Phenotypic and functional changes associated with antigen recognition by memory 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 in vitro activated anti-HA CD4⁺ TCR transgenic T cells intravenously. One month later, half the animals in each group were challenged with 1 × 10⁶ A20HA tumor cells given intravenously. 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). 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. (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 or was not 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 < .01). Shown is a representative experiment of 3 independent experiments with similar results.