Mycobacterium tuberculosisAntigens Induce the Differentiation of Dendritic Cells from Bone Marrow

Abstract

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-γ 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-α, 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-κB, thereby indicating its role during MTSA-induced differentiation of DCs.

Impaired Generation of Reactive Oxygen Species during Differentiation of Dendritic Cells (DCs) byMycobacterium tuberculosisSecretory Antigen (MTSA) and Subsequent Activation of MTSA-DCs by Mycobacteria Results in Increased Intracellular Survival

“…We have shown that MTSA and many other secretory Ags activate DCs by inducing their differentiation and maturation (reviewed in Refs. [13][14][15]. However, a challenge of these DCs with either M. tuberculosis cell extract (CE) (16) or live M. bovis BCG (our unpublished data) down-regulates primary and recall Th1 responses.…”

Cross-regulation of CD86 by CD80 differentially regulates T helper responses fromMycobacterium tuberculosissecretory antigen-activated dendritic cell subsets

“…For measuring antigen-specific T cell responses, BALB/c mice were immunized subcutaneously at the base of tail with MTSA (50 g/mouse) in incomplete Freund's adjuvant for 7 days as described previously [20]. Inguinal lymph nodes from these mice were removed, and T cells were enriched as described above.…”

“…When required, these BM-derived DCs (hereafter called BMDCs) were matured with 20 g/ml MTSA or 20 ng/ml TNF-␣ [21]. For up-regulating CD86, cytokine-or antigen-activated DCs were stimulated with 50 g/ml purified anti-CD80 for 24 h. Cells at the end of incubation in all sets were analyzed for the levels of surface molecules by flow cytometry as described before [20] or cocultured with naïve, allogeneic or antigen-primed, syngeneic T cells as described below. All flow cytometric analyses were performed on CD11c ϩ -gated cells.…”

“…MTSA was recombinantly expressed in Escherichia coli using the pQE31 vector (Qiagen, Valencia, CA) followed by purification over Ni-NTA columns as described earlier [20,21]. The recombinant protein has earlier been shown to contain negligible amounts of endotoxins.…”

“…Purification of antigen-specific T cells was done as described previously [20,21]. Briefly, inguinal lymph nodes from immunized BALB/c mice were first depleted of adherent cells by panning over plastic plates.…”