Figure 3. Effective Combination Immunotherapy Elicits Protective Antitumor Immune Memory.
(A) Survival curves for mice treated with FcIL2 + TA99 and IFNα 48 h later that rejected initial challenge with 106 B16F10 melanoma cells s.c. and were rechallenged on day ~100 with 105 B16F10 cells s.c. As a control, the survival of naïve mice challenged with 105 B16F10 s.c. was also monitored. n = 12–19 per group.
(B) Percentages of peripheral blood CD8+ T cells expressing IFNγ following B16F10 tumor rechallenge. On day 8 post rechallenge, blood was collected from mice treated as described in (A) and incubated for 6 h in the presence of brefeldin A and monensin with PMA/ionomycin restimulation prior to flow cytometric analysis. Background IFNγ expression levels detected using controls incubated without PMA/ionomycin were subtracted from the corresponding samples. n = 3–10 per group.
(C) ELISPOT analysis of B16F10-specific IFNγ production by splenocytes isolated from mice treated as described in (A) on day 6 post rechallenge. 106 splenocytes and 2.5×104 irradiated tumor cells were co-incubated for 24 h prior to analysis. Nonspecific responses were quantified by co-incubation with the unrelated TC-1 tumor cell line. Background IFNγ expression levels detected using splenocytes incubated in the absence of tumor cells were subtracted from the corresponding samples. n = 3–7 per group.
(D) Endogenous antitumor antibody response following B16F10 tumor rechallenge as measured by immunoblot. 3–5 weeks post rechallenge, sera were obtained from mice treated as described in (A) and analyzed for antibodies reactive against B16F10 cell lysate. A control immunoblot using TA99 antibody against B16F10 cell lysate was also performed. Each lane represents pooled sera from three mice (naïve) or serum from one individual mouse (FcIL2 + TA99, IFNα 48 h).
Data represent mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001 between the indicated pairs or versus the corresponding color group in the legend. See also Figure S4.