(A) Splenocytes were stained with anti CD4 and TNFR2 Abs and analyzed by FACS. (B) Splenocytes were stained with anti CD3, CD4, CD25 and TNFR2 Abs. The expression of TNFR2 by CD4⁺CD25⁺ cells (grey histogram) and CD4⁺CD25⁻ cells (solid line histogram) was analyzed by FACS, gating on CD3⁺ cells. Dashed line represents isotype control. (C) Splenocytes were stained with anti CD3, CD4, CD25 and TNFR2. Expression of CD25 and TNFR2 was analyzed with FACS by gating on CD3⁺CD4⁺ cells. (D) Splenocytes were stained with anti CD4, CD25, TNFR2 and CD45RB, or CD62L, or CD44, or CD69, or CD103, or GITR, or CTLA-4, or FoxP3 Abs. CTLA-4 and FoxP3 were stained intracellularly. Expression of phenotypic markers was analyzed with FACS by gating on indicated CD4 subsets. (E) Cells from indicated lymphoid tissues and peripheral blood were stained with anti CD3 (or CD8 for thymocytes), CD4, CD25 and TNFR2 Abs. The percentage of TNFR2⁺ cells was analyzed with FACS by gating on CD3⁺CD4⁺CD25⁺ population (except for thymocytes which were gated on CD8⁻CD4⁺CD25⁺ cells). Error bars indicate SE derived from 3 mice (n=3). Numbers in the quadrants represent percentage of positive staining cells (%).The numbers in the histograms show percentage of positive cells (%) and MFI. Data shown are representatives of at least 3 separate experiments with similar results.

(A) LN cells, spleen cells (SPL) and TILs from LLC tumor bearing mice were stained with CD4, CD25 and TNFR2. Expression of TNFR2 was analyzed with FACS by gating on CD4⁺CD25⁺ or CD4⁺CD25⁻ cells from LLC bearing mouse TIL (LLC TIL) or spleen (LLC SPL) or LNs (LLC LN) or from tumor free C57BL/6 mouse spleen (Ctrl SPL) or LNs (Ctrl LN). (B-C) CD4⁺CD25⁻ T cells from normal control mouse LNs (5×10⁴ cells/well) were labeled with CFSE and cultured alone or co-cultured with indicated source of Tregs at desired ration. The percentage of CFSE-diluted cells was shown in the histograms. Data shown are representatives of 3 separate experiments with similar results.

Flow-sorted CD4⁺CD25⁻TNFR2⁻ or CD4⁺CD25⁻ T cells (5×10⁴ cells/well) were labeled with CFSE and cultured alone or co-cultured with indicated number or ratio of flow-sorted CD4 subsets from spleen and LNs of normal C57BL/6 mice. The percentage of CFSE-diluted cells was shown in the histograms. (A), (C) and (D) show a representative data of at least 3 separate experiments with similar results and (B) shows summary of percent inhibition of replication (%) from 6 separate experiments.