in the absence of any T cell addition. CFU in macrophages GSK461364 price incubated with T cells that were activated by L-type 19111597 and R-type VGCC-blocked-BCG-infected CFP10-DCs, showed a significant decrease when compared 11465152 to CFU from either M. tuberculosis infected resting or activated macrophages or when compared with T cells activated by CFP10-DCs in the absence of VGCC blocking. A similar decrease in CFU were seen when T cells activated by L-type and Rtype VGCC-blocked-BCG-infected GM-CSF-DCs were employed. These results indicate that blocking L-type and R-type VGCC in DCs activated T cells that subsequently mediated effective killing of virulent M. tuberculosis inside macrophages. T cells activated by VGCC-blocked-DCs mediate killing of M. tuberculosis in macrophages To investigate the functional relevance of the results obtained so far, we next tested the ability of T cells activated by VGCC-blocked DCs to kill mycobacteria inside macrophages as recently carried out with chemokine and cytokines conditioned CFP10-DCs. We first ensured that blocking L-type and R-type VGCC in CFP10DCs and GM-CSF-DCs activated T cells that secreted high levels of IFN-c and low levels of IL-10. Next, BCG infected DCs were co-cultured with T cells enriched from BCG immunized mice. Form the DC-T cell co-culture, T cells were separated by MACS and incubated with M. tuberculosis H37Rv infected macrophages. Control groups included M. tuberculosis infection of Blocking L-type and R-type VGCC in macrophages and PBMCs kills M. tuberculosis Next it was relevant to investigate whether blocking L-type and R-type VGCC in mouse macrophages or human PBMCs would mediate effective killing of M. tuberculosis. We first monitored whether blocking 
L-type and R-type VGCC induced calcium influx in macrophages and PBMCs. BCG stimulation of macrophages or PBMCs did not induce appreciable levels of calcium influx. However, blocking either L-type or R-type VGCC induced a significant increase in calcium mobilization in both macrophages and PBMCs Ca Channels and Mycobacteria . PBMCs of patients with active TB disease express high levels of L-type and R-type VGCC Ca Channels and Mycobacteria were very similar to or in some cases even lower than healthy controls. Similar results were obtained in the other 6 patients with active disease and patients who had taken 2 months of therapy, wherein PBMCs of patients with active TB expressed higher levels of L-type and R-type VGCC when compared with patients who had taken treatment. These results are consistent with the data obtained in Blocking VGCC in vivo reduces bacterial burden in mice In order to test proof of principle, we next investigated the role of L-type and R-type VGCC in regulating M. tuberculosis infection in mice. To this end, we first infected mice with M. tuberculosis H37Rv. Post-exposure we followed it up by injection of antibodies to VGCC. We first investigated whether antibody treatment would indeed increase intracellular calcium in vivo. As shown in 7 Ca Channels and Mycobacteria 8 Ca Channels and Mycobacteria were also reduced by 50% upon injection of VGCC specific antibodies when compared with mice that received non-specific antibody. These results indicate that blocking VGCC in vivo results in reduction of M. tuberculosis infection as a result of increased calcium influx in infected cells. Discussion M. tuberculosis has devised numerous ways to evade protective immune responses by modulation of host factors. These include downmodulati
