Tumor-induced CD4⁺ regulatory cells contain both CD25⁺ and CD25^– cells, whereas antigen-specific suppression resides exclusively in a GITR⁺ subset. Spleen cells were pooled from vaccinated TM mice that had previously received CFSE-labeled Tg CD4⁺Thy1.1⁺ T cells as described in Figure 1. CFSE_low cells were further separated into CD25⁺ and CD25^– subsets. (A) Proliferation of CD25-separated and unseparated TMTregs. CFSE_low effector cells from vaccinated NT mice were included for comparison. (B) In vitro suppression assay. Rag2^–/– 6.5CD4⁺ responder T cells were mixed with CD25-separated TMTregs at the indicated ratios and assayed as in Figure 2A. (C) CFSE CD4⁺Thy1.1⁺ low cells from vaccinated TM mice were also sorted into GITR^high and GITR^low subsets. GITR-fractionated cells were stimulated with irradiated fresh BALB/c splenocytes and varied concentration of HA peptide. Cell proliferation (C) and IFN-γ production (D) were measured as described in Figure 1C. (E) qRT-PCR analysis of GITR-fractionated cells. mRNA frequencies of the indicated genes were normalized to HPRT. (A-E) Results are shown as mean ± SE of triplicate cultures or reactions. (F) GITR^high subset exclusively suppresses responder cells in vitro. Thy1.2⁺CD4⁺ responder cells from Rag2^–/– 6.5 Tg mice were CFSE labeled and cultured with irradiated BALB/c (Thy1.1⁺/+ background) splenocytes and HA peptide, either alone or with equal number of GITR-fractionated cells (Thy1.1^+/+). Three days later, cells were stained with anti-Thy1.2 and either anti-CD25 or anti-CD69 mAb. Plots shown are gated on Thy1.2⁺ responder cells. The data shown are representative of 2 separate experiments with similar results. Numbers indicate the percentage of cells in each quadrant.