Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11b hi Ia lo DCs secreting high levels of TGF-␤, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. IntroductionDendritic cells (DCs) play crucial roles in the initiation and regulation of immune responses. 1,2 The ability of DCs to initiate immune responses or induce tolerance is strictly dependent on their maturation state or subsets. It has been reported that immature DCs that are deficient of costimulatory molecules can induce T-cell anergy, generate regulatory T (Treg) cells, and promote alloantigen-specific tolerance. Several types of DCs with negative regulatory functions have been reported. 3 Most regulatory DCs are prepared in vitro using immunosuppressive cytokines, such as IL-10 and TGF-␤. [4][5][6] However, this may not reflect the real differentiation of regulatory DCs in the immune microenvironment in vivo.The development of hematopoietic cells in vivo occurs in the context of microenvironment or niche, 7 which consists of many types of stromal cells, such as fibroblasts, macrophages, endothelium cells, and adipose cells. The microenvironment provides various signals for hematopoietic cell development. 7 Different microenvironments support different types of cell differentiation. The spleen is an important lymphoid organ, and the mouse spleen maintains hematopoietic function throughout life. 8 Furthermore, hematopoietic stem cells (HSCs) are found in spleens of adult mice. 8 Therefore, it is conceivable that HSCs in the spleen may differentiate into different immune cells in situ.Splenic stromal cells cultured in vitro could mimic the splenic microenvironment in vivo to some extent, despite their differences in some constituents. There is evidence that long-term cultured splenic stromal cells can support the development of dendritic-like cells in the absence of exogenous cytokines, and the dendritic-like cells have the phenotype and function of DCs, 9-13 strongly suggesting that splenic microenvironment could induce hematopoietic progenitors to differentiate directly into DCs. Stromal cells cultured in vitro consist of multiple components. Purification of the various components will help to study the role of specific cell type in the induction of DCs. We established the method of preparing endothelial splenic stroma cells (ESSCs) and i...

Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2αβ, hence is designated as MIP-2γ. Mouse MIP-2γ was identified by electrocloning and is highly homologous to human MIP-2γ. Northern blotting revealed that MIP-2γ was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2γ was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2γ expression by monocyte-derived DC. Recombinant MIP-2γ from 293 cells is about 9.5 kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2γ is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2γ does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.

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IntroductionThe Th1 cellular immune response is crucial for antitumor and antimicrobial immunity, 1,2 and powerful immunomodulatory adjuvants that induce Th1 polarization are an important facet of vaccination strategies. 3,4 While traditional adjuvants, such as aluminum salts and oil emulsions, mainly evoke Th2 responses, characterized by production of antibodies specific for conformational antigenic determinants, 5 new generation adjuvants, including CpG-rich motifs, monophosphoryl lipid A (MPA), and quil a saponin (QS21) promote Th1 polarization. 6 These effects are thought to result from the activation of antigen-presenting cells (APCs), in particular, dendritic cells (DCs). 7,8 In recent years the molecular chaperone heat shock protein (HSP) has been revealed to interact with APCs, and its considerable capacity to induce antigen-specific CD8 ϩ cytotoxic T lymphocyte (CTL) and Th1 responses has attracted much attention. 9,10 Extracellular HSPs can interact with APCs, exhibiting potent adjuvant functions in stimulating the host immune response. 10,11 This interaction triggers a cascade of events, including re-presentation of the chaperoned peptides by the major histocompatibility complex (MHC), secretion of proinflammatory cytokines, and maturation of DCs. [12][13][14] These properties combine to make HSPs a potent adjuvant, eliciting significant immune responses against associated antigens.The Hsp70 subfamily is one of the most important HSP subfamilies. Hsp70 prepared from tumor cells or virus-infected cells are capable of eliciting potent antigen-specific CD8 ϩ CTL responses 10,15 ; these responses are CD4 ϩ T-cell-independent and have been attributed to antigenic peptides bound to the HSP. 16 It also has been shown that Hsp70 complexed with ovalbumin (OVA) antigen-specific epitopes can activate DCs and induce OVAspecific CTL responses. 17 Hsp70 can bind to CD91, CD14, and TLR2/4 receptors on the surface of APCs, activating APCs and facilitating the re-presentation of HSP-associated antigen via a TAP-dependent or TAP-independent route. 13,14,18,19 These findings demonstrate the adjuvant effects of Hsp70 and encourage the potential application of Hsp70 in vaccination.We report here a new member of the Hsp70 subfamily cloned from a human DC cDNA library, Hsp70-like protein 1 (Hsp70L1). Recombinant Hsp70L1 can bind DCs, resulting in DC maturation and activation. While Hsp70L1 shares common receptors (CD91, TLR2, TLR4) on DCs with Hsp70, there are functional differences between Hsp70L1 and Hsp70. Hsp70L1 can induce DCs to secrete IP-10, a cytokine vital for Th1 polarization, but Hsp70 cannot. Moreover, Hsp70L1 is more potent than Hsp70 in the stimulation of IL-12p70, MIP-1␣, MIP-1␤, RANTES, CCR7, and CXCR4 expression by DCs. Hsp70L1 may therefore polarize Th1 responses more effectively than Hsp70. We used OVA as a model antigen to evaluate the adjuvant effects of Hsp70L1 in vivo, finding that Hsp70L1-OVA 257-264 hybrid peptide immunization could strongly Materials and methods Cell linesE.G7-OVA, an OVA-express...