Dendritic cells generated in the presence of GM-CSF plus IL-15 prime potent CD8+ Tc1 responses in vivo

Dendritic cells (DC) comprise a system of professional antigen-presenting cells, which induce the stimulation of very rare antigen-specific naive T cells. DC progenitors can be stimulated to differentiate into immature DC by various growth factors, including GM-CSF and IL-4. Here we show that IL-15, in combination with GM-CSF, is a growth factor for murine DC. Murine bone marrow cells, depleted of T cells, B cells, I-A+ cells and Gr-1+ granulocytes, and cultured in the presence of GM-CSF plus IL-15 (IL-15 DC), yielded DC expressing high levels of CD11c and MHC class II molecules, as well as CD11b. These cells expressed significant levels of CD40, CD80 and CD86, and could stimulate allogeneic CD4+ T cells efficiently. Interestingly, IL-15 DC were far superior to DC generated with GM-CSF plus IL-4 in stimulating allogeneic CD8+ T cells in vitro. Consistent with this, IL-15 DC induced much more potent antigen-specific CD8+ T cell responses with high levels of Th1 cytokines in vivo, compared to DC generated with GM-CSF plus IL-4, or with GM-CSF plus TGF-beta, or with GM-CSF alone. Together, these data suggest that IL-15 promotes the development of DC, which induce potent Th1 and Tc1 responses in vivo. This suggests potential roles for these IL-15 DC cells in the immunotherapy of tumors and infectious diseases.     

Dendritic cells generated in the presence of interferon-alpha stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells

Dendritic cells (DCs) generated in the presence of IFN-alpha (IFN-DCs) exhibit high expression of major histocompatibility and co-stimulatory molecules and a potent ability to stimulate CD8(+) T-cell responses. Here, we found that IFN-DCs were more potent stimulators of bulk and purified CD8(+) T-cell proliferation, as compared with IL-4-DCs. In contrast, IFN-DCs were less efficient than IL-4-DCs in stimulating allogeneic CD4(+) T-cell proliferation, due to a weak induction of naive CD4(+)CD45RO(-) T-cell proliferation by these DCs. However, both DC populations induced similar levels of proliferation of memory CD4(+)CD45RO(+) T cells. IFN-DCs and IL-4-DCs exhibited a similar phenotype and production of IL-10 following maturation induced by CD40 ligation. In contrast, IFN-DCs produced higher levels of IL-10 during the first days of differentiation. In addition, neutralization of IL-10 during the differentiation of DCs increased the expression of DC-LAMP and MHC class II by IFN-DCs, and the ability of IFN-DCs to stimulate allogeneic CD4(+) T-cell proliferation at similar levels, than IL-4-DCs. Independently of IL-10 production, IFN-DCs were found to induce higher levels of CD4(+)T-cell apoptosis, this effect being more sticking on naive T cells. Finally, we demonstrated that IFN-DCs induced a differentiation bias of naive CD4(+) T cells towards Th1 and Tr1 cells, compared to IL-4-DCs. Taken together, these results indicate that, despite the induction of Tr1 cells and enhanced apoptosis of naive CD4(+) T cells, IFN-DCs are potent stimulators of CD8(+) and memory CD4(+) T cells, and induce a strong polarization of naive CD4(+) T cells towards Th1 cells, further supporting their use in immune-based therapy.     

Leukemic plasmacytoid dendritic cells share phenotypic and functional features with their normal counterparts

This work aims to further characterize the newly described leukemic plasmacytoid dendritic cells (LPDC), for which we had previously demonstrated their normal, PDC-like ability to produce IFN-alpha. In addition, LPDC also express the specific antigens BDCA-2 and BDCA-4. Importantly, they become fully competent antigen-presenting cells (APC) after a short maturation induced by IL-3 + CD40L or virus, exhibiting a characteristic APC phenotype (high expression of CD83 and of the costimulatory molecules CD40, CD80, CD86). Whereas IL-3 + CD40L-activated LPDC prime naive CD4(+) T cells towards a Th2 pathway (IL-4-secreting T cells), virus-activated LPDC drive a Th1 profile (IFN-gamma-secreting T cells). Moreover, we show in one case that LPDC are able to capture, process and present exogenous antigens, leading to the activation of both CD4(+) and CD8(+) T cell clones in an antigen-specific manner. This study further characterizes the phenotype and immunological functions of LPDC.     

