Altered primary CD8+ T cell response to a modified virus Ankara(MVA)-vectored vaccine in the absence of CD4+ T cell help

T cell receptor-transgenic F5 mice were used to assess primary CD8+ T cell responses to a modified virus Ankara (MVA)-vectored vaccine in the absence of CD4+ T cell help. Naive, CD8-enriched, CFSE-labelled F5 cells were transferred into normal or CD4+ cell-depleted mice and the mice were vaccinated with MVA.HIVA-NP. At different time points during the primary response, F5 cells were re-isolated and analysed on divisional basis for a number of parameters. We demonstrated that the primary CD8+ T cell response in the absence of CD4+ T cell help differed from that in normal CD4+ cell-undepleted mice. While in the absence of CD4+ T cell help, the initial migratory progress from the local response to a systemic one was not grossly affected, the proportion of dying F5 cells during the expansion phase was markedly increased and resulted in an overall smaller expansion and significantly decreased frequency of CD8+ T cell memory after contraction. T cells primed without help displayed accelerated proliferation and activation, while expression of interferon-gamma remained similar. These phenomena were observed in the lymph nodes draining the MVA.HIVA-NP immunization site and were similar, but delayed by 2-3 days in spleen and non-draining lymph nodes.     

CD4+ CD25+ regulatory T cells control the magnitude of T-dependent humoral immune responses to exogenous antigens

CD4+ CD25+ T reg cells are critical for peripheral tolerance and prevention of autoimmunity. Here we show that CD4+ CD25+ T reg also regulate the magnitude of humoral responses against a panel of T-dependent antigens of foreign origin during both primary and secondary immune responses. Depletion of CD4+ CD25+ T cells leads to increased antigen-specific antibody production and affinity maturation but does not affect T-independent B cell responses, suggesting that CD4+ CD25+ T reg exert a feedback mechanism on non-self antigen-specific antibody secretion by dampening the T cell help for B cell activation. Moreover, we show that CD4+ CD25+ T reg also suppress in vitro B cell immunoglobulin production by inhibiting CD4+ CD25- T cell help delivery, and that blockade of TGF-beta activity abolishes this suppression.     

Mast cells are required for optimal autoreactive T cell responses in a murine model of multiple sclerosis

Once considered to be of sole importance in allergy and parasitic infections, the role of mast cells in other pathologic and protective immune responses is becoming increasingly evident. We previously demonstrated that mast cells contribute to the severity of EAE, the rodent model of multiple sclerosis. Here we show that one mode of mast cell action is through effects on the autoreactive T cell response. Early indices of both peripheral CD4 and CD8 T cell activation, including IFN-gamma production and increases in CD44 and CD11a expression, are attenuated in mast cell-deficient (W/Wv) mice after myelin oligodendrocyte glycoprotein(35-55) priming when compared to WT animals. Reduced infiltrates of activated T cells in the central nervous system are also observed. Importantly, selective repletion of the mast cell compartment restores most T cell responses in the lymph nodes and the central nervous system, correlating with reconstitution of severe disease. The adoptive transfer of WT-derived encephalitogenic T cells results in significantly less severe disease in W/Wv recipients, indicating that mast cells also exert potent effects after the initial T cell response is generated. Our data provide the first in vivo evidence that mast cells can significantly influence T cell responses and suggest that mast cells exacerbate disease during both the inductive and effector phases.     

Prevention of toxic epidermal necrolysis by regulatory T cells

To analyze immunoregulation of autoreactive T cells specific for epidermal skin antigens, we crossed transgenic mice expressing ovalbumin selectively in keratinocytes under the keratin 5 promoter (K5-mOVA) with mice expressing a K(b)-restricted OVA-specific T cell receptor transgene (OT-I). In athymic double-transgenic mice, OT-I cells developed extrathymically and, at 8-12 weeks of age, initiated severe epidermal damage mimicking toxic epidermal necrolysis (TEN). In contrast, euthymic double-transgenic mice showed thymic deletion of OT-I cells, had few of these cells in the periphery, and never developed skin changes mimicking TEN. Adoptive transfer of OT-I cells isolated from euthymic double-transgenic mice induced TEN in athymic K5-mOVA single-transgenic mice. This spontaneous disease in athymic double-transgenic mice was prevented by transferring lymph node cells from euthymic mice, but was not prevented when CD4(+) or CD25(+) cells were depleted from this population. Although purified CD4(+)CD25(+) cells scarcely prevented the skin disease induced by adoptive transfer of OT-I cells, they efficiently prevented the disease when co-transferred with CD11c(+) dendritic cells. These results suggested that thymus-derived regulatory T cells cooperate with CD11c(+) dendritic cells to prevent life-threatening skin damage such as TEN.     

