Infected Macrophages as Vaccine Figure 3. sIM-1.6.a provides significantly better protection than BCG. Mice were immunized and aerosol challenged as mentioned in Figure 1. Mycobacterial load in the lungs was enumerated by CFU plating of diluted lung homogenates. A, data are represented as log10 CFU (n = 6 animals/group) where each triangle indicates single mouse. B, photomicrographs (106) of formalin fixed lungs sections stained with H & E. ‘+’ indicates active TB lesion with edematous follicular reaction while ‘R’ shows small or large developing follicular granulomas. Data are representative of two independent experiments. ‘**’ and ‘***’indicate p,0.01 and p,0.001 respectively. doi:10.1371/journal.pone.0016097.g003 One of the cardinal features of a successful vaccine is its ability to elicit long-lasting memory. It is worth to mention here that even though BCG successfully protects against the infant TB, it fails in preventing the adult manifestation of the disease, reflecting poor induction of long-lasting T cell memory [29]. Over the recent years, studies have demonstrated a non-redundant role of cytokines in differentiation and maintenance of memory T cells [12,13]. Common c-chain and proinflammatory cytokines are known to induce and support the persistent T cell memory [12,14]. These cytokines act directly on both naive and memory T cells and help in their survival and expansion [30]. Further, they also assist in the recruitment of antigen presenting cells at the site of vaccine administration. Hence, in the light of above-mentioned facts, the present study was conducted. We evaluated the role of co-administration of common c-chain (IL-7+IL-15) or proinflammatory (IL-1+IL- 6+TNF-a) cytokines, with a cell based vaccine prepared using macrophages infected with M. tb, in generating enduring T cell memory and protection against TB. Mice were challenged with M. tb after 240 days of vaccination to ensure the legitimate long-term memory T cell responses [18]. The following major findings have emerged from this study: IM-1.6.a significantly (i) inducted a robust immune response that resulted in enduring memory CD4 and CD8 T cells; (ii) elicited a predominant Th1 immune response; (iii) reduced mycobacterial load; (v) alleviated the lung pathology; (vi) protected better than conventional BCG vaccine. More than 90%–95% of the 2 billion individuals exposed to M. tb never develop active disease [2], indicating that the bacillus inhabiting the macrophages secretes unique molecules that stimulate the immune system for efficient control of the disease. Keeping this fact in the mind, we prepared a vaccine by infecting macrophages with live M. tb simulating a natural scenario. The preparation was made safe by killing the bacteria with isoniazid and c-irradiated to induce apoptosis [5,26]. Interestingly, incorporation of IL-1+IL-6+TNF-a with the vaccine significantly enhanced enduring T cell memory response. Although this approach effectively protected the immunized mice from TB, the concern was its applicability to humans. Preparing syngeneic macrophage vaccines for humans may not be feasible. We had earlier demonstrated that major histocompati- bility complex (MHC)–mismatched macrophages infected with mycobacterium can also be used successfully for vaccination [5,25]. Interestingly, co-administration of IL-1+IL-6+TNF-a with xenogeneic vaccine (xIM-1.6.a) also elicited long-term immunity against M. tb and response was comparable to sIM-1.6.a (Figure S7). We have also demonstrated the proof of principle of how this vaccine may eventually prime the immune system. Apoptotic bodies are likely to be generated by the infected macrophage vaccine. Such apoptotic bodies could be avidly taken up in vivo by DCs. Interestingly, we could locate apoptotic bodies loaded with mycobacterial antigens inside the DCs (Figure 7). Further, DCs but not macrophages can cross-present antigen of apoptotic cells to T cells because they express avb5 integrin, which plays a critical role in the trafficking of exogenous antigens [31,32]. Hence, DCs that have phagocytosed apoptotic bodies containing mycobacterial antigens are likely to prime both na¨ıve CD4 T cells (via exogenous pathway) and CD8 T cells (via cross presentation) to generate PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e16097