Splenic T cells in the mESC-derived TEP-treated allo-BM transplant recipients are functional and have a diverse TCR repertoire. (A-C) Lethally irradiated C57BL/6 mice were injected intrathymically with mESC-derived EpCAM1⁺, EpCAM1⁻ cells, or PBS, and intravenously with TCD-BM from BALB/c mice as in Figure 2. Thirty days after BMT, (A) splenocytes were stimulated with anti-CD3 and anti-CD28 antibodies, and cell proliferation was determined by [³H] thymidine incorporation. (B-C) Splenocytes were stimulated with phorbol myristate acetate and ionomycin and stained for cell surface markers and intercellular cytokines with the use of H-2^d, CD4, CD8, IL-2, IFN-γ, and TNFα. The percentages of IL-2–, IFN-γ–, and TNFα-positive cells in donor origin CD4⁺ and CD8⁺ T cells were determined by flow cytometry. (D) Groups of BM transplant recipients were vaccinated with 50 μg of OVA emulsified in complete Freund adjuvant. Two weeks later, freshly isolated splenic CD3⁺ cells were cultured in the presence of irradiated normal splenocytes (from BALB/c mice) ± OVA for 4 days. T-cell proliferation was then determined by [³H] thymidine incorporation. (E) Lethally irradiated 129B6F2 mice were injected intrathymically with mESC-derived EpCAM1⁺ cells and intravenously with TCD BM from B6 mice. On day 60 after BMT, splenocytes were analyzed for the TCR Vβ families on donor origin CD3⁺ T cells by flow cytometry. Means ± SDs are presented (n = 5-6). ***P < .001. The data are representative of 3 independent experiments.