HLA-G1-expressing antigen-presenting cells induce immunosuppressive CD4+ T cells

We recently reported that HLA-G1-transfected antigen-presenting cells (HLA-G1+ APCs) were capable of inhibiting alloproliferative responses. The aim of the present work was to further study the function and the mechanisms of action of HLA-G1+ APCs. We show here that HLA-G1+ APCs are immunoinhibitory cells that (i) inhibit the proliferation of CD4+ T cells, (ii) shed HLA-G1 molecules that might provide extra, non-antigen-specific, inhibitory or proapoptotic signals, (iii) induce CD4+ T cell anergy, or at least long-term unresponsiveness, and (iv) cause the differentiation of CD4+ T cells into suppressive cells. Thus, HLA-G+ APCs might (i) be involved in the direct suppression of immune responses and (ii) contribute to long-term efficient immune escape or tolerance.     

Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells

To exploit a novel strategy to regulate T cell-mediated immunity, we established human and murine modified dendritic cells (DCs) with potent immunoregulatory properties (designed as regulatory DCs), which displayed moderately high expression levels of major histocompatibility complex (MHC) molecules and extremely low levels of costimulatory molecules compared with their normal counterparts. Unlike human normal DCs, which caused the activation of allogeneic CD4(+) and CD8(+) T cells, human regulatory DCs not only induced their anergic state but also generated CD4(+) or CD8(+) regulatory T (Tr) cells from their respective naive subsets in vitro. Although murine normal DCs activated human xenoreactive T cells in vitro, murine regulatory DCs induced their hyporesponsiveness. Furthermore, transplantation of the primed human T cells with murine normal DCs into severe combined immunodeficient (SCID) mice enhanced the lethality caused by xenogeneic graft-versus-host disease (XGVHD), whereas transplantation of the primed human T cells with murine regulatory DCs impaired their ability to cause XGVHD. In addition, a single injection of murine regulatory DCs following xenogeneic or allogeneic transplantation protected the recipients from the lethality caused by XGVHD as well as allogeneic acute GVHD. Thus, the modulation of T cell-mediated immunity by regulatory DCs provides a novel therapeutic approach for immunopathogenic diseases.     

Stressed apoptotic tumor cells stimulate dendritic cells and induce specific cytotoxic T cells

We have previously reported that stressed apoptotic tumor cells are more immunogenic in vivo than nonstressed ones. Using confocal microscopy we have confirmed our previous observation that heat-stressed apoptotic 12B1-D1 leukemia cells (BCR-ABL(+)) express HSP60 and HSP72 on their surface. To explore how the immune system distinguishes stressed from nonstressed apoptotic tumor cells, we analyzed the responses of dendritic cells to these 2 types of apoptotic cells. We found that nonstressed and heat-stressed apoptotic 12B1-D1 cells were taken up by dendritic cells in a comparable fashion. However, when stressed apoptotic 12B1-D1 cells were coincubated with immature dendritic cells for 24 hours, this resulted in greater up-regulation of costimulatory molecules (CD40, CD80, and CD86) on the surface of dendritic cells. Moreover, stressed apoptotic 12B1-D1 cells were more effective in stimulating dendritic cells to secrete interleukin-12 (IL-12) and in enhancing their immunostimulatory functions in mixed leukocyte reactions. Furthermore, we demonstrated that immunization of mice with stressed apoptotic 12B1-D1 cells induced the secretion of T helper-1 (T(H)1) profile of cytokines by spleen cells. Splenocytes from mice immunized with stressed apoptotic cells, but not nonstressed ones, were capable of lysing 12B1-D1 and the parental 12B1 line, but not a B-cell leukemia line, A20. Our data indicate that stressed apoptotic tumor cells are capable of providing the necessary danger signals, likely through increased surface expression of heat shock proteins (HSPs), resulting in activation/maturation of dendritic cells and, ultimately, the generation of potent antitumor T-cell responses.     

树突状细胞诱导的抗肿瘤免疫诱导移植瘤细胞凋亡并抑制其增殖

目的研究树突状细胞(DC)体外诱导的细胞免疫能否抑制裸鼠移植瘤生长及其机制.方法联合应用粒/巨噬细胞集落刺激因子(GM-CSF)及白介素-4(IL-4)直接从肝癌患者外周血中培养出DC,以源于人肝癌细胞系HepG2肿瘤细胞的肿瘤抗原粗提物刺激DC,DC激活同源的T淋巴细胞产生细胞毒性T淋巴细胞(CTL),建立裸鼠人肝癌细胞系HepG2移植瘤模型.以CTL治疗裸鼠HepG2移植瘤并观察治疗效果,检测移植瘤标本肿瘤细胞凋亡情况.结果DC诱导的CTL通过诱导肿瘤细胞凋亡并抑制其增殖而抑制移植瘤生长.结论经肿瘤抗原激发的DC有可能在肿瘤的治疗中发挥重要作用.     

