Activation-induced expression of murine CD83 on T cells and identification of a specific CD83 ligand on murine B cells

Human CD83 is a cell surface protein expressed predominantly by dendritic cells (DC) and lymphoid cells. So far, there exists no information on the function and distribution of mCD83. Here we demonstrate that mCD83 is moderately expressed on resting T cells and DC, but strongly increases in its expression on T cells following activation with antigenic peptides or T cell receptor-specific mAb. When returning to the resting state, T cells down-regulate CD83 again. Ig fusion proteins which express the extracellular part of the mCD83 molecule (mCD83-Ig) specifically inhibit antigen-specific T cell proliferation and IL-2 secretion in spleen cell cultures from DO11.10 T cell receptor transgenic mice. Staining of spleen cells from BALB/c, XID and mu MT (B cell) knockout mice with mCD83-Ig proteins reveals the presence of a CD83 ligand predominantly expressed most likely by B220(+) cells since spleen cells from mu MT knockout mice do not bind mCD83-Ig. CD83, besides its established expression on human dendritic cells, thus, also represents a new marker molecule on activated T cells which with its specific ligand is involved in the regulation of T cell responses.     

The soluble form of CD83 dramatically changes the cytoskeleton of dendritic cells

CD83 is the best-known surface marker for mature dendritic cells (DC) and recently we could show that a soluble form of CD83 inhibits DC maturation. In addition, this soluble form inhibits DC-mediated T cell proliferation in vitro and in vivo. Furthermore, several viruses induce CD83 degradation or shedding in infected DC. A soluble form of CD83 was also found in plasma and serum of healthy individuals and interestingly at highly elevated levels in a number of haematological malignancies. Thus, CD83 also has functional implications for the immune response. However, the molecular mechanism is not well defined. Here we describe for the first time that soluble CD83 completely changed the cytoskeleton (analysed using phalloidin-, tubulin- and fascin-specific antibodies) when administered at a concentration of 10 microg/ml to mature DC. The cells rounded off and had only short, truncated, or no veils at all. Furthermore, soluble CD83-treated cells were completely inhibited in their ability to form clusters with T cells, an absolute prerequisite in order to stimulate T cells.     

Enhanced CD40 and ICOSL expression on dendritic cells surface improve anti-tumor immune responses; effectiveness of mRNA/chitosan nanoparticles

Abstract

Objective: To improve dendritic cells (DCs) function, we targeted DCs to over express CD40 and inducible costimulator ligand (ICOSL) costimulatory molecules along with total messenger RNA (mRNA) of tumor cells to achieve a safe and effective system for treatment of tumor.

Materials and methods: We generated CD40 and ICOSL mRNA in vitro and manipulated DCs using chitosan nanoparticles and also lipofectamine transfection system then examined in vitro and in vivo.

Results: Mice bone marrow derived DCs pulsed with total tumor mRNA/CD40 mRNA or ICOSL mRNA showed higher expression of DCs maturation markers (CD40, ICOSL, CD86, and MHC-II) and accelerated secretion of pro-inflammatory cytokines. Co-culture of DCs with T cells enhanced proliferation of T cells and shift toward stronger Th1 cytokine responses especially in presence of CD40 over expressed DCs. Intra-tumor administration of manipulated DCs to 4T1 tumor mice model showed delay in growth of tumor volume, trend to increase in mice survival, and stronger anti-tumor cytokines production in splenocytes of mice model (with higher efficacy of mRNA/chitosan nanoparticle system).

Conclusions: Hence, we suggest that targeting intra-tumor DCs to elicit expression of CD40 and ICOSL and present broad range of tumor antigens could yield effective anti-tumor responses. In this regard, CD40 molecule manipulation trigger stronger functions, while mRNA/chitosan nanoparticles system could provide a high potent tool for targeting strategies.     

