Tolerant Des-TCR CD8 T cells fail to acquire effector function after LIP. (A) Seven to 10 days after transfer of 2 × 10⁶ splenocytes from thymectomized Des-TCR × 2.4KerIV-K^b.RAG2^−/− (tolerant) and thymectomized Des-TCR.RAG2^−/− (reactive) donor mice into RAG2^−/− mice host splenocytes were restimulated in vitro for 6 hours with anti-CD3 antibody (4 μg/mL). Des-TCR CD8 T cells were analyzed for intracellular IFN-γ production by flow cytometry. Shown are relative log fluorescence intensities for CD8 and IFN-γ. Representative diagrams are depicted; numbers indicate mean percentages of IFN-γ–positive CD8 T cells (0.5% ± 0.7% versus 9.3% ± 7.6%; P < .01) and were pooled from 3 independent experiments (n = 8). (B) In vivo kill assay of K^b-tolerant and K^b-reactive Des-TCR CD8 T cells (2 × 10⁶ splenocytes per mouse) analyzed 10 days after T-cell transfer into RAG2^−/− mice. Splenocytes from CBA and CBK mice were used as targets (control = RAG2^−/− mice without transfer). The data were pooled from 2 separate experiments (*P < .05; n = 8 per group). (C) Splenocytes (2 × 10⁶) from K^b-tolerant and K^b-reactive donor mice were transferred intravenously into RAG2^−/− mice, which were challenged subcutaneously with P815.K^b.B7 tumor cells 3 days later (see Table 1). Representative tumor growth curves of 4 individual mice per group are shown. Data are representative for 3 independent experiments. (D) Splenocytes (2 × 10⁶) from tolerant and reactive donor mice (as above) were transferred intravenously into RAG2^−/− mice. Ten days later recipient splenocytes were restimulated in vitro with αCD3 (4 μg/mL), αCD3, and αCD28 (2 and 4 μg/mL) or irradiated C57BL/6 splenocytes (B6), and ³H-thymidine incorporation was measured after 4 days of culture.