Virus-stimulated plasmacytoid dendritic cells induce CD4+ cytotoxic regulatory T cells

Immune responses to pathogens need to be maintained within appropriate levels to minimize tissue damage, whereas such controlled immunity may allow persistent infection of certain types of pathogens. Interleukin 10 (IL-10) plays an important role in such immune regulation. We previously showed that HSV-stimulated human plasmacytoid dendritic cells (pDCs) induced naive CD4+ T cells to differentiate into interferon gamma (IFN-gamma)/IL-10-producing T cells. Here we show that HSV-stimulated pDCs induce allogeneic naive CD4+ T cells to differentiate into cytotoxic regulatory T cells that poorly proliferate on restimulation and inhibit proliferation of coexisting naive CD4+ T cells. IL-3-stimulated pDCs or myeloid DCs did not induce such regulatory T cells. Both IFN-alpha and IL-10 were responsible for the induction of anergic and regulatory properties. High percentages of CD4+ T cells cocultured with HSV-stimulated pDCs, and to a lesser extent those cocultured with IL-3-stimulated pDCs, expressed granzyme B and perforin in an IL-10-dependent manner. CD4+ T cells cocultured with HSV-stimulated pDCs accordingly exhibited cytotoxic activity. The finding that virus-stimulated pDCs are capable of inducing CD4+ cytotoxic regulatory T cells suggests that this DC subset may play an important role in suppressing excessive inflammatory responses and also in inducing persistent viral infection.     

Abnormal presence of semimature dendritic cells that induce regulatory T cells in HIV-infected subjects

Dendritic cells (DCs), because they orchestrate the immune response to microbes, represent an ideal target for pathogens attempting to evade the immune system. We hypothesized that interactions between human immunodeficiency virus (HIV) and DCs lead to the development of a semimature state, in which DCs migrate to lymph nodes but induce tolerance in T cells, rather than immunity. We found that lymph nodes from untreated HIV-infected subjects contained an abundance of semimature DCs, the disappearance of which correlated with the initiation of highly active antiretroviral therapy (HAART). Such lymph nodes also contained an abundance of T cells that had a regulatory phenotype and that persisted after HAART. Lymph node DCs from untreated HIV-infected subjects cultured with normal allogeneic T cells induced these T cells to adopt the phenotype of regulatory T cells, an ability that was lost after HAART. We conclude that HIV infection correlates with the presence of semimature DCs that stimulate T cell tolerance rather than immunity. These regulatory T cells may contribute to the lack of effective HIV immune responses.     

Autoantigens signal through chemokine receptors: uveitis antigens induce CXCR3- and CXCR5-expressing lymphocytes and immature dendritic cells to migrate

We tested the hypothesis that interaction between autoantigens and chemoattractant receptors may be an important step in the development of autoimmunity. The retinal autoantigens S-antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP) can induce autoimmune uveitis in rodent models. We evaluated the chemotactic activity of S-Ag and IRBP and found that both induced migration of human and mouse immature dendritic cells (iDCs) and lymphocytes, but not neutrophils, monocytes, or mature DCs. Cross-desensitization studies and single-receptor transfected cells revealed that subfamily of alpha chemokine receptors CXCR5 and CXCR3 mediated the chemotactic effect of IRBP, while only CXCR3 was required for the chemotactic response to S-Ag. Examination of the relationships between chemoattraction and the ability to elicit pathology at the protein or peptide levels in the mouse uveitis model revealed dissociation of the capacity to induce uveitis, lymphocyte proliferation, and chemoattraction. These studies suggest that IRBP and S-Ag can initiate innate and, in sensitive individuals, adaptive immune response by attracting iDCs and T and B cells expressing CXCR3 and CXCR5.     

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFβ, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP− cells. Remarkably, CD25+GARP− T cells expanded in culture contained 3–5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25−GARP− cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) −infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation.     

Antigen-bearing immature dendritic cells induce peptide-specific CD8(+) regulatory T cells in vivo in humans

Regulatory T cells (T(R)s) can suppress the function of other effector T cells in the setting of autoimmunity, transplantation, and resistance to tumors. The mechanism for the induction of T(R)s has not been defined. We previously reported that an injection of immature dendritic cells (DCs) pulsed with influenza matrix peptide (MP) led 7 days later to antigen-specific silencing of effector T-cell function in the blood of 2 healthy human subjects. Here, we found that interferon-gamma-producing effectors return by 6 months. Importantly, in mixing experiments, CD8(+) T cells from the sample obtained 7 days after injection could suppress MP-specific effectors obtained before injection and those in recovery samples. This suppression or regulation was specific for the immunizing peptide (MP) and cell-dose dependent, and it required contact between the 2 samples. These data show the capacity of immature DCs to induce antigen-specific regulatory CD8(+) T cells in humans.     