Granulocyte colony-stimulating factor promotes the generation of regulatory DC through induction of IL-10 and IFN-alpha

We have recently demonstrated that G-CSF promotes the generation of human T regulatory (T(REG)) type 1 cells. In this study, we investigated whether the immunomodulatory effects of G-CSF might be mediated by DC. CD14(+) monocytes were cultured with serum collected after clinical administration of G-CSF (post-G), which contained high amounts of IL-10 and IFN-alpha. Similar to incompletely matured DC, monocytes nurtured with post-G serum acquired a DC-like morphology, expressed high levels of costimulatory molecules and HLA-DR, and exhibited diminished IL-12p70 release and poor allostimulatory capacity. Importantly, post-G DC-like cells were insensitive to maturation stimuli. As shown by neutralization studies, IFN-alpha and, even more pronounced, IL-10 contained in post-G serum inhibited IL-12p70 release by post-G DC-like cells. Furthermore, phenotypic and functional features of post-G DC-like cells were replicated by culturing post-G monocytes with exogenous IL-10 and IFN-alpha. Post-G DC-like cells promoted Ag-specific hyporesponsiveness in naive allogeneic CD4(+) T cells and orchestrated a T(REG) response that was dependent on secreted TGF-beta 1 and IL-10. Finally, neutralization of IL-10 and IFN-alpha contained in post-G serum translated into abrogation of the regulatory features of post-G DC-like cells. This novel mechanism of immune regulation effected by G-CSF might be therapeutically exploited for tolerance induction in autoimmune disorders.     

HIV-1 matrix protein p17 prevents loss of CD28 expression during IL-2-induced maturation of naïve CD8(+) T cells

Na?ve CD8(+) T cells differentiate into effectors secreting various cytokines that modulate immune functions. A striking finding for most HIV-1-infected patients is that they accumulate CD8(+) T cells belonging to early and intermediate differentiated elements. Structural HIV-1 proteins, and among these the matrix protein p17, have been associated with loss of functional competence by different immune cells. We therefore evaluated the influence of p17 on na?ve CD8(+) T-cell activation and maturation. Anti-CD3 mAb preactivation and subsequent IL-2 stimulation are able to drive human naive CD(+) T cells to an effector phenotype characterized, among other features, by downregulation of the co-stimulatory molecule CD28. Strikingly, however, IL-2-induced downmodulation of CD28 was completely prevented by p17, and cells derived from p17-stimulated cultures showed a strong Tc1 polarization that was fourfold higher than that observed in IL-2-stimulated cultures.Moreover, p17 preserved a markedly high proportion of CD8(+) T cells that were able to respond to CD28 triggering with a proinflammatory cytokine storm. Our evidence suggests that p17 has important effects on cytokine polarization and phenotype of terminally differentiated CD8(+) T cells, and that new p17-based therapeutic approaches could control or prevent HIV-1-related immune disorders.     

Differential effect of CD8(+) and CD8(-) dendritic cells in the stimulation of secondary CD4(+) T cells

Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4(+) and primary CD8(+) T cells; the myeloid-related CD8alpha(-) DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8alpha despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4(+) T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8(-) DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8(+) DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8(+) DC, but was related to the levels of IL-2 induced. The deficiency in the CD8(+) DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8(+) splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4(+) T cells to sustain their proliferation.     

