) class II antigens and can produce and release interleukin-1 (IL-1). We have obtained several T cell clones specific for Mesocestoides corti antigens and used T cell hybridoma specific for ovalbumin (OVA) to test this hypothesis. Granulocyte-macrophage colony-stimulating factor-activated pure eosinophils (99.9%), express class II antigens and are able to present M. corti antigens to specific T cell clones or OVA to T cell hybridoma 3DO 11.10, inducing the proliferation of T cell clones and IL-2 release by the T cell hybridoma. Proliferation of T cells clones is dependent on the number of eosinophils used as APC. We have compared the efficiency of the same number of macrophages and eosinophils as APC, and have found that macrophages are more efficient than eosinophils. Lysosomotropic agents, such as chloroquine and ammonium chloride, that inhibit antigen processing, impaired eosinophil presentation. This presentation is restricted by MHC class II and inhibited by anti-I-Ad monoclonal antibody. The present study provides clear evidence of APC function for eosinophils. Our investigation points to a new role for eosinophils in the immune response.     

Cytotoxic human CD4(+) T cells

The induction of adaptive immune responses critically depends on helper signals provided by CD4(+) T cells. These signals not only license antigen presenting cells (APC) to activate na?ve CD8(+) T cells leading to the formation of vast numbers of cytotoxic T lymphocytes but also support the differentiation of B cells into immunoglobulin-secreting plasma cells. Next to these helper functions, a subpopulation of CD4(+) T cells can also directly function as effector cells by executing cytotoxicity in a peptide-specific and MHC class II-restricted manner. Cytotoxic CD4(+) T cells may function in combating pathogens but additionally their presence has been associated with autoimmune disease and vascular damage. On the contrary, the induction of cytotoxic CD4(+) T cells may be a future target for vaccine strategies.