Phenotypic and functional changes associated with antigen recognition by naive CD4⁺ T cells in the presence or absence of cross-presentation of tumor antigens. Three months after bone marrow reconstitution, H-2^d SCID→ H-2^dxb (left panel) or H-2^b SCID→ H-2^dxb (right panel) chimeras received 1 × 10⁶ naive anti-HA CD4⁺ TCR transgenic T cells intravenously. Four weeks later, half the animals in each set of chimeras were challenged with 1 × 10⁶ A20HA tumor cells given intravenously. In a cohort of tumor-bearing and tumor-free chimeras, BrdU was added to the drinking water on day 14 after tumor injection. Three weeks after tumor challenge, all the animals were killed, and T cells were purified from their spleens, as described in “Materials and methods.” (A) Purified splenic T cells were analyzed by 2-color flow cytometry staining for CD4 compared with anti-HA TCR clonotype (mAb 6.5). Three mice were included per group. Values represent mean ± SE of percentage of T cells expressing the clonotypic TCR for 3 mice/group. Shown is a representative experiment of 3 independent experiments with similar results. (B) Representative FACS profile of BrdU labeling on anti-HA CD4⁺ T cells isolated from H-2^d SCID→ H-2^dxb or H-2^b SCID→ H-2^dxb tumor-bearing chimeras. Purified splenic T cells were stained with PE-conjugated anti-CD4 and biotinylated rat anti-clonotypic TCR mAb 6.5 followed by APC-conjugated streptavidin. Cells were then fixed, permeabilized, and stained for BrdU using the BrdU Flow Kit, and 50 000 gated events were collected on FACScalibur and were analyzed using FlowJo software. (C) Purified T cells (4 × 10⁴/well) from the mice in panel A were mixed with fresh splenocytes (8 × 10⁴/well) from F1 mice, to which 12.5 μg HA peptide was added. Forty-eight hours later, supernatants were collected and assayed for IL-2 by ELISA. Data represent mean ± SE of IL-2 production (pg/mL) per well from 3 mice in each group. ^*Statistically significant difference between tumor-bearing and tumor-free mice (P < .05). Shown is a representative experiment of 3 independent experiments with similar results.