responses against cancer, infectious diseases, etc. [9,10,11]. Like- wise, this also encouraged us to generate a vaccine by culturing live M. tb in host macrophages. Our assumption that this approach may be effective, stems from the fact that the bacterium in its natural habitat (macrophages) is likely to secrete unique antigens that may eventually help in generating protective immune response [5]. The preparation was made safe for immunization, by drug treatment [5]. Noteworthy observations obtained on vaccination with the infected macrophages was that it augmented T cell proliferation, IFN-c production and reduction in mycobac- terial load, the parameters that are crucial for protection against M. tb. This vaccination strategy worked successfully not only for TB but also against S. typhimurium [5]. This study demonstrated significant protection against both the intracellular pathogens in a short-term study (30 days). However, no CD8 T cell responses were examined and efficacy of vaccine was also not compared with BCG. The vaccine failed to generate protective immunity in long- term study (240 days). Immunological memory can be enhanced by the use of selected cytokines [12,13]. Hence, supplementing cytokines with vaccines to bolster T cell memory can be an attractive approach. Role of inflammatory and common c-chain cytokines has been highlighted in the literature in enhancing T cell memory response. Cytokines like IL-1, IL-6 and TNF-a help in the expansion and survival of memory T cells [14,15] whereas common c-chain cytokines, IL-7 and IL-15 have dominant roles in generation and homeostasis of memory T cells [12,16,17]. The present study was conducted to check whether vaccination with M. tb infected macrophages supplemented with T cell memory enhancing cytokines could generate enduring T cell memory. We vaccinated mice with M. tb infected macrophages vaccine with IL-1+IL-6+TNF-a (IM-1.6.a) and IL-7+IL-15 (IM- 7.15) to study the generation and sustenance of long-term (240 days) protective immunity against M. tb [18]. Interestingly, IM-1.6. a demonstrated considerable augmentation in both CD4 and CD8 T cell memory pool, as evidenced by significant improvement in immune response and protection against M. tb. Importantly, the vaccine showed better efficacy than BCG and hence can be a potent future vaccination strategy against TB. Results Vaccination with sIM-1.6.a elicits enduring Th1 response Induction of long-lasting immunity is a cardinal feature of a successful vaccine. Hence in the present study, we investigated how enduring T cell memory can be elicited by M. tb infected syngeneic macrophages (sIM). It is reported that combination of either IL- 1+IL-6+TNF-a or IL-7+IL-15 can induce long-term T cell memory response [14,16,19]. Therefore, we co-administered these cytokines with the vaccine and studied the sustenance of long-lasting memory T cells. After vaccination, the mice were rested for sufficiently long- duration (240 days) before challenge with M. tb, to establish bona fide T cell memory response [18]. Interestingly, co-administration of IL- 1+IL-6+TNF-a with M. tb infected syngeneic macrophages (sIM- 1.6.a) considerably improved the immune response (Figure 1). The T cell response observed with sIM-1.6.a immunization in response to PPD (purified protein derivative from M. tb), was significantly better than BCG (p = 0.0253) and sIM alone (p = 0.0176) (Figure 1A). Cells obtained from the control groups inoculated with PBS (110206643 cpm), uninfected syngeneic macrophages (65136417 cpm), and 1.6.a alone (69816293 cpm) failed to improve T cell response. Further, these results were substantiated using two other M. tb antigens, short-term culture filtrate (STCF- H37Rv) and infected macrophages cytosolic (IMC) antigenic Infected Macrophages as Vaccine proteins (Figure S1A and Figure S2A). However, we did not observe any change when IL-7 and IL-15 were administered along with sIM (data not shown). Hence, subsequent experiments were conducted with vaccine supplemented with IL-1+IL-6+TNF-a. Th1 cells are known to play crucial role in protection against TB [20]. Hence we next monitored the Th1 cytokines (IL-2 and IFN-c) elicited by the vaccine. As compared to BCG and sIM, vaccination with sIM-1.6.a showed predominance of Th1 immune response as evident by significantly higher production of IFN-c (p = 0.0071; p = 0.0071 respectively) and IL-2 (p = 0.0057; p = 0.0052 respectively) on in vitro stimulation with either PPD (Figure 1B, C) or STCF-H37Rv (Figure S1B, C) or IMC (Figure S2B, C). It is a well established fact that B cells produce mainly IgG2a and IgG1 on interaction with Th1 and Th2 cells respectively [21]. Interestingly, we detected primarily M. tb specific IgG2a-isotypes (BCG vs sIM-1.6.a, p = 0.0037) (Figure 1D). This further substantiates the predominance of Th1 response elicited by sIM-1.6.a. Adaptive immune response is initiated in the local lymph nodes, effectors subsequently migrate to the site of infection [22,23,24]. Since the appearance of adaptive immunity in the lungs play a seminal role in imparting protection against M. tb, it was imperative to monitor the local immunity as well. Like lymphoid organs, mainly Th1 response was also observed in the lymphocytes isolated from the lungs (Figure 1E, 1F; Figure S1D, S1E; Figure S2D, S2E). No change was observed in the secretion of IL-4 and IL-5 (Figure S3). The proliferation and cytokines data signify that vaccination with sIM-1.6.a significantly evokes long- lasting Th1 immunity against M. tb. sIM-1.6.a induces enduring CD4 and CD8 T cell memory response We next monitored the expression of memory T cell markers viz. CD44, CD62L and IL-7R (CD127), on the cells isolated from spleen and lymph nodes of the immunized mice (Figure 2). The cells were in vitro stimulated with PPD. sIM-1.6.a substantially expanded pool of CD44 and IL-7R expressing CD4 T cells (13.1 vs 51.0%) compared to sIM vaccine (Figure 2A). Similar results were exhibited by CD8 T cells (16.1 vs 41.5%) (Figure 2C). Further, this observation was also supported by improvement in the central (CD44hi CD62Lhi) as well as effector (CD44hi CD62Llo) memory T cells (Figure 2A–D). Enhancement in total number of cells was also observed (Figure 2B, D). Overall these findings specify that immunization with sIM-1.6.a can induce long-lasting memory response in both CD4 and CD8 T cells. Vaccination with sIM-1.6.a imparts better protection than BCG sIM-1.6.a elicited enduring CD4 Th1 and CD8 T cell memory, the parameters favorable for a successful TB vaccine. Hence, we next checked the protective efficacy of sIM.1.6.a in M. tb aerosol challenged mice. Interestingly, vaccination with sIM-1.6.a con- siderably reduced the lung mycobacterial burden compared to sIM (p = 0.0004) and BCG (p = 0.0067) (Figure 3A). Control groups inoculated with placebo; PBS (log10 CFU 5.1760.16) (Figure 3A), uninfected syngeneic macrophages alone (log10 CFU 5.3760.029), and 1.6.a alone (log10 CFU 4.6660.064) (Figure S4) failed to show any significant decline in CFU (p.0.05 for PBS vs 1.6.a or uninfected syngeneic macrophages). Interestingly, neither sIM-7.15 nor BCG-1.6.a could provide better protection than controls syngeneic uninfected macrophages (sMac) or BCG respectively (Figure S4). Further, protection imparted by sIM- 1.6.a vaccination was reflected by qualitative analysis of lungs PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16097