Frequencies of LLO296–304 specific CD8+ T cells in spleens of B16F10 treated mice or non-treated after LMWT therapy.

aC57BL/6 mice non-treated with murine melanoma were inoculated with saline i.p for 7 days and next injected i.p with LMWT for 3 days as described in Materials and Methods. Splenocytes from mice treated with LMWT were incubated with recombinant dimeric H-2Kb: Ig fusion protein (BD Biosciences) loaded with LLO296–304 peptide. The staining cocktail contained the dimeric fusion protein loaded with the peptides, CD8 and anti-IFN-gamma antibodies. CD8+ cells were gated for anti-IFN-gamma staining (% Gated dimer-CD8) to calculate the frequencies of CD8+-LLO296–304. Results are expressed as percentages of triplicate samples ± SD. PbB16F10 murine melanoma were inoculated i.p into C57BL/6 mice for 7 days and next injected i.p with LMWT for 3 additional days as described in Materials and Methods. Splenocytes from mice were incubated with recombinant dimeric H-2Kb: Ig fusion protein as in a. PcB16F10 murine melanoma pre-infected with LMWT was inoculated into C57BL/6 mice for 7 days as described in Materials and Methods. Splenocytes from mice were incubated with recombinant dimeric H-2Kb: Ig fusion protein as in a. PFrequencies of LLO296–304 specific CD8+ T cells in spleens of B16F10 treated mice or non-treated after LMWT therapy.