Antigen-dependent suppression of alloresponses by Foxp3-induced regulatory T cells in transplantation

Adoptive transfer of polyclonal CD4+CD25+ regulatory T cells (Treg) can tolerize transplantation alloresponses. Treg are activated via their specific TCR, but the antigen specificity of wild-type Treg remains elusive, and therefore controlling potency and duration of Treg activity in the transplantation setting is still not feasible. In this study, we used murine graft-versus-host disease (GVHD) as a model system to show that antigen-specific Treg suppress the response of T effector cells to alloantigens in vitro and prevent GVHD in vivo. The suppressive potential of antigen-specific Treg was much greater than that of polyclonal Treg. To acquire large numbers of antigen-specific Treg, we transduced CD4+CD25- cells with foxp3, and found that these foxp3-induced Treg suppress alloresponses in vitro and prevent GVHD in vivo as effectively as naturally derived CD4+CD25+ Treg. Furthermore, we used an antigen-specific CD4 Th1 clone as a source of foxp3-induced Treg after transduction with foxp3, and found those Treg to effectively prevent GVHD in an antigen-dependent manner. The findings of this study provide a basis for the concept that the onset and potency of the suppression by Treg can be regulated, and suggest a novel approach to enhance the feasibility and effectiveness of inducing tolerance by Treg as an adoptive immunotherapy in transplantation.     

Development and activation of regulatory T cells in the human fetus

There is an increasing amount of knowledge on the functional properties of regulatory T cells (Treg) in the adult immune system, but data on the generation and function of these cells during human embryonic development are scarce. In this study, we show that in the fetal thymus, double-positive cells initiate expression of CD25, GITR, CTLA4 and CD122 during their transition from the CD27- to the CD27+ stage. Moreover, CD4+CD25+ fetal thymocytes already have the potential to suppress proliferation of CD25- cells. After leaving the thymus, FoxP3+CD4+CD25+ Treg enter the fetal lymph nodes and spleen, where they acquire a primed/memory phenotype. A model is proposed for the development of human fetal Treg that encompasses two sequential maturation steps: initiation of a regulatory phenotype and suppressive activity in the thymus; and subsequent activation within the peripheral lymphoid organs. Upon activation, FoxP3+CD4+CD25+ Treg suppress potentially deleterious responses by autoreactive lymphocytes and maintain homeostasis within the developing fetus.     

Suppressive properties of human CD4+CD25+ regulatory T cells are dependent on CTLA-4 expression

It has been demonstrated that T cells with regulatory properties are present within the peripheral blood CD4(+)CD25(+) T cell compartment. Here, we describe an original method to purify human CD4(+)CD25(+)CD152(+) T lymphocytes as living cells by forcing the exportation of CTLA-4 molecules stored in intracellular vesicules at the cell surface. By doing so, we demonstrate that CD4(+)CD25(+) T cells contain a smaller and more homogeneous population enriched in cells with in vitro regulatory activity. Moreover, we show that this enrichment in regulatory T cells is associated with an increased expression of Foxp3 and that CD4(+)CD25(+)CD152(+) T lymphocytes display a much stronger suppressive activity in controlling in vitro proliferation of alloantigen-specific T cells than CD4(+)CD25(+)CD152(-) T lymphocytes purified in parallel. Lastly, by purifying such cells expressing CTLA-4, we demonstrate that indeed CTLA-4 is involved in CD4(+)CD25(+)CD152(+) T cell regulatory activity, while suppressive cytokines are not.     

CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative

We investigated the mechanisms of immune tolerance raised by tumors by comparing immunogenic and tolerogenic tumor cell clones isolated from a rat colon carcinoma. When injected into syngeneichosts, the immunogenic REGb cells yield tumors that are rejected, while the tolerogenic PROb cells yield progressive tumors and inhibit the regression of REGb tumors. We show here that PROb tumor volume is correlated with an expansion of CD4(+)CD25(+) regulatory T lymphocytes in lymphoid tissues. These cells delay in vivo the rejection of REGb tumors and inhibit in vitro T cell-mediated immune responses against REGb cells through a mechanism that requires cell contact between effector and regulatory T cells and involves TGF-beta. While total T cells fromPROb tumor-bearing rats yield no apparent anti-tumor immune response, depletion of CD25(+) T cells restores this reactivity. A single administration of cyclophosphamide depletes CD4(+)CD25(+) T cells in PROb tumor-bearing animals, delays the growth of PROb tumors, and cures rats bearing established PROb tumors when followed by an immunotherapy which has no curative effect when administered alone. These results demonstrate the role of CD4(+)CD25(+) regulatory T cells in tumor-induced immune tolerance and the interest of regulatory T cell depletion to sensitize established tumors to immunotherapy.     

The fate of heterologous CD4+ T cells during Leishmania donovani infection

Little is currently understood about the consequences of chronic parasitic infection for the fate of memory CD4+ T cells that recognize heterologous antigens, e.g. resulting from prior infections or vaccination. Here, we address how Leishmania donovani infection affected the fate of non-cross-reactive (OVA)-specific memory CD4+ T cells. DO11 cells were adoptively transferred into naive recipient mice, which were then immunized to generate memory DO11 cells. After 6 weeks, mice were infected with L. donovani and the fate of DO11 cells was determined. L. donovani infection stimulated an approximately threefold expansion in the total number of CD4+ T cells and DO11 cells, compared to that observed in uninfected mice. DO11 T cells were more actively dividing in infected mice, as judged by 5-bromo-2' deoxyuridine labeling, whereas their rate of apoptosis in control and infected mice was identical. Both CD45RBhiCD44lo naive T cells and to a greater extent CD45RBloCD44hi memory DO11 cells increased in number in the spleens of infected mice, whereas no changes occurred to DO11 cell number or phenotype in the draining lymph nodes. These data indicate that heterologous CD4+ T cells may actively divide during chronic infectious diseases, with important implications for how chronic infection may impact on heterologous immunity.     

Role of Foxp3-positive regulatory T cells during infection

Surviving an infection requires the generation of an immune response that controls the invading pathogen while limiting collateral damage to self tissues that may result from an exuberant immune response. Various populations of regulatory cells, including Foxp3+ Treg, have been shown to play a central role in the establishment of these controlled immune responses. In this review, I discuss current hypotheses and points of polemic associated with the origin, mode of action and antigen specificity of Foxp3+ Treg during infection.     

Skin-derived TSLP stimulates skin migratory dendritic cells to promote the expansion of regulatory T cells

Therapeutic strategies that enhance regulatory T (Treg) cell proliferation or suppressive function hold promise for the treatment of autoimmune and inflammatory diseases. We previously reported that the topical application of the vitamin D3 analog MC903 systemically expands Treg cells by stimulating the production of thymic stromal lymphopoietin (TSLP) from the skin. Using mice lacking TSLP receptor expression by dendritic cells (DCs), we hereby show that TSLP receptor signaling in DCs is required for this Treg expansion in vivo. Topical MC903 treatment of ear skin selectively increased the number of migratory DCs in skin-draining lymph nodes (LNs) and upregulated their expression of co-stimulatory molecules. Accordingly, DCs isolated from skin-draining LNs but not mesenteric LNs or spleen of MC903-treated mice showed an enhanced ability to promote Treg proliferation, which was driven by co-stimulatory signals through CD80/CD86 and OX40 ligand. Among the DC subsets in the skin-draining LNs of MC903-treated mice, migratory XCR1⁻CD11b⁺ type 2 and XCR1⁻CD11b⁻ double negative conventional DCs promoted Treg expansion. Together, these data demonstrate that vitamin D3 stimulation of skin induces TSLP expression, which stimulates skin migratory DCs to expand Treg cells. Thus, topical MC903 treatment could represent a convenient strategy to treat inflammatory disorders by engaging this pathway.     

