Regulation of immune responses to Mycobacterium tuberculosis secretory antigens by dendritic cells.

Towards elucidating the immune responses induced by antigens from the Mycobacterium tuberculosis (M. tb) RD-1 region, we have been characterizing their interactions with dendritic cells (DCs) and their precursors. We have shown that incubation of bone marrow DC precursors with M. tb antigens induces the differentiation of DC precursors and also the maturation of various DC subsets. While MTSA differentiated DCs were immature, MTSA matured DCs were terminally mature. However, regardless of their maturation status M. tb secretory antigen-activated DCs down-regulated pro-inflammatory T helper cell responses to a subsequent challenge with M. tb cell extract (CE) while increasing regulatory responses. Investigations into the underlying mechanisms showed that stimulation with M. tb CE changed the polarization of antigen-activated DCs from DC1 to DC2. This resulted in secretion of high levels of IL-10 and TGF-beta together with increased surface expression of CD86. Blocking either IL-10 or TGF-beta or CD86 restored Th1 responses to CE antigens. Conversely, treatment of antigen-activated DCs with IL-12 and/or IFN-gamma fully restored Th1 responses of CE antigens. These results indicate that M. tb strategically secretes antigens from infected macrophages to down-regulate pro-inflammatory immune responses at sites of infection.

Functional characterization of the MENTAL domain.

Human metastatic lymph node (MLN) 64 is composed of two conserved regions. The amino terminus contains a conserved membrane-spanning MENTAL (MLN64 NH(2)-terminal) domain shared with an unique protein called MENTHO (MLN64 NH(2)-terminal domain homologue) and targets the protein to late endosome. The carboxyl-terminal domain is composed of a cholesterol binding steroidogenic acute regulatory-related lipid transfer domain exposed to the cytoplasm. MENTHO overexpression leads to the accumulation of enlarged endosomes. In this study, we show that MLN64 overexpression also induces the formation of enlarged endosomes, an effect that is probably mediated by the MENTAL domain. Using an in vivo photocholesterol binding assay, we find that the MENTAL domain of MLN64 is a cholesterol binding domain. Moreover, glutathione S-transferase pull-down or co-immunoprecipitation experiments demonstrate that this domain mediates homo- and hetero-interaction of MLN64 and MENTHO. In living cells, the expression of paired yellow fluorescent and cyan fluorescent fusion proteins show MENTHO homo-interaction and its interaction with MLN64. These data indicate that within late-endosomal membranes, MLN64 and MENTHO define discrete cholesterol-containing subdomains. The MENTAL domain might serve to maintain cholesterol at the membrane of late endosomes prior to its shuttle to cytoplasmic acceptor(s).

Cross-regulation of CD86 by CD80 differentially regulates T helper responses from Mycobacterium tuberculosis secretory antigen-activated dendritic cell subsets.

We report that stimulation of Mycobacterium tuberculosis secretory antigen- and tumor necrosis factor alpha-matured BALB/c mouse bone marrow dendritic cells (BMDCs) with anti-CD80 monoclonal antibody up-regulated CD86 levels on the cell surface. Coculture of these BMDCs with naïve, allogeneic T cells now down-regulated T helper cell type 1 (Th1) responses and up-regulated suppressor responses. Similar results were obtained with splenic CD11c(+)/CD8a(-) DCs but not to the same extent with CD11c(+)/CD8a(+) DCs. Following coculture with T cells, only BMDCs and CD11c(+)/CD8a(-) DCs and not CD11c(+)/CD8a(+) DCs displayed increased levels of surface CD86, and further, coculturing these DCs with a fresh set of T cells attenuated Th1 responses and increased suppressor responses. Not only naïve but even antigen-specific recall responses of the Th1-committed cells were modulated by DCs expressing up-regulated surface CD86. Further analyses showed that stimulation with anti-CD80 increased interleukin (IL)-10 and transforming growth factor-beta-1 levels with a concomitant reduction in IL-12p40 and interferon-gamma levels from BMDCs and CD11c(+)/CD8a(-) DCs and to a lesser extent, from CD11c(+)/CD8a(+) DCs. These results suggest that cross-talk between costimulatory molecules differentially regulates their relative surface densities leading to modulation of Th responses initiated from some DC subsets, and Th1-committed DCs such as CD11c(+)/CD8a(+) DCs may not allow for such modulation. Cognate antigen-presenting cell (APC):T cell interactions then impart a level of polarization on APCs mediated via cross-regulation of costimulatory molecules, which govern the nature of subsequent Th responses.

Down-regulation of T helper 1 responses to mycobacterial antigens due to maturation of dendritic cells by 10-kDa mycobacterium tuberculosis secretory antigen.

Interactions of 10-kDa Mycobacterium tuberculosis secretory antigen (MTSA) with dendritic cells (DCs) were investigated to elucidate the role of secretory antigens in regulating immune responses to M. tuberculosis early in the course of infection. MTSA induced the maturation of different DC subsets. The cytokine profiles of these DCs were characteristic to each DC subset. Of interest, coculture of M. tuberculosis whole-cell extract (CE)-pulsed, MTSA-matured DCs with CE-specific T cells led to a marked reduction in interleukin (IL)-2 and interferon (IFN)-gamma production, thereby down-regulating proinflammatory responses to mycobacterial antigens. Attenuation of IL-2 and IFN-gamma levels of CE-specific T cells also was obtained when M. tuberculosis culture filtrate protein-activated DCs were employed as antigen-presenting cells, which suggests that MTSAs induce maturation of DCs at sites of infection, probably to down-regulate proinflammatory immune responses to mycobacteria that may subsequently be released from infected macrophages.

Mycobacterium tuberculosis antigens induce the differentiation of dendritic cells from bone marrow.

We show in this study that incubation of freshly isolated bone marrow cells with Mycobacterium tuberculosis (M. tb) secretory Ag (MTSA), in the absence of any growth or differentiation-inducing factor, differentiates them into dendritic cell (DC)-like APCs. These DCs expressed moderate to high levels of various markers typical of DCs. These included T cell costimulatory molecules CD80, CD86, CD40, and CD54 and high levels of surface MHC class I and II on CD11c(+) cells. The levels and the kinetics of up-regulation of these molecules were comparable with those of GM-CSF-differentiated DCs. Furthermore, these DCs exhibited morphology characteristics to DCs like the presence of dendritic processes. These DCs were also potent stimulators of allogeneic T cells and preferentially induced the secretion of IFN-gamma over IL-10 from the interacting T cells. Interestingly, the differentiation of bone marrow cells into DC-like APCs was obtained with many other M. tb Ags, including whole cell extract of M. tb. Further characterization of MTSA-differentiated DCs showed that they were immature in nature, as stimulation of these DCs with TNF-alpha, anti-CD40, or LPS further up-regulated the surface levels of various molecules together with an increase in their T cell stimulatory capacity. The Ag-specific T cell responses of MTSA-differentiated DCs were mainly contributed by the CD4(+) subset, indicating that MTSA was largely MHC II restricted. Furthermore, stimulation of bone marrow cells with MTSA induced the nuclear translocation of the transcription factor NF-kappaB, thereby indicating its role during MTSA-induced differentiation of DCs.