Large TRP-2_180-188–specific CD8⁺ T-cell responses can be generated by vaccination after BMT. (A) Large TRP-2_180-188–specific CD8⁺ T-cell responses, but minimal OVA_257-264–specific responses, were detected in mice that received TRP-2_180-188–containing vaccines. Mice underwent BMT as described in Figure 1. On day 14 after BMT, 100 000 B16F1 cells were injected. On days 14, 17, and 20 after BMT, priming vaccines containing TRP-2_180-188 + CpG + IFA were administered. IL-2 was given on days 21 to 23. On day 28 after BMT, a boost vaccine that was identical to the priming vaccines was administered, and IL-2 was given on days 29 to 32. On day 33 after BMT, the mice were killed. Splenocytes were stimulated ex vivo for 6 hours with EL4 cells pulsed with either TRP-2_180-188 or OVA_257-264, and ICCS for IFNγ was performed. Plots are gated on CD3⁺ lymphocytes. The percentage of CD3⁺CD8⁺ cells that were positive for IFNγ is shown on each plot. Results from one representative mouse are shown. (B) In mice that received vaccines containing TRP-2_180-188 + CpG and systemic IL-2, a mean of 9.3% of CD3⁺CD8⁺ splenocytes produced IFNγ in response to TRP-2_180-188, but only 0.1% of CD3⁺CD8⁺ splenocytes produced IFNγ in response to the negative control peptide OVA_257-264 (n = 10 mice per group). (C) TRP-2_180-188–specific CD8⁺ T-cell responses were detected 33 days after BMT by TRP-2_180-188-K^b tetramers in mice treated as described in (A). A representative example of 7 mice is shown. (D) TRP-2_180-188–specific CD8⁺ T-cell responses were detected 25 days after the final vaccination in mice that were vaccinated and treated with IL-2 as described in (A). A representative example of 6 mice tested by ICCS assay is shown. (E) Mice underwent BMT, were vaccinated with peptide + CpG–containing vaccines, and received IL-2 as described in (A). One group was vaccinated with TRP-2_180-188–containing vaccines and another group was vaccinated with OVA_257-264–containing vaccines. Both groups had B16F1 injected on the same day as the first vaccination (day 14). On day 33, when all mice had palpable tumors, the mice were killed. Splenocytes were stimulated for 6 hours with either TRP-2_180-188 or OVA_257-264, and ICCS was performed. A representative example of CD8⁺ T-cell responses detected after ex vivo TRP-2_180-188 stimulation of splenocytes from tumor-bearing TRP-2_180-188–vaccinated mice is shown. Representative examples are also shown of CD8⁺ T-cell responses detected in splenocytes from tumor-bearing OVA_257-264–vaccinated mice after ex vivo stimulation with either TRP-2_180-188 or OVA_257-264. Ten TRP-2_180-188–vaccinated and eight OVA_257-264–vaccinated mice were tested. Plots are gated on CD3⁺ lymphocytes. The percentage of CD3⁺CD8⁺ cells that produced IFNγ is shown on each plot. (F) Mice underwent BMT as described in Figure 1. The mice were then divided into 2 groups. One group was injected with 100 000 B16F1 cells on day 13 after BMT and the other group was not injected with tumor cells. Both groups then received TRP-2_180-188 + CpG + IFA vaccines on days 13, 16, 19, and 27 after BMT. IL-2 was administered on days 20 to 22 and days 28 to 31 after BMT. On day 32 after BMT, TRP-2_180-188–specific CD8⁺ T-cell responses were measured by ICCS assay as described in panel A. The presence of B16F1 did not affect the number of TRP-2_180-188–specific CD8⁺ T cells generated by the vaccination regimen (n = 8 mice per group).