Infected Macrophages as Vaccine Figure 2. sIM-1.6.a induces enduring CD4 and CD8 T cells memory response. Mice were immunized and aerosol challenged as mentioned in Figure 1. Lymphocytes were stimulated in vitro with PPD (50 mg/ml) for 48 h and later stained with fluorochrome conjugated antibodies and analyzed by flowcytometry. A, flow contours depict percentage of gated CD4 T cells expressing IL-7R and CD44 (upper panel); CD62L on CD4 T cells (lower panel). C, gated CD8 T cells expressing IL-7R and CD44 (upper panel) and CD62L on CD8 T cells (lower panel). Bar diagrams show total number of CD4 (B) and CD8 (D) T cells expressing CD44hi, CD62Lhi and CD62Llo. Data shown are mean 6 SEM of two independent experiments with n = 3 animals per group. ‘*’ and ‘**’ indicate p,0.05 and p,0.01 respectively. doi:10.1371/journal.pone.0016097.g002 lung pathology was observed (Figure 6B) and these results were comparable with sIM-1.6.a vaccine (Figure 3A, B and Figure 6A, B). These data very categorically establish that syngeneic macrophage is not a pre-requisite for this vaccination strategy, rather both syngeneic and xenogeneic macrophages can be employed with equal efficiently. Mechanism of priming of immune system by the vaccine We hypothesize two possibilities which may explain generation of immune response by the vaccine. Firstly, M. tb infected syngeneic macrophages may directly present antigens to T cells. Secondly, infected macrophages (syngeneic and xenogeneic) on c- irradiation may undergo apoptosis [5,26] and resulting apoptotic vesicles contain processed mycobacterial antigens [27,28]. These apoptotic vesicles can be avidly taken up by Dendritic cells (DCs), which then process and present antigens to CD4 T cells through MHC-II or cross present to CD8 T cells via MHC-I pathway [27,28]. Therefore to demonstrate the antigenic transfer by apoptotic bodies to bystander DCs, Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled macrophages were infected with Texas Red labeled M. tb (Figure 7A), followed by c-irradiation to induce apoptosis [26]. This infected macrophage preparation was in vitro co-cultured with bone marrow derived dendritic cells (BMDCs). Interestingly, we observed the presence of apoptotic vesicles containing mycobacterial antigens (orange fluorescence) in DCs (Figure 7B), demonstrating that M. tb antigens from infected macrophages can get transported to DCs. These DCs can then process and present antigens to T cells and thereby imparting optimum protection by eliciting immune response (Figure 1–6). Discussion Recently, cell based vaccines have gained considerable impetus following their success against cancer [11]. We had earlier demonstrated a novel strategy of vaccination by utilizing infected macrophages [5]. This vaccination approach offered significant protection not only against M. tb but also S. typhimurium [5]. However, it failed to induce long-lasting protection against M. tb. PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16097