CD4+CD25+调节性T细胞在约氏疟原虫感染早期作用机制的实验研究

目的探讨CD4^＋CD25^＋调节性T细胞在约氏疟原虫感染早期的免疫调节机制。方法用抗CTLA-4 mAb和抗CD25 mAb分别与约氏疟原虫感染早期的小鼠脾细胞共同培养后,以ELISA法检测其培养上清的IL-10、TGF-β1及IFN-γ的含量。结果与抗CTLA-4 mAb共同培养后,脾细胞上清中IL-10的水平明显降低,但TGF-β1和IFN-γ水平未出现有意义的变化;与抗-CD25 mAb共同培养后,IL-10、TGF-β1和IFN-γ水平均未出现有意义的变化,但IL-10水平有一定的降低趋势。结论约氏疟原虫感染早期,IL-10可能是介导CD4^＋CD25^＋调节性T细胞参与免疫调节的关键性细胞因子,CD4^＋CD25^＋调节性T细胞可能通过CTLA-4发挥免疫抑制作用。     

The C terminus of T cell-specific adapter protein (TSAd) is necessary for TSAd-mediated inhibition of Lck activity

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, is expressed in activated T cells. The function of TSAd is as yet unknown. We previously showed that TSAd may modulate T cell receptor-triggered signaling events. TSAd contains a Src homology (SH)2 domain, ten tyrosines and a C-terminal proline-rich region. Here, we show that human TSAd interacts with Lck through the Lck SH2 and SH3 domains and is a substrate for Lck. The TSAd C terminus, including the proline-rich region and five tyrosines, is both necessary and sufficient for TSAd interaction with and phosphorylation by Lck. Expression of TSAd in Jurkat TAg cells results in hyperphosphorylation of endogenous Lck on Y394 and to an even larger extent on Y505, resulting in a reduced Y394/Y505 phosphorylation ratio in these cells. Furthermore, full-length TSAd, but not TSAd lacking the C terminus, inhibits the hyperactive Lck Y505F mutant when both are expressed in Jurkat T cells. In contrast, expression of the TSAd C terminus alone is sufficient to inhibit Lck Y505F in phosphorylating its substrates in Jurkat T cells. Our results indicate that the TSAd C terminus is essential for inhibition of Lck activity by TSAd, and suggest a mechanism for how TSAd may inhibit early T cell activation events.     

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+ T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

Immature dendritic cells (DC) infected with an endotheliotropic (Huv(+)) and leukotropic (Leuk(+)) human cytomegalovirus (HCMV) strain were used as a stimulus to determine functional HCMV-specific CD4(+) and CD8(+) T cells. Infected DC were co-cultured with autologous peripheral blood mononuclear cells and both arms of T cell activation were determined by intracellular flow cytometry analysis of IFN-gamma production. Efficient stimulation of HCMV-specific CD4(+) and CD8(+) T cell responses was achieved using DC productively infected with Huv(+) Leuk(+) VR1814 strain. On the contrary, a negligible CD8(+) T cell response was obtained when HCMV strains unable to infect DC, or DC pulsed with inactivated viral antigen, were used. HCMV specificity of the T cell response was confirmed in 46 HCMV-seropositive and 8 HCMV-seronegative healthy subjects. A cut-off was established to discriminate between immune and nonimmune subjects. The novel ex vivo assay enables the simultaneous evaluation of HCMV-specific CD4(+) and CD8(+) T cell responses and may be a useful tool for monitoring HCMV-specific T cell activity in immunocompromised transplanted patients.     

Involvement of CD8+ T cells in protective immunity against murine blood‐stage infection with Plasmodium yoelii 17XL strain

When developing malaria vaccines, the most crucial step is to elucidate the mechanisms involved in protective immunity against the parasites. We found that CD8⁺ T cells contribute to protective immunity against infection with blood‐stage parasites of Plasmodium yoelii. Infection of C57BL/6 mice with P. yoelii 17XL was lethal, while all mice infected with a low‐virulence strain of the parasite 17XNL acquired complete resistance against re‐infection with P. yoelii 17XL. However, the host mice transferred with CD8⁺ T cells from mice primed only with P. yoelii 17XNL failed to acquire protective immunity. On the other hand, the irradiated host mice were completely resistant to P. yoelii 17XL infection, showing no grade of parasitemia when adoptively transferred with CD8⁺ T cells from immune mice that survived infection with both P. yoelii XNL and, subsequently, P. yoelii 17XL. These protective CD8⁺ T cells from immune WT mice had the potential to generate IFN‐γ, perforin (PFN) and granzyme B. When mice deficient in IFN‐γ were used as donor mice for CD8⁺ T cells, protective immunity in the host mice was fully abrogated, and the immunity was profoundly attenuated in PFN‐deficient mice. Thus, CD8⁺ T cells producing IFN‐γ and PFN appear to be involved in protective immunity against infection with blood‐stage malaria.     