可溶性CD83与免疫抑制

CD83是树突状细胞(DC)上重要的表面标志,同时也是重要的功能分子。对CD83功能的认识是从对可溶性CD83(sCD83)研究开始的。sCD83主要通过膜结合型CD83(mCD83)蛋白水解形成,存在于正常人血清中,具有免疫抑制活性。sCD83通过影响细胞骨架抑制DC的功能。在某些血液病和病毒感染患者血清内浓度升高,并引起免疫抑制,导致血液肿瘤细胞和病毒的免疫逃逸。sCD83能够显著抑制实验性自身免疫病病变的发生,在治疗自身免疫病方面展示了广阔的前景。     

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca(2+) release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter-receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte-derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down-modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN-gamma > CD40 cross-linking). Although weak, IFN-gamma consistently induces DC maturation. De novo-synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF-kappa B activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti-CD38 mAb induces up-regulation of CD83 expression and IL-12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL-12 secretion and MDDC-induced allogeneic T cell proliferation.     

Human dendritic cells activate T lymphocytes via a CD40: CD40 ligand-dependent pathway

The CD40: CD40 ligand (CD40L) interaction provides T lymphocyte-mediated help for B lymphocyte and monocyte function but has also been shown to serve as a co-stimulus for T lymphocyte activation. In this report, we studied the regulation of CD40 expression and its functional relevance for the human dendritic cell (DC) stimulation of T lymphocytes. Only a small subpopulation of directly isolated blood DC expressed CD40. However, CD40 was rapidly up-regulated by culture, and its expression was further enhanced by interleukin (IL)-1α, IL-1β, IL-3, tumor necrosis factor-α and granulocyte/macrophage-colony-stimulating factor. Expression of CD40L on DC was not detected. The proliferation of T lymphocytes in an allogeneic mixed leukocyte reaction, stimulated by blood DC or epidermal Langerhans cells, was significantly reduced in the presence of the CD40 immunoglobulin (CD40Ig) fusion protein or CD40L monoclonal antibodies. Cross-linking of CD40 on directly isolated DC with mouse CD40L trimer (mCD40LT) markedly augmented CD80 and CD86 up-regulation. Nevertheless, the same cross-linking mCD40LT inhibited DC stimulated T lymphocyte proliferation. When CD40Ig was added simultaneously with CTLA-4Ig, only minimal and variable additional inhibition of DC-stimulated allogeneic T lymphocyte proliferation and IL-2 secretion was observed, compared to each fusion protein alone. These results suggest that both CD80/CD86-dependent and -independent components of DC-T lymphocyte CD40: CD40L co-stimulation exist and further emphasize that the majority of blood DC have to differentiate or be activated to express co-stimulatory molecules.     

CD83 on murine APC does not function as a costimulatory receptor for T cells

The transmembrane glycoprotein CD83 is rapidly upregulated on murine and human DC upon maturation and therefore a costimulatory function for T cell activation has been suggested. Studies employing human APC indeed showed that CD83 expression was positively correlated to the stimulatory capacity of the APC. Murine APC that were CD83 deficient however, did not display a reduced capacity to activate T cells. To elucidate this contradiction, we thoroughly compared the stimulatory capacity of CD83-overexpressing and CD83-deficient APC. Here we show that CD83 expression levels on APC did not affect the capacity of the APC to activate CD8(+) T cells. CD83 expression levels did not significantly affect CD4(+) T cell activation in vivo, but a weak positive correlation of CD83 expression with CD4(+) T cell activation was observed in vitro under suboptimal stimulation conditions. As CD83 expression also positively correlated with MHC-II but not with MHC-I expression, this differential stimulation specifically of CD4(+) T cells could be explained by a higher density of MHC-II peptide complexes on the APC surface. Taken together, our results strongly suggest that CD83 does not deliver crucial costimulatory signals to murine T cells.     

A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera.

CD83 is an inducible glycoprotein expressed predominantly by dendritic cells (DC) and B lymphocytes. Expression of membrane CD83 (mCD83) is widely used as a marker of differentiated/activated DC but its function and ligand(s) are presently unknown. We report the existence of a soluble form of CD83 (sCD83). Using both a sCD83-specific ELISA and Western blotting, we could demonstrate the release of sCD83 by mCD83(+) B cell and Hodgkin's disease-derived cell lines, but not mCD83(-) cells. Inhibition of de novo protein synthesis did not affect the release of sCD83 during short-term (2 h) culture of cell lines although mCD83 expression was significantly reduced, suggesting sCD83 is generated by the release of mCD83. Isolated tonsillar B lymphocytes and monocyte-derived DC, which are mCD83(low), released only low levels of sCD83 during culture. However, the differentiation/activation of these populations both up-regulated mCD83 and increased sCD83 release significantly. Analysis of sera from normal donors demonstrated the presence of low levels (121 +/- 3.6 pg/ml) of circulating sCD83. Further studies utilizing purified sCD83 and the analysis of sCD83 levels in disease may provide clues to the function and ligand(s) of CD83.     

