Fig. 2. Immunophenotyping of CD25⁺ and CD25⁻ naïve CD4⁺ T cells.

(A) Gating strategy used to define the CD25⁺ and CD25⁻ subsets of human peripheral blood CD4⁺, CD45RA⁺, CD45RO⁻, CCR7⁺, CD62L⁺ naïve T cells (representative of 20 donors). (B) Representative examples of flow cytometric analyses of CD25⁺ and CD25⁻ naïve CD4⁺ T cell subsets: overlaid histograms show the expression of the indicated molecule in three CD4⁺ T cell subsets defined as memory CD45RA⁻ CD45RO⁺ (grey solid histogram), naïve CD45RA⁺ CD45RO⁻ CD62L⁺ CD25⁻ (blue histogram) and naïve CD45RA⁺ CD45RO⁻ CD62L⁺ CD25⁺ (red histogram), representative example of between 5 and 20 donors. (C) Gating strategy to assess the proportion of cells in cycle (Ki-67⁺) within various CD4⁺ T cell subsets: CD25^−/+ CD45RA⁺ FOXP3⁻ naïve CD4 T cells, CD25^−/+ CD45RA⁻ FOXP3⁻ memory CD4 T cells and CD25^high Tregs. (D) Combined analysis of Ki-67⁺ T cell frequency in five CD4 T cell subsets in 16 donors.