Infected Macrophages as Vaccine Figure 4. Vaccination with xIM-1.6.a evokes immune response. Mice were vaccinated with xIM-1.6.a with similar immunization protocol as mentioned in Figure 1. Lymphocytes pooled from spleen and lymphnodes of immunized animals were stimulated in vitro with PPD (50 mg/ml) for 48 h and later T cell proliferation was monitored by 3H-thymidine incorporation (A); secretion of IFN-c (B) and IL-2 (C) in culture SNs; PPD specific IgG2a/IgG1 antibodies in the serum (10006 dilution) (D) were estimated by ELISA. Production of IFN-c (E) and IL-2 (F) was estimated in the culture SNs of the lymphocytes isolated from lungs, in vitro stimulated with PPD (50 mg/ml). Data shown as mean 6 SEM are representative of two experiments, n = 3 animals per group. ‘*’ and ‘**’ indicate p,0.05 and p,0.01 respectively. doi:10.1371/journal.pone.0016097.g004 protective immune response against M. tb. Furthermore, as DCs secrete IL-12, they are capable of skewing immune response towards IFN-c secreting CD4 and CD8 T cells [33]. The proinflammatory stimuli is also necessary for diverting the immune response towards protective Th17 cells, which is reported to be important for the rapid recruitment of Th1 effectors to the lungs [34,35]. Moreover, DCs trap foreign antigen (in this case mycobacterium antigen) and act as reservoir, slowly releasing the antigen in the system for the activation of T cells and maintenance of memory cells [36]. Hence, incorporating IL-1+IL-6+TNF-a with vaccine, promotes the generation of long-term memory T cells. Based on our findings, we envisage that the strategy of utilizing MHC-mismatched macrophages can work as a promiscuous vaccine for humans. Importantly, the preparation requires no adjuvant and shows better protection than BCG. Therefore, we conclude that IM-1.6.a may be a possible vaccine candidate against TB, based on its uniqueness of utilizing novel antigens (secreted within the natural environment of the macrophages which may be of paramount importance for evoking protection) and presence of IL-1+IL-6+TNF-a (for induction and sustenance of enduring CD4 and CD8 T cell memory responses). Materials and Methods Experimental animals BALB/c mice (female, 6–8 weeks old) were procured from National Institute of Pharmacological Education and Research, Mohali, India and National Institute of Immunology, New Delhi, India. Animals were kept in Biosafety Level-3 facility of Institute of Microbial Technology (IMT), Chandigarh, India and National JALMA Institute for Leprosy and Other Mycobacterial Diseases PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16097