The ability of murine dendritic cell subsets to direct T helper cell differentiation is dependent on microbial signals

Dendritic cells (DC) initiate T cell responses and direct the class of T cell immunity through the production of Th-polarizing cytokines. In the mouse, immunization with CD8alpha(+) DC has led to Th1 priming whereas immunization with CD8alpha(-) DC has been associated with Th2 induction. Here, we use a direct T cell priming assay in vitro to re-examine the Th-directing potential of total DC or purified CD4(+) DC, CD8alpha(+) DC or CD4(-) CD8alpha(-) (double-negative; DN) DC subsets from mouse spleen. We show that the default Th effector phenotype induced by priming with DC depends on the protocol used for T cell purification, the T cell:antigen-presenting cell ratio and the antigen dose but is only marginally affected by DC subtype. All DC subsets can direct increased Th1 development in response to microbial stimuli known to elicit IL-12 production. Similarly, all subsets can suppress Th1 development and allow Th2 cellsto expand upon exposure to IL-10-inducing microbial agents. The flexibility of DC in directing Th development in function of microbial signals argues against the notion of pre-determined "DC1" and "DC2" subsets and suggests that multiple DC subtypes can direct an appropriate Th response to different classes of infectious agents.     

Notch1 expression on T cells is not required for CD4+ T helper differentiation

Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.     

Environmental pollutant tributyltin promotes Th2 polarization and exacerbates airway inflammation

It has been shown that a relatively high dose of tributyltin (TBT), which is recognized as a particularly notable environmental pollutant, exerts immunotoxic effects such as thymic atrophy via induction of T cell apoptosis. However, the effect of low doses of TBT on the immune responses remains unknown. Here we show that environmentally relevant doses of TBT promoted strong Th2 polarization via suppression and augmentation of Th1 and Th2 development, respectively, from naive CD4(+) T cells primed with anti-CD3 and splenic antigen-presenting cells (APC). TBT-induced Th2 polarization was indirect, working through APC via suppression of IL-12 production by macrophages/DC and the augmentation of IL-10 production by B cells. Th2 polarization was also induced in mice treated with TBT and immunized with OVA or infected with Nippostrongylus brasiliensis. Furthermore, airway inflammation in mice sensitized and challenged with OVA was exacerbated by the administration of TBT with concomitant augmentation of Th2-type immunity. Our results highlight the fact that an important environmental pollutant TBT may present significant risk for the induction of allergic diseases via promotion of Th2 polarization.     

Instruction of naive CD4+ T-cell fate to T-bet expression and T helper 1 development: roles of T-cell receptor-mediated signals

Using T-cell receptor (TCR) transgenic mice, we demonstrate that TCR stimulation of naive CD4(+) T cells induces transient T-bet expression, interleukin (IL)-12 receptor beta2 up-regulation, and GATA-3 down-regulation, which leads to T helper (Th)1 differentiation even when the cells are stimulated with peptide-loaded I-A(b)-transfected Chinese hamster ovary cells in the absence of interferon-gamma (IFN-gamma) and IL-12. Sustained IFN-gamma and IL-12 stimulation augments naive T-cell differentiation into Th1 cells. Intriguingly, a significant Th1 response is observed even when T-bet(-/-) naive CD4(+) T cells are stimulated through TCR in the absence of IFN-gamma or IL-12. Stimulation of naive CD4(+) T cells in the absence of IFN-gamma or IL-12 with altered peptide ligand, whose avidity to the TCR is lower than that of original peptide, fails to up-regulate transient T-bet expression, sustains GATA-3 expression, and induces differentiation into Th2 cells. These results support the notion that direct interaction between TCR and peptide-loaded antigen-presenting cells, even in the absence of T-bet expression and costimulatory signals, primarily determine the fate of naive CD4(+) T cells to Th1 cells.     

