Infected Macrophages as Vaccine Figure 5. Vaccination with xIM-1.6.a induces CD4 and CD8 T cells memory response. Mice were immunized and aerosol challenged as mentioned in Figure 1. Pooled lymphocytes from spleen and lymphnodes of immunized animals were stimulated in vitro with PPD (50 mg/ml) for 48 h and later stained with fluorochrome tagged antibodies and analyzed by flowcytometry. A, flow contours depict percentage of CD4 T cells expressing IL-7R and CD44 (upper panel); CD62L expression on CD4 T cells (lower panel). C, CD8 T cells expressing IL-7R and CD44 (upper panel); 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. ‘*’ indicates p,0.05. doi:10.1371/journal.pone.0016097.g005 (NJIL&OMD), Agra, India. The animals were offered pellet feed and water ad libitum. Ethics Statement All animal experiments were approved by the Institutional Animal Ethics Committees of IMT and NJIL&OMD. All animal experiments were performed according to the National Regulatory Guidelines issued by CPSEA (Committee for the Purpose of Supervision of Experiments on Animals), Ministry of Environment and Forest, Govt. of India. Reagents and antibodies Middlebrook 7H9, 7H11, ADC and OADC were procured from Difco Laboratories (Sparks, MD). Recombinant mouse IL-1, IL-6, TNF-a, IL-7 and IL-15 from Peprotech (Rocky Hill, NJ). Fluorochrome labeled anti-mouse antibodies, FITC-conjugated CD4 (H129.19), IgG2a isotype control (R35-95); PE-conjugated CD62L (MEL-14), IgG2b isotype control (R35-95), IgG2c isotype control (A23-1); PE-Cy5-conjugated CD44 (IM7), Sv-PE-Cy5; Pacific Blue conjugated CD4 and IgG2a isotype control (R35-95); APC-Cy7-conjugated CD8 (53-6.7) and IgG2a isotype control (R35- 95); biotin-conjugated CD4 (H129.19) and IgG2a isotype control (R35-95) and pair matched antibodies for ELISA, JES6-1A12, JES6- 5H4 (IL-2); 11B11, BVD6-24G2 (IL-4); TRFK5, TRFK4 (IL-5); R4-6A2, XMG1.2 (IFN-c), A85-1 (IgG1-Biotin), R19-15 (IgG2a- biotin) procured from BD Biosciences (San Diego, CA). FITC- conjugated CD8 (KT15) and IgG2a isotype control were from AbD Serotec (Langford Lane, Kidlington, UK). Tissue culture grade plastic-wares were purchased from BD Biosciences (Bedford, MA) and Nunc (Rochester, NY). Texas Red succinimidyl ester was procured from Invitrogen (Eugene, OR). All other standard reagents were purchased from Sigma (St. Louis, MO) unless mentioned. PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16097