Dose dependence and MHC class II restriction of responses to the RplJ epitope following M. smegmatis immunization. (A) C57BL/6 (B6) mice were immunized with 5 × 107 CFU IKE or IKEPLUS i.v., 107 CFU mc2155 i.v., or 5 × 106 CFU BCG-Danish s.c. At 2 weeks postimmunization, CD4+ T cells were isolated from immunized splenocytes and cultured in the presence of APCs and the rplJTB146–160 (10 μg/ml) and TB9.8 (10 μg/ml) peptides. Responses of immune CD4+ T cells to peptides were quantified by an IFN-γ ELISPOT assay. *, P < 0.05 compared to BCG-immunized and naive mice; **, P < 0.0005 compared to BCG-immunized and naive mice; ***, P < 0.0001 compared to all other groups (ANOVA). (B) Dose-dependent responses of IKEPLUS-immunized splenocytes to the rplJTB146–160 peptide were assayed by an IFN-γ ELISPOT assay in comparison to BCG-immunized splenocytes. (C) The requirement for MHC class II presentation was assessed by an ELISPOT assay using APCs from wild-type mice or MHC class II−/− mice. Data shown represent mean values and standard errors for triplicate (A) or quadruplicate (B and C) samples. All data shown are representative of results from at least two independent experiments.