IL-17/IFN-γ double producing CD8+ T (Tc17/IFN-γ) cells: a novel cytotoxic T-cell subset converted from Tc17 cells by IL-12

It has been reported that IFN-γ-producing CD8(+) T (Tc1) cells express cytotoxic molecules such as perforin and granzyme B to exhibit higher cytotoxicity against tumor cells compared with Tc2 cells. However, the critical role of IL-17-producing CD8(+) T (Tc17)-cell subsets in tumor immunity remains unclear. Tc17 cells differentiated from naive CD8(+) T cells did not possess cytotoxic molecules and exhibited no strong cytotoxicity. However, when Tc17 effector cells were further cultured with IL-12, they converted into IFN-γ-producing Tc17 cells, which mainly consisted of IL-17/IFN-γ double-producing cells (Tc17/IFN-γ). IL-12-converted Tc17 cells also acquired cytotoxic function in addition to IFN-γ producibility. Moreover, they showed strong anti-tumor activity both in vitro and in vivo as well as Tc1 cells. Among four distinct subsets in IL-12-converted Tc17 cell populations, the isolated Tc17/IFN-γ cells exhibited cytotoxicity as well as IFN-γ-producing Tc1-like cells. Thus, we first indicate direct evidence that Tc17/IFN-γ cells, which were plastically converted from non-cytotoxic Tc17 cells by IL-12, exhibited strong anti-tumor activity as well as Tc17 cell-derived Tc1-like cells.     

The Toll-like receptor ligand MALP-2 stimulates dendritic cell maturation and modulates proteasome composition and activity

A 2-kDa synthetic derivative of the macrophage-activating lipopeptide (MALP-2) from Mycoplasma fermentans is a potent inducer of monocytes/macrophages and improves the immunogenicity of antigens co-administered by systemic and mucosal routes. Dendritic cells (DC) are the most potent antigen-presenting cells, which are able to prime naive T cells in vivo. To elucidate the underlying mechanisms of MALP-2 adjuvanticity, we analyzed its activity on bone marrow-derived murine DC. In vitro stimulation of immature murine DC with MALP-2 resulted in the induction of maturation with up-regulated expression of MHC class II, costimulatory (CD80, CD86) and adhesion (CD40, CD54) molecules. MALP-2 also enhances the secretion of cytokines (IL-1alpha, IL-6 and IL-12), and increases DC stimulatory activity on naive and antigen-specific T cells. Further studies demonstrated that MALP-2 treatment of DC results in a dose-dependent shift from the protein pattern of proteasomes to immunoproteasomes (up-regulation of LMP2, LMP7 and MECL1), which correlates with an increased proteolytic activity. Thus, the adjuvanticity of MALP-2 can be mediated, at least in part, by the stimulation of DC maturation, which in turn leads to an improved antigen presentation. Therefore, MALP-2 is a promising molecule for the development of immune therapeutic or prophylactic interventions.     

Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-γ production by T helper 1 cells

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-γ (IFN-γ), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell-mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, Northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-γ bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 bymurine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral blood mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-γ production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.     

尖锐湿疣患者外周血Thl/Th2及Tc1/Tc2亚群的检测及意义

目的 探讨辅助性T淋巴细胞(Th细胞)和细胞毒性T淋巴细胞(Tc细胞)的极化情况在尖锐湿疣(CA)发病机理中的作用及其与疾病复发的关系.方法 采用三色荧光抗体染色流式细胞术检测细胞内细胞因子的方法 ,检测30例CA患者和20名健康对照者外周血CD3+CD8-/IFN-γ+(Th1)、CD3+CD8-/IL4+(Th2)、CD3+CD8+/IFN-γ+(Tc1)和CD3+CD8+,IL-4+(Tc2)细胞比例.结果 与健康对照组比较,CA患者外周血中Thl细胞含量显著减少(P<0.01),Tc1细胞含量、Th1/Th2比值、Tc1/Tc2比值均减少(P<0.05);其中15例复发CA患者的Th1/Th2比值较正常对照显著减少(P<0.01),而Te1/Te2比值与健康对照组比较差异无统计学意义.结论 CA患者外周血Th1和Tc1细胞数减少,而Th2和Tc2细胞相对占优势,复发的CA患者这种失衡更趋严重.CA患者机体免疫状态可能存在Th1→Th2、Tc1→Tc2方向的漂移,这种漂移可能是人乳头瘤病毒(HPV)不易被有效清除或CA反复发生的一种免疫机制.     

