(A and B) Specific peptide pools restricted in H-2^d (A) and H-2^b (B) haplotypes. 1 × 10⁶ splenocytes from BALB/c (A) or C57BL/6 (B) mice vaccinated with Tem1-TT were cultured with the 4 TEM1 peptide pools and tested by ELISpot and ICS for IFN-γ secretion. (A) Splenocytes from Tem1-TT–vaccinated BALB/c mice recognized pool C (left), which induced CD3⁺CD4⁺ (middle) and CD3⁺CD8⁺ (right) T cell responses. (B) Splenocytes from Tem1-TT–vaccinated C57BL/6 mice recognized pool D (left). By ICS, there was a specific CD3⁺CD8⁺ response (right). Data are mean ± SD (n = 5 per group) from 1 of 3 experiments. (C and D) Characterization of TEM1-derived peptides specific for H-2^d (C) and H-2^b (D) haplotypes. Peptides from pool C (C) or pool D (D) were divided into 7 minipools of 15 mers, so each individual peptide was present in 2 minipools and could be identified through an experimental matrix design. (C) Reactivity to pool C was present in minipools f, g, and 5. (D) Reactivity to pool D was present in minipools a and 6. Data are from 1 of 3 independent experiments. (E) TEM1_696–710 induced IFN-γ secretion and cytotoxic effector activity. Splenocytes from TT-, Tem1-, or Tem1-TT–vaccinated mice was used in peptide-stimulated cultures and tested in an IFN-γ secretion ELISA (left) and CTL assay (right). Tem1-TT exclusively primed and expanded antigen-specific CD8⁺ T cells that killed ECs in the context of MHC class I/TEM1_696–710 peptide, but not control peptides.