mTOR抑制剂一雷帕霉素对T细胞功能调控的研究进展

哺乳动物雷帕霉素靶蛋白（mTOR）作为信号通路的调节分子参与多条重要的信号转导通．路,能形成细胞对多种刺激的应答,mTOR至少存在两种功能性多蛋白复合物形式：mTORCl和mTORC2,其可发挥不同的生物学作用。雷帕霉素作为mTOR的特异性抑制剂可阻断mTOR信号通路信息的传导,调节T细胞的分化、发育、失能以及调节性T细胞（Treg）的增殖和功能,影响生长因子和细胞因子等生物活性物质的分泌,表现出有效的免疫抑制作用。     

Function and recruitment of mucosal regulatory T cells in human chronic Helicobacter pylori infection and gastric adenocarcinoma

CD4(+)CD25(high) FOXP3-expressing regulatory T cells (Treg) can suppress immune responses to infections and tumors, thereby promoting microbial persistence and tumor progression. However, little is known about the phenotype and function of human mucosal Treg. Therefore, we analyzed the suppressive activity and homing phenotype of Treg in gastric mucosa of Helicobacter pylori-infected gastric adenocarcinoma patients. We found increased numbers of CD4(+)FOXP3(+) Treg in the tumor compared to tumor-free gastric mucosa. Gastric Treg cells were able to suppress H. pylori-induced T cell proliferation and IFN-gamma production. Furthermore, gastric Treg expressed increased levels of l-selectin and CCR4, compared to non-Treg cells, suggesting that these receptors contribute to Treg recruitment. The presence of functional antigen-specific Treg in H. pylori-infected gastric mucosa supports an important role for these cells in suppression of mucosal effector T cell responses, which probably contribute to bacterial persistence and possibly also to gastric tumor progression.     

Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4+ Foxp3+ regulatory T cells

Recently, we demonstrated elevated numbers of CD4⁺ Foxp3⁺ regulatory T (Treg) cells in Plasmodium yoelii‐infected mice contributing to the regulation of anti‐malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus‐derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3⁺ Treg cells expressing neuropilin‐1 (Nrp‐1) decreased at early time‐points during P. yoelii infection, whereas percentages of Helios⁺ Foxp3⁺ Treg cells remained unchanged. Both Foxp3⁺ Nrp‐1⁺ and Foxp3⁺ Nrp‐1^? Treg cells from P. yoelii‐infected mice exhibited a similar T‐cell receptor Vβ chain usage and methylation pattern in the Treg‐specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3^? T cells adoptively transferred to P. yoelii‐infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3⁺ Treg cells.     

CD8+ T cell responses to human immunodeficiency viruses type 2 (HIV-2) and type 1 (HIV-1) gag proteins are distinguishable by magnitude and breadth but not cellular phenotype

The mechanisms underlying the relatively slow progression of human immunodeficiency virus type 2 (HIV-2) compared with HIV-1 infection are undefined and could be a result of more effective immune responses. We used HIV-2 and HIV-1 IFN-gamma enzyme-linked immunospot assays to evaluate CD8(+) T cell responses in antiretroviral-naive HIV-2- ('HIV-2(+)') and HIV-1-infected ('HIV-1(+)') individuals. Gag-specific responses were detected in the majority of HIV-2(+) and HIV-1(+) subjects. Overlapping gag peptide analysis indicated a significantly greater magnitude and breadth of responses in the HIV-1(+) cohort, and this difference was attributable to low responses in HIV-2(+) subjects with undetectable viral load (medians 2107 and 512 spot-forming units per 10(6) PBMC, respectively, p=0.007). We investigated the phenotype of viral epitope-specific CD8(+) T cells identified with HLA-B53- and HLA-B58-peptide tetramers (8 HIV-2(+), 11 HIV-1(+) subjects). HIV-2-specific CD8(+) T cells were predominantly CD27(+) CD45RA(-), and only a minority expressed perforin. The limited breadth and low frequency of CD8(+) T cell responses to HIV-2 gag in aviremic HIV-2(+) subjects suggests that these responses reflect antigen load in plasma, as is the case in HIV-1 infection. Immune control of HIV-2 does not appear to be related to the frequency of perforin-expressing virus-specific CD8(+) T cells.