Figure 2

TSA-treated B16 vaccine elicits cytotoxic and IFN-γ-producing lymphocytes and requires T and NK cells in immunity. A) T-cell-enriched splenic lymphocytes isolated from immune or control mice after in vitro re-stimulation were analyzed in triplicate for cytotoxic activity using ⁵¹Cr-labeled untreated B16 or EL4 as targets. B) Splenocytes isolated from immune, B16 tumor-bearing or naive B6 mice were cultured in anti-IFN-γ coated plates with B16 cell lysate or melanoma antigen mgp100_25–33 peptide and IFN-γ secretion was detected by ELISpot assay. *, p <0.0008 (cell lysate stimulation) and **, p <0.03 (peptide stimulation) compared with splenocytes from tumor-bearing mice. PMA and Ionomycin treated splenocytes served as positive control. These experiments (A and B) were repeated 3 times with similar results. C) Reduced incidence of TSA-treated (500 nM for 48 h) B16 tumor rejection in MHC deficient mice that lack CD4⁺ and/or CD8⁺ T cells compared with immunocompetent mice. The Kaplan-Meier plot shows tumor-free survival of class II^-/- (n = 16), class I^-/- (n = 18), class I^-/--II^-/- (n = 18) and immunocompetent B6 (n = 15) mice after s.c. vaccination with TSA-treated B16 (1 × 10⁵ cells). D) Depletion of CD4⁺, CD8⁺ T cells and NK cells reduces immunity generation by TSA-treated B16 cells. B6 mice were depleted of CD4⁺ (n = 16), CD8⁺ (n = 8) T cells or NK cells (n = 8) by repeated injection of respective antibodies (see Methods). Undepleted controls (n = 16) received IgG. All mice were observed for tumor growth after s.c. vaccination with TSA-treated B16 cells in the trunk.