Infected Macrophages as Vaccine Figure 1. sIM-1.6.a elicits a long-lasting Th1 immune response. Mice were vaccinated with sIM-1.6.a and sIM. After 240 days, they were aerosol challenged with M. tb and were sacrificed 35 days later. Lymphocytes Pooled from spleen and lymphnodes of immunized mice were stimulated in vitro with PPD (50 mg/ml) for 48 h. T cell proliferation was monitored by 3H-thymidine incorporation (A); secretion of IFN-c (B) and IL-2 (C) in the culture SNs by ELISA. Ratio of PPD specific IgG2a/IgG1 antibodies were estimated in serum (10006dilution) (D); production of IFN-c (E) and IL-2 (F) was estimated in the culture SNs of lymphocytes isolated from lungs, in vitro stimulated with PPD (50 mg/ml). Data shown are mean 6 SEM and representative of two experiments with n = 3 animals per group. ‘*’, ‘**’ and ‘***’ indicate p,0.05, p,0.01 and p,0.001 respectively. doi:10.1371/journal.pone.0016097.g001 pathology. Alleviation in pathology with normal alveolar structure was observed with minimum lungs tissue involvement. In contrast, mice inoculated with BCG and placebo (PBS) revealed granulo- matous infiltrations with active TB lesions (Figure 3B). Pragmatic approach to utilize IM-1.6.a The requirement of syngeneic macrophages is a major concern in extrapolating mice data for practical human application. We showed earlier that vaccination with syngeneic, allogeneic and xenogeneic macrophages infected with M. tb and S. typhimurium or pulsed with ovalbumin could successfully mount an immune response against the entrapped antigens [5,25]. Therefore we next ascertained whether IL-1+IL-6+TNF-a with infected xenogeneic (THP-1) macrophages (xIM-1.6.a) could provide similar outcome as observed in the case of syngeneic infected macrophages (sIM- 1.6.a) (Figure 1, 2, 3). The cells isolated from spleen and lymph nodes of the immunized mice were in vitro stimulated with PPD. As observed in the case of sIM-1.6.a, similar pattern of enhancement of T cell proliferation (p = 0.004), IFN-c (p = 0.0066), IL-2 (p = 0.0046) and IgG2a (p = 0.0317) secretion than control xIM alone was observed with xIM-1.6.a (Figure 4A–D). Further, like lymphoid organs, lymphocytes isolated from lungs also showed significantly better yield of IFN-c (p = 0.0355) and IL-2 (p = 0.0164) (Figure 4E, F). Furthermore, no difference was observed in the production of IL-4 and IL-5 (Figure S6). The results were reproduced with all the tested antigenic preparations (PPD, STCF-H37Rv) (Figure 4, Figure S5). Furthermore, like sIM-1.6.a, augmentation in both central (CD44hi CD62Lhi) and effector (CD44hi CD62Llo) memory CD4 and CD8 T cells was noticed on vaccination with xIM-1.6.a (Figure 5). Enhancement in both percentage (Figure 5A, C) and total number of cells (Figure 5B, D) was observed. Lastly, we also checked the protective efficacy of xIM-1.6.a against M. tb. Mice vaccinated with xIM-1.6.a showed significantly better reduction in mycobacterial load in the lungs compared to uninfected control xenogeneic macrophages (p = 0.0015) and xIM (p = 0.0495) (Figure 6A) and its protective efficacy of was found to be similar with that of sIM-1.6.a (Figure S7). Further, alleviated PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16097