Figure 2.

Sin1 deficiency increases the number of thymic natural regulatory T cells

a) Splenocytes from Sin1^+/+ (WT) or Sin1^−/− (KO) chimeric mice were analyzed for percentages of CD4 and CD8 T cells. The FACS plots shown are pre-gated on CD45.2⁺ donor cells.

b) Splenocytes from Sin1^+/+ (WT) or Sin1^−/− (KO) chimeric mice were stimulated with PMA and ionomycin for 4 hours in the presence of Golgi-Stop and assayed for IFN-γ, IL-17A, and IL-4 expression by flow cytometry. The FACS plots shown are pre-gated on donor CD4⁺CD8⁻ CD45.2⁺ cells.

c) The expression of CD62L and CD44 was measured on splenic CD4⁺ T cells from Sin1^+/+ or Sin1^−/− chimeric mice by flow cytometry. The mean fluorescent intensity (MFI) of CD62L in the CD44^lowCD62L^hi gate are as follows: Sin1^+/+ MFI=8520 and Sin1^−/− MFI=17400. The FACS plots shown are pre-gated on donor CD4⁺CD8⁻ CD45.2⁺ cells.

d) The expression of IL-7R (CD127) on splenic donor CD4⁺CD45.2⁺ T cells from Sin1^+/+ or Sin1^−/− chimeric mice was determined by flow cytometry.

e) The thymic (upper panel) and splenic (lower panel) donor CD4⁺Foxp3⁺CD45.2⁺ T cells in Sin1^+/+ or Sin1^−/− chimeric mice were determined by flow cytometry.

f) Chimeric mice containing host WT and Sin1^−/− (KO) T cells were generated by transferring Sin1^−/− fetal liver cells into sub-lethally irradiated WT (CD45.1⁺) hosts. The percent of thymic WT CD4⁺Foxp3⁺ T cells (CD45.1⁺) and donor Sin1^−/− CD4⁺Foxp3⁺ T cells (CD45.2⁺) in a chimeric mouse was determined by flow cytometry.

Numbers in the plots indicate percentages of the gated populations. All flow cytometry plots shown are representative of Sin1^+/+ (n=3) and Sin1^−/− (n=4) fetal liver chimeric mice.