CD40分子在树突状细胞抗人多发性骨髓瘤免疫应答中的作用及其机理

目的为研究CD40分子在人树突状细胞(DC)抗多发性骨髓瘤(MM)中的作用及其机制。方法采用CD40配体(CD40L)转基因细胞及抗CD40的激发型单克隆抗体在体外激发DC,并通过DC细胞计数、形态学和细胞表型分析、IL-12定量检测、DC对MM抗原的摄取及混合淋巴细胞反应等手段对其进行研究。结果CD40分子配基化可促进DC的体外增殖和分化,使DC增加分泌IL-12,并下调DC摄取抗原的能力,促进DC的成熟,同时赋予DC激发自体CD8+细胞增殖的作用,使后者对MM细胞产生特异性的杀伤作用。结论CD40分子激发不仅有利于DC的增殖、分化和增强激发T细胞增殖,而且Th细胞表达的CD40L是DC获得直接激发抗MM抗原特异性CD8+T细胞的关键分子。     

Priming of CD8+ T cell responses by pathogens typically depends on CD70-mediated interactions with dendritic cells

The CD27/CD70-interaction has been shown to provide a costimulatory and survival signal for T cells in vitro and in vivo. Recently, CD70 expression by DC was found to be important for the priming of CD8+ T cells. We show here that blocking CD70 interactions has a significant impact on priming of CD8+ T cell responses by vaccinia virus (VV), Listeria monocytogenes and vesicular stomatitis virus (VSV) in mice. However, the priming of specific CD8+ T cells upon infection with lymphocytic choriomeningitis virus (LCMV) was only marginally reduced by CD70-blockade. Blocking of CD70 prevented CD8+ T cell priming in DIETER mice, a model in which presentation of LCMV-derived epitopes can be induced selectively in dendritic cells (DC). In contrast, CD70-CD27 interactions were not important for the priming of VSV-specific CD4+ T cells or class switch of neutralizing antibodies. As we show that priming of CD8+ T cells by the pathogens used here is dependent on antigen presentation by DC and that infection results in up-regulation of CD70 on DC, we conclude that CD70 expression on DC plays an important role in the priming of CD8+ T cells by pathogens. Moreover, the lack of CD70 cannot be completely compensated for by other costimulatory molecules.     

Increased expression of DC-SIGN+IL-12+IL-18+ and CD83+IL-12-IL-18- dendritic cell populations in the colonic mucosa of patients with Crohn's disease

Dentritic cells (DC) as antigen-presenting cells are most likely responsible for regulation of abnormal T cell activation in Crohn's disease (CD), a chronic inflammatory bowel disease. We have analyzed the expression of activation and maturation markers on DC in the colon mucosa from patients with CD compared with normal colon, using immunohistochemical techniques. We found two distinct populations of DC present in CD patients: a DC-specific ICAM-3 grabbing non-integrin (DC-SIGN)(+) population that was present scattered throughout the mucosa, and a CD83(+) population that was present in aggregated lymphoid nodules and as single cells in the lamina propria. In normal colon the number of DC-SIGN(+) DC was lower and CD83(+) DC were detected only in very few solitary lymphoid nodules. Co-expression of activation markers and cytokine synthesis was analyzed with three-color confocal laser scanning microscopy analysis. CD80 expression was enhanced on the majority of DC-SIGN(+) DC in CD patients, whereas only a proportion of the CD83(+) DC co-expressed CD80 in CD as well as in normal tissue. Surprisingly, IL-12 and IL-18 were only detected in DC-SIGN(+) DC and not in CD83(+) DC. A similar pattern of cytokine production was observed in normal colon albeit to a much lesser extent. The characteristics of these in-situ-differentiated DC markedly differ from the in-vitro-generated DC that simultaneously express DC-SIGN, CD83 and cytokines.     

