Heterogeneity of tumor-specific CD4⁺ T cells. (A) A total of 2.5 × 10⁶ CFSE-labeled HA-specific CD4⁺Thy1.1⁺ T cells were transferred into BALB/c recipients (Thy1.2^+/+) either tumor free (NT) or inoculated with 1 × 10⁶ A20HA 10 days earlier (TM). Sixteen days after T-cell transfer, some mice were immunized with vacHA and analyzed 5 days later. Mice were killed at indicated time points. The frequency of transferred Thy1.1⁺CD4⁺ cells in the spleen was measured by FACS analysis. Percentage of the gated population is displayed in each dot plot. CFSE profiles of the gated cells are shown. The percentage of the divided cells in the gated population is indicated in each histogram. (B) Absolute number of divided donor cells recovered from spleen (total splenocyte count × percent CD4⁺Thy1.1⁺ × percent donor cells that are CFSE_low). For unvaccinated NT mice, only CFSE_high donor cells were counted. Each group had a minimum of 3 mice. Results are shown as mean ± SE. (C) Cells were sorted based on cell division status as indicated in panel A. Sorted cells (2 × 10⁴) were stimulated with 10 μg/mL HA_110-120 peptide in the presence of 2 × 10⁵ irradiated BALB/c splenocytes. Cell proliferation was measured by ³H-thymidine incorporation. Supernatants were analyzed by ELISA for detection of IL-2 and IFN-γ. (D) qRT-PCR analysis of sorted cells. Purified CD4⁺ T cells from 6.5 Tg mice were included as naive controls. Each symbol represents data from one mouse. Each sample was run in triplicate for each gene, with HPRT as internal reference. mRNA abundance of the target gene was normalized to HPRT and represented as relative mRNA frequency.