CD8(+) T cells regulate immune responses in a murine model of allergen-induced sensitization and airway inflammation

The role of CD8(+) T cells in the development of allergic airway disease is controversial. On the one hand, CD8(+) T cells are known to inhibit the development of airway hyperreactivity (AHR) in murine models of asthma. In humans, IL-10-producing CD8(+) T cells were shown to act as regulatory cells, inhibiting both proliferation and cytokine secretion of T cells. On the other hand, CD8(+) T cells can promote IL-5-mediated eosinophilic airway inflammation and the development of AHR in animal models. To examine this, we investigated the role of CD8(+) T cells during the induction of allergen-induced AHR and demonstrated a protective effect of CD8(+) T cells. Depletion of CD8(+) T cells prior to the immunization led to increased Th2 responses and increased allergic airway disease. However, after development of AHR, CD8(+) T cells that infiltrated the lungs secreted high levels of IL-4, IL-5 and IL-10, but little IFN-gamma, whereas CD8(+) T cells in the peribronchial lymph nodes or spleen produced high levels of IFN-gamma, but little or no Th2 cytokines. These data demonstrate protective effects of CD8(+)T cells against the induction of immune responses and show a functional diversity of CD8(+) T cells in different compartments of sensitized mice.     

抗原肽浓度对初始T细胞向Th1/Tc1、Th2/Tc2分化影响的研究

目的 研究不同浓度抗原肽(OVA)对初始CD4~+T细胞向Thl/Th2、初始CD8~+T细胞向Tc1/Tc2分化偏向性的影响.方法 采用系列浓度TCR特异性抗原肽与小鼠脾脏树突状细胞联合培养,然后刺激小鼠初始CD4~+或CD8~+T细胞.流式细胞术检测细胞内因子IFN-γ和IL-4的表达水平,同时用CFSE检测活化T细胞的增殖水平.结果 低浓度抗原肽刺激初始CD4~+T向Th2、初始CD8~+T向Tc2分化;而高浓度抗原肽刺激初始CD4~+T向Th1、初始CD8~+T向Tc1分化.Th细胞比Tc细胞受影响程度要明显.结论 抗原肽在很大浓度范围内均可以刺激初始T细胞的活化,但随着浓度的升高,分化方向从Th2、Tc2逐渐向Th1、Tc1倾斜.这一结论为动物实验中疫苗免疫剂量的控制提供了重要的参考价值.     

CD40L-expressing CD8 T cells prime CD8alpha(+) DC for IL-12p70 production

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.     

Dendritic cells differentiated in the presence of a single-stranded viral RNA sequence conserve their ability to activate CD4 T lymphocytes but lose their capacity for Th1 polarization

Monocyte-derived dendritic cells (DCs) differentiate in the presence of Toll-like-receptor (TLR) ligands in the course of ongoing infections. A single-stranded RNA (ssRNA) sequence, corresponding to the sequence of the U5 region of human immunodeficiency virus type 1 RNA, was used to mimic viral activation of TLR7 in human DCs. We determined the effector potential of DCs differentiated in the presence of this ssRNA molecule (ssRNA-DCs). ssRNA-DCs phenotypically resembled mature DCs. In contrast, their capacity to allostimulate naive CD4(+) T cells resembled that of conventional immature DCs and could be increased by TLR4 stimulation. Th1 polarization of CD4(+) T cells and production of interleukin 12p70 (IL-12p70) by ssRNA-DCs were selectively abrogated in response to a late TLR4, but not in response to a CD40, maturation signal. Inhibition of p38 mitogen-activated protein kinase partially restored IL-12p70 secretion but did not restore Th1 polarization, whereas addition of exogenous IL-12 led to recovery of Th1 polarization. In contrast to lipopolysaccharide, ssRNA induced IL-12p70 production at the very earliest stages of DC differentiation, indicating a particular role for TLR7 in monocyte-derived DCs recently engaged in differentiation. These data demonstrate generation of phenotypically mature DCs with the ability to expand CD4(+) T lymphocytes lacking Th1/2-polarizing capacity.