Ex vivo induction of viral antigen-specific CD8 T cell responses using mRNA-electroporated CD40-activated B cells

Cell-based immunotherapy, in which antigen-loaded antigen-presenting cells (APC) are used to elicit T cell responses, has become part of the search for alternative cancer and infectious disease treatments. Here, we report on the feasibility of using mRNA-electroporated CD40-activated B cells (CD40-B cells) as alternative APC for the ex vivo induction of antigen-specific CD8(+) T cell responses. The potential of CD40-B cells as APC is reflected in their phenotypic analysis, showing a polyclonal, strongly activated B cell population with high expression of MHC and co-stimulatory molecules. Flow cytometric analysis of EGFP expression 24 h after EGFP mRNA-electroporation showed that CD40-B cells can be RNA transfected with high gene transfer efficiency. No difference in transfection efficiency or postelectroporation viability was observed between CD40-B cells and monocyte-derived dendritic cells (DC). Our first series of experiments show clearly that peptide-pulsed CD40-B cells are able to (re)activate both CD8+ and CD4(+) T cells against influenza and cytomegalovirus (CMV) antigens. To demonstrate the ability of viral antigen mRNA-electroporated CD40-B cells to induce virus-specific CD8+ T cell responses, these antigen-loaded cells were co-cultured in vitro with autologous peripheral blood mononuclear cells (PBMC) for 7 days followed by analysis of T cell antigen-specificity. These experiments show that CD40-B cells electroporated with influenza M1 mRNA or with CMV pp65 mRNA are able to activate antigen-specific interferon (IFN)-gamma-producing CD8(+) T cells. These findings demonstrate that mRNA-electroporated CD40-B cells can be used as alternative APC for the induction of antigen-specific (memory) CD8(+) T cell responses, which might overcome some of the drawbacks inherent to DC immunotherapy protocols.     

Induction,regulation, and function of soluble TRAP (CD40 ligand) during interaction of primary CD4+ CD45RA+ T cells with dendritic cells

To assess the induction, regulation, and the relative roles of cell surface tumor necrosis factor-related activation protein (TRAP; CD40 ligand) and the soluble form of TRAP (sTRAP) in the initial phase of T cell activation, primary CD4⁺ CD45RA⁺ (naive) T cells were co-cultured with mature Langerhans' cells (mLC) in the presence of superantigen. In this cell system, TRAP was very efficiently induced in T cells at both the mRNA and protein levels. After appearing on the cell surface, TRAP was rapidly down-regulated by a mechanism triggered through interaction of TRAP with CD40 on mLC. Co-culture of T cells with mLC led to the release of sTRAP, an 18-kDa protein capable of binding to CD40. Experimental data strongly suggest that sTRAP is not released by proteolytic cleavage of TRAP on the cell surface, but is generated in an intracellular compartment. Release of sTRAP and induction of TRAP cell surface expression were found to be regulated independently. In terms of function, sTRAP cannot compete with cell surface TRAP for ligation of CD40 on mLC, indicating that sTRAP release is not a mechanism for termination of the TRAP/CD40 interaction. However, sTRAP on its own rapidly down-regulates CD40 expression on mLC and has long-lasting anti-apoptotic effects on dendritic cells. Thus, we infer from our results obtained in vitro that primary activation of CD4⁺ T cells by dendritic cells in the lymphoid tissues leads to release of sTRAP, which may act on CD40⁺ bystander cells in a cytokine-like fashion.     

