Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.
During acquired immunity to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in mice, dendritic cells (DCs) present mycobacterial antigens to naive T cells to prime an immune response. Complement C5a (anaphylatoxin) secreted by mycobacteria-infected macrophages regulates IL-12p70 production. As IL-12p70 regulates Th1 immunity against mycobacteria in mice, we examined the effects of C5a on IL-12p70 secretion by murine DCs and Th1 immunity. DCs cultured from C5-deficient (C5(-/-)) and -sufficient (C5(+/+)) mice were infected with BCG in the presence or absence of the C5a peptide. ELISA showed that C5(-/-) DCs secreted less IL-12p70 (600 pg/mL vs. 100 pg/mL) than C5(+/+) DCs, and they secreted more IL-10. Using immunophenotyping, reduced CD40 expression was found on C5(-/-) DCs after BCG infection. BCG-primed DCs were then cocultured with naive or BCG-immune T cells to differentiate them into IFN-gamma-secreting Th1 T cells. Coincident with increased IL-12p70 levels, BCG-primed C5(+/+) DCs cocultured with naive or immune C5(+/+) T cells showed a larger increase in CD4+ IFN-gamma/CD8+ IFN-gamma+ T cells compared with cocultured DCs and T cells from C5(-/-) mice. Thus, BCG-primed C5(+/+) DCs were better able to drive a Th1 response. Furthermore, BCG aerosol-infected C5(-/-) mice showed reduced CD4 and CD8 IFN-gamma-secreting T cells in the lungs, concurrent with an increased growth of BCG. Thus, C5a, an innate peptide, appears to play an important role in the generation of acquired immune responses in mice by regulating the Th1 response through modulation of IL-12p70 secretion from DCs.
Complement C5a anaphylatoxin is a potent activator of macrophages, neutrophils, and dendritic cells (DC) and binds the C5a receptor (C5a-R; CD88). Although C5a is chemotactic for T cells, expression of C5a-R on murine T cells has been disputed. We report here that naïve, Con A-activated, and cytokine (IL-12, IL-18)-stimulated murine CD3+ T cells from three strains of mice [C57Bl/6, B10.nSn (C5+/+), B10.on (C5-/-)] lacked C5a-R, as evaluated by immunophenotyping with an anti-C5a-R mAb. Ligation of CD3 induced a modest up-regulation with 3% of CD3+ T cells expressing cell surface C5a-R. T cells primed by APC differentiate into effector T cells. Activation of mycobacteria [bacillus Calmette-Guerin (BCG)]-sensitized T cells through MHC II and TCR interactions via BCG-infected macrophages enhanced the expression of C5a-R with approximately 14% of CD3+ T cells positive for C5a-R. Comparable expression was found in C5+/+ as well as C5-/- strains of mice (14% and 15%, respectively). Furthermore, anti-CD3-activated T cells were primed by BCG-infected DC, and a larger proportion of the primed T cells expressed C5a-R (30-40%). Finally, mice infected with BCG showed significant numbers of CD3+ T cells expressing C5a-R in the spleens during infection. As APC, such as macrophages and DC, can secrete C5 and cleave C5 to C5a and C5b through a peptidase, we suggest that macrophage and DC-T cell interactions can up-regulate C5a-R on T cells through MHC II-TCR and provide a C5a peptide for additional local activation of T cells via C5a-R.
Dendritic cells (DCs) are under investigation as immunotherapeutic agents in the treatment of cancer and infectious diseases. One of the important factors in skewing the immune response toward clinically beneficial TH1-type immunity is interleukin-12p70. IL-12p70 is synthesized and secreted in response to inflammatory cytokines, bacterial/viral components, and CD40 ligation. This study investigated the production of IL-12 by DCs at the single-cell level using a sensitive intracellular cytokine flow cytometry-based assay system. The authors observed that immature DCs could be stimulated with several compounds to produce IL-12, but that IL-12 production was a feature of a minority of activated DCs. IL-12+ DCs were characterized as being partially matured (ie, absent or low CD83 expression, with variable expression of CD1a and CD64). Interestingly, activated DCs lacked expression of the CD16 and CD64 Fc gammaR, which may have important implications for exogenous antigen-loading strategies.
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