Phenotypic changes associated with antigen recognition on CD4⁺ TCR clonotype⁺ T cells is only seen in H-2^d→H-2^dxb tumor-bearing chimeras.

T cells from the mice in Figure 4 were isolated from their spleen or lymph nodes as described in “Materials and methods.” Cells 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 CD45RB. Live gating on CD4⁺ T cells was set, and 100 000 events collected per sample. (A) Mean fluorescence intensity ± SE is shown for CD45Rb expressed by CD4⁺TCR clonotype-positive T cells isolated from the spleen of tumor-free (No tumor) or A20HA-bearing chimeric mice (3 mice per group). (B) Representative FACS profile of CD45Rb expression by anti-HA CD4⁺ T cells isolated from lymph nodes. Inguinal and axillary lymph nodes were harvested from tumor-free and tumor-bearing chimeric mice. Lymph node cells from 3 animals per group were pooled and T cells were isolated and stained as described above. Shaded histograms in each figure correspond to clonotype-positive T cells from tumor-free mice. Open histograms correspond to clonotype-positive T cells from tumor-bearing mice. MF indicates mean fluorescence intensity. Shown is a representative experiment of 3 independent experiments with similar results.