CD5− dendritic epidermal T cells are derived from CD5+ precursor cells

Murine Thy-1⁺, TcR Vγ3/Vδ⁺ dendritic epidermal T cells (DETC) differ from most other T cell subsets by the absence of CD4 and CD8 antigens as well as the lack of CD5 expression. To see whether negativity for those antigens is an intrinsic feature of a given T cell population or if such triple-negative T cells go through a maturational stage where they express these antigens, we determined the phenotype of TcR Vγ3⁺ fetal thymocytes which are the precursor cells of DETC. We found that TcR Vγ3⁺ fetal thymocytes phenotypically differ from mature DETC in that they are CD5⁺, mostly CD8⁺ and partly CD4⁺. The injection of fetal thymic suspensions containing TcR Vγ3⁺/CD5⁺ (but not TcR Vγ3⁺/CD5^?) thymocytes into Thy-1-disparate athymic nude mice resulted in the appearance of donor-type TcR Vγ3⁺/CD5^? dendritic cells in the recipients' epidermis, indicating that TcR Vγ3⁺ thymocytes are indeed the precursors of CD5^? DETC. Tracing CD5 expression on DETC precursors during their intrathymic maturation and their migration to the fetal skin, we found that (i) the earliest DETC precursor cells as defined by TcR Vγ3 expression express high levels of CD5 antigen (day 15 of gestation), (ii) after day 16 of gestation 70% of TcR Vγ3⁺ thymocytes express high and 30% express intermediate levels of CD5, (iii) TcR Vγ3⁺ cells in the fetal blood express low levels of CD5, (iv) the first TcR Vγ3⁺ cells entering the epidermis express very low levels of this antigen and (v) TcR Vγ3⁺ epidermal cells later than day 19 of gestation are CD5^?. A similar down-regulation of CD5 expression on DETC precursors was also noted when TcR Vγ3⁺ cells were cultured in vitro. Even the addition of PMA and ionomycin, which up-regulates CD5 expression on TcR α/β-bearing thymocytes and lymph node T cells, could not prevent down-regulation on DETC precursors. The described cell system may serve as a useful tool in further experiments aimed to clarify the function of the CD5 glycoprotein as well as the mechanism(s) regulating its expression.     

CD40配基化调节小鼠树突状细胞上B7-H3分子表达的研究

目的 探讨CD40配基化对小鼠骨髓来源树突状细胞上B7-H3分子表达的调节作用及其生物学意义。方法 采用GM-CSF和IL-4联合方案体外诱导小鼠髓系DC，并利用mCD40-CHO和TNF-α分别刺激凋亡肿瘤细胞负载的Dc制备成熟DC；采用间接免疫荧光标记法检测成熟Dc上B7-H3分子的表达；RT-PCR检测B7-H3 mRNA转录水平；混合淋巴细胞反应（MLR）和B7-H3单抗阻断实验分析CD40配基化的DC表面B7-H3分子在T细胞活化中的作用；^3H-TdR掺入试验检测DC对T淋巴细胞的促增殖效应；ELISA测定各组MLR反应和DC培养上清中IFN-γ分泌水平。结果 B7-H3分子在DC不同分化发育阶段均有表达，CD40配基化能显著上调凋亡肿瘤细胞负载的DC中B7-H3表达，TNF-α激发的DC弱表达（P〈0．05）；阻断CD40配基化的DC上B7-H3分子能抑制T细胞增殖和IFN-γ分泌；CD40配基化促进凋亡肿瘤细胞负载的DC分泌IFN-γ量也明显高于TNF-α组（P〈0．05）。结论 体外CD0配基化DC的B7-H3分子上调性表达有助于其刺激T细胞增殖和IFN-γ的产生。     

CD2+/CD14+ monocytes rapidly differentiate into CD83+ dendritic cells

Since denditric cells (DC) represent the main players linking innate and adaptive immunity, their prompt generation from blood cells would be instrumental for an efficient immune response to infections. Consistent with this, CD2+ monocytes were found to express the DC maturation marker CD83, along with acquisition of high antigen-presenting activity, after a surprisingly short time in culture. This rapid process is associated with expression of IFN-alpha/beta genes and secretion of low levels of pro-inflammatory cytokines. Exposure of monocytes to IFN-alpha, but not to IL-4, induced persistence of CD2+/CD83+ cells, which were fully competent in stimulating primary responses by naive T cells. These results unravel the natural pathway by which infection-induced signals rapidly transform pre-armed monocytes into active DC.     