Antidiabetogenic MHC class II promotes the differentiation of MHC-promiscuous autoreactive T cells into FOXP3+ regulatory T cells

Polymorphisms in MHC class II molecules, in particular around β-chain position-57 (β57), afford susceptibility/resistance to multiple autoimmune diseases, including type 1 diabetes, through obscure mechanisms. Here, we show that the antidiabetogenic MHC class II molecule I-A^b affords diabetes resistance by promoting the differentiation of MHC-promiscuous autoreactive CD4⁺ T cells into disease-suppressing natural regulatory T cells, in a β56–67-regulated manner. We compared the tolerogenic and antidiabetogenic properties of CD11c promoter-driven transgenes encoding I-A^b or a form of I-A^b carrying residues 56–67 of I-Aβ^g7 (I-A^b-g7) in wild-type nonobese diabetic (NOD) mice, as well as NOD mice coexpressing a diabetogenic and I-A^g7–restricted, but MHC-promiscuous T-cell receptor (4.1). Both I-A transgenes protected NOD and 4.1-NOD mice from diabetes. However, whereas I-A^b induced 4.1-CD4⁺ thymocyte deletion and 4.1-CD4⁺Foxp3⁺ regulatory T-cell development, I-A^b-g7 promoted 4.1-CD4⁺Foxp3⁺ Treg development without inducing clonal deletion. Furthermore, non–T-cell receptor transgenic NOD.CD11cP-I-A^b and NOD.CD11cP-IA^b-g7 mice both exported regulatory T cells with superior antidiabetogenic properties than wild-type NOD mice. We propose that I-A^b, and possibly other protective MHC class II molecules, afford disease resistance by engaging a naturally occurring constellation of MHC-promiscuous autoreactive T-cell clonotypes, promoting their deviation into autoregulatory T cells.     

Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients

CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg's) play an important role in the maintenance of immune tolerance. The mechanisms controlling the induction and maintenance of Treg's in humans need to be defined. We find that human myeloid dendritic cells (DCs) are superior to other antigen presenting cells for the maintenance of FOXP3(+) Treg's in culture. Coculture of DCs with autologous T cells leads to an increase in both the number of Treg's, as well as the expression of FOXP3 protein per cell both in healthy donors and myeloma patients. DC-mediated expansion of FOXP3(high) Treg's is enhanced by endogenous but not exogenous interleukin-2 (IL-2), and DC-T-cell contact, including the CD80/CD86 membrane costimulatory molecules. DCs also stimulate the formation of Treg's from CD25(-) T cells. The efficacy of induction of Treg's by DCs depends on the nature of the DC maturation stimulus, with inflammatory cytokine-treated DCs (Cyt-DCs) being the most effective Treg inducers. DC-induced Treg's from both healthy donors and patients with myeloma are functional and effectively suppress T-cell responses. A single injection of cytokine-matured DCs led to rapid enhancement of FOXP3(+) Treg's in vivo in 3 of 3 myeloma patients. These data reveal a role for DCs in increasing the number of functional FOXP3(high) Treg's in humans.     

Vasoactive intestinal peptide generates human tolerogenic dendritic cells that induce CD4 and CD8 regulatory T cells

Induction of antigen-specific tolerance is critical for autoimmunity prevention and immune tolerance maintenance. In addition to their classical role as sentinels of the immune response, dendritic cells (DCs) play important roles in maintaining peripheral tolerance through the induction/activation of regulatory T (T(reg)) cells. The possibility of generating tolerogenic DCs opens new therapeutic perspectives in autoimmune/inflammatory diseases. Characterizing endogenous factors that contribute to the development of tolerogenic DCs is highly relevant. We here report that the immunosuppressive neuropeptide vasoactive intestinal peptide (VIP) induces the generation of human tolerogenic DCs with the capacity to generate CD4 and CD8 T(reg) cells from their respective naive subsets. The presence of VIP during the early stages of DC differentiation from blood monocytes generates a population of IL-10-producing DCs unable to fully mature after the effects of inflammatory stimuli. CD4 T(reg) cells generated with VIP-differentiated DCs resemble the previously described Tr1 cells in terms of phenotype and cytokine profile. CD8 T(reg) cells generated with tolerogenic VIP DCs have increased numbers of IL-10-producing CD8(+)CD28(-)-CTLA4(+) T cells. CD4 and CD8 T(reg) cells primarily suppress antigen-specific T(H)1-mediated responses. Therefore, the possibility of generating or expanding ex vivo tolerogenic DC(VIPs) opens new therapeutic perspectives for treating autoimmune diseases and graft-versus-host disease after allogeneic transplantation in humans.     

Superoxide anions induce the maturation of human dendritic cells

Dendritic cells play a key role in immune responses. There is growing evidence that reactive oxygen species participate in signaling pathways involving nuclear factor (NF)-kappaB, leading to expression of important immune system genes. We found that, unlike H2O2, reactive oxygen species generated by the reaction of oxidase on xanthine induced early phenotypic maturation of dendritic cells by upregulating specific markers CD80, CD83, and CD86 and downregulating mannose receptor-mediated endocytosis. Maturation induced by xanthine oxidase was prevented by allopurinol, an inhibitor of xanthine oxidase activity, and by N-acetylcysteine. The proteasome inhibitor MG-132, which blocks NF-kappaB activation, also inhibited CD86 upregulation, but not endocytosis downregulation by reactive oxygen species. Finally, xanthine-xanthine oxidase enhanced or blocked antigen presentation by dendritic cells depending on whether they had been prepulsed or not with the antigen. Taken together, these results demonstrate that oxidative stress induces phenotypic and functional maturation of dendritic cells, partly through an NF-kappaB-dependent mechanism.