Induction of tumor antigen–specific CD4⁺ T-cell tolerance requires presentation by BM-derived APCs.

Three months after BM reconstitution, H-2^dSCID→H-2^dxb (left panel) or H-2^bSCID→H-2^dxb chimeras (right panel) received 1 × 10⁶ anti-HA CD4⁺ TCR transgenic T cells IV. One day later, half the animals in each set of chimeras were challenged with 1 × 10⁶ A20HA tumor cells given IV. On day +22 after tumor challenge, all the animals were killed and T cells were purified from their spleens as described. (A) Purified splenic T cells were analyzed by 2-color flow cytometry staining for CD4 versus anti-HA TCR clonotype as in Figure 4. Values represent mean ± SE of percentage of T cells expressing the clonotypic TCR for 3 mice per group. (B) T cells from tumor-free or tumor-bearing chimeras were stained with cychrome-labeled anti–mouse CD4, biotinylated anti–TCR clonotype MoAb 6.5 followed by PE-labeled streptavidin and FITC-conjugated anti–mouse CD62 as in Figure 5. Mean fluorescence intensity ± SE is shown (2 mice per group). (C) In vitro proliferative response of clonotype-positive T cells to stimulation with HA_110-120 peptide. Purified T cells (4 × 10⁴/well) from tumor-free (no tumor, ●), tumor-bearing chimeras (A20HA, ■), or from F1 naive mice (no T-cell transfer, ▴) were mixed with fresh splenocytes (8 × 10⁴/well) from F1 mice to which different concentrations of HA peptide were added. ³H incorporation was assayed after 3 days of incubation, and is shown as counts per minute (cpm) from which values for medium alone were subtracted. Values represent the mean of the cpm/100 clonotype-positive T cells per well from 2 to 3 mice in each group. (D) Purified T cells (4 × 10⁴ /well) from the mice in panel C were mixed with fresh splenocytes (8 × 10⁴/ well) from naive F1 mice to which HA peptide (100 μg/mL) was added. Forty-eight hours later, supernantants were collected and assayed for IL-2 by ELISA. Data represent mean ± SE of the cpm/100 clonotype-positive T cells per well from 2 mice in each group.