纳米粒子-siRNA复合物抑制脐血树突状细胞SOCS1基因表达的实验研究

目的:构建复合纳米粒子载体,传递siRNA序列,抑制脐血树突状细胞SOCS1基因表达.方法:构建PEI包覆的以SiO2和Fe3O4为主要成分的复合纳米粒子载体,以LipofectamineTM2000为对照,琼脂糖凝胶电泳检测纳米粒子-siRNA的结合效率;荧光显微镜和流式细胞术检测纳米粒子-SOCS1-siRNA序列被细胞摄入的效率;Western blot检测纳米粒子-siRNA复合物转染脐血树突状细胞后对SOCS1基因表达的抑制.MTT法检测纳米粒子-SOCS1-siRNA处理的脐血DCs对肿瘤细胞的杀伤活性.结果:纳米粒子可以高效结合siRNA序列,纳米粒子-SOCS1-siRNA序列可被脐血树突状细胞摄取,但是单独的纳米粒子抑制SOCS1基因表达效率比较低,在外加磁场的作用下,基因沉默作用显著增强,抗肿瘤作用也相应提高.结论:纳米粒子载体传递siRNA安全性好,效率高,可以高效沉默脐血树突状细胞SOCS1基因表达,能为设计更有效的以树突状细胞为基础的抗肿瘤疫苗提供新思路和实验依据.     

CD4+ CD25+调节性T细胞对树突状细胞的杀伤作用

目的 探讨CD4^＋CD25^＋调节性T细胞是否对树突状细胞发挥免疫调节作用及其可能的机制。方法 用MACS（magnetic cell sorting）从BALB／c小鼠静息T细胞分离纯化CD4^＋CD25^＋T细胞，体外细胞增殖实验观察其对CD4^＋CD25^＋T细胞的免疫抑制作用；GM-CSF／IL-4培养自体小鼠骨髓来源DC，FACS（fluorescence-activated cell sorting）鉴定其表面分子特性；以CD3／CD28单克隆抗体活化CD4^＋CD25^＋调节性T细胞，FACS体外杀伤实验研究其对自体DC的调节作用，并观察穿孔素抑制剂EGTA对上述作用的影响。结果 用MACS法成功分离出CD4^＋CD25^＋T细胞，纯度可达98％，特异性表达而Faxp3基因，能明显抑制CD4^＋CD25^＋T细胞的体外增殖；骨髓来源的DC表达CDllc、MHCⅡ及少量协同刺激分子CD80、CD86；FACS体外杀伤实验证实以CD3／CD28抗体体外活化的CD4^＋CD25^＋调节性T细胞对自体DC有显著杀伤作用（P〈0．05），穿孔素抑制剂EGTA能部分抑制该杀伤效应（P〈0．05）。结论 CD4^＋CD25^＋调节性T细胞可通过杀伤作用对自体DC发挥免疫调节作用，穿孔素／颗粒酶杀伤途径可能参与其中。     

Interleukin 10 regulates cell surface and soluble LIR-2 (CD85d) expression on dendritic cells resulting in T cell hyporesponsiveness in vitro

Dendritic cells (DC) are unique in their ability to stimulate naive T cells to proliferate and to differentiate into effector T cells. DC, however, can also inhibit T cell activation and play a role in central and peripheral tolerance. IL-10 has been shown to render DC tolerogenic by unknown mechanisms. Using a combined monoclonal antibody/retroviral expression cloning approach, we show here that the inhibitory receptor LIR-2 (leukocyte immunoglobulin-like receptor-2, CD85d) is specifically up-regulated by IL-10 on maturing human DC. LPS-stimulated, LIR-2-transfected DC inhibited the proliferation of T cells in autologous, as well as allogeneic culture systems in vitro. In addition, overexpression of LIR-2 on resting T cells, which lack LIR-2 expression, inhibited T cell proliferation induced by TCR activation. A novel soluble form of LIR-2 was detected in culture supernatants of maturing DC. IL-10 treatment of DC potently inhibited the production of soluble LIR-2. Recombinant soluble LIR-2 was able to completely restore the proliferation of T cells activated with LPS-plus IL-10-treated DC. Thus, IL-10 renders DC hypostimulatory by up-regulating cell surface LIR-2 and by inhibiting soluble LIR-2 in vitro.