PTPN22 Alters the Development of Regulatory T Cells in the Thymus

CD25 expression on thymic Tregs and Treg precursors. (A) Mean fluorescent intensity (MFI) of CD25 on Tregs (CD4⁺Foxp3⁺CD25⁺) in the thymus of WT, Het, and KO mice. (B) Representative histogram of CD25 expression on Tregs in the thymus of WT (filled plot) and KO (unfilled plot) mice (Het plot was not included for clarity). (C) MFI of CD25 on Treg precursors (CD4⁺Foxp3⁻CD25⁺).

Signaling differences in the thymus of PTPN22-deficient versus WT mice. (A) Calcium flux in double positive and CD4⁺CD25⁺ thymocytes derived from WT (thin line) or KO (thick line) mice. Cells were prelabeled with anti-CD3 biotin Ab and stimulated at 30 s with streptavidin. Exogenous calcium was added at 120 s and ionomycin at 300 s. For the experiment shown, WT cells were labeled with Cy5 and mixed with unlabeled KO cells to stimulate under identical conditions. During the same experiment, the Cy5 labeling was reversed to ensure it did not interfere with Ca flux. This is an example of one of four separate experiments (WT, n = 8; KO, n = 8). (B) CD69 expression on double positive thymocytes from WT (filled plot), Het (unfilled, thin line), and KO (unfilled, thick line) mice either from unstimulated thymocytes or CD3/CD28 stimulated thymocytes (overnight stimulation). (C) Normalized mean fluorescent intensity (MFI) of CD69 on CD3/CD28 stimulated DP thymocytes. *p < 0.05.

PTPN22 deficiency results in increased peripheral Tregs. Spleens were removed from WT, Het, and KO mice and stained for CD4, CD8, Foxp3, and CD25. (A) Representative CD4 versus CD8 plots for the three genotypes of mice. (B) Representative dot plots showing CD25 and Foxp3 expression of the CD4 gate on WT, Het, and KO splenocytes. (C) Treg/CD4 ratio comparing the CD4⁺Foxp3⁺ cells to CD4⁺Foxp3⁻ cells in the spleen of the three groups. (D) Treg/effector CD4 ratio in the spleen of the three genotypes. Effector CD4 cells are defined as CD4⁺CD44^hiFoxp3⁻. *p < 0.05, **p < 0.01.

CD25 expression on peripheral Tregs of PTPN22-deficient mice. (A) Mean fluorescent intensity (MFI) of CD25 on Tregs (CD4⁺Foxp3⁺) in the spleen of WT, Het, and KO mice. (B