Figure 2.

CD28 is involved in the generation of Foxp3⁻ Treg cell precursors. (A) Decreased frequency of cytokine responsive Treg cell precursors in CD28 KO and AYAA mice. CD25^hiFoxp3⁻ CD4SP cells were FACS purified from WT and indicated strains, cultured in vitro with hIL-2 (50 U/ml), and analyzed at 24 hours for Foxp3 expression by flow cytometry. Data were normalized to that of WT from the same experiment to account for inter-experimental variation. Each dot represents data from an individual experiment. Bars indicate the average normalized percentage of Treg cell precursors (n=8 WT and KO; n=5 AYAA, n=6 Y170F). (B) The effect of CD28 on the generation of Treg cells is cell-intrinsic. Lethally irradiated congenic hosts (CD45.1) were reconstituted with an equal mixture of bone marrow cells from WT (CD45.1) and WT, KO, or AYAA (all CD45.2) mice. Foxp3 and CD25 expression in CD45.2⁺CD4SP cells were analyzed by flow cytometry six weeks post-reconstitution. (C) Percentages of CD25^hiFoxp3⁻ and Foxp3⁺ cells within the CD45.2⁺CD4SP subset are summarized (n=12 WT and KO; n=8 AYAA; n=6 Y170F). (D) The effect of CD28 on the generation of Treg cell precursors is cell-intrinsic. FACS purified CD45.2⁺ Foxp3⁻ CD4SP cells were cultured in vitro with hIL-2 (50 U/ml) and Foxp3 expression was analyzed 24 hours later by flow cytometry. Data shown were normalized to that of WT cells in the same experiment. Each dot represents data from an individual mouse (n=6 mice, 4 independent experiments) and the bars indicate the average normalized percentage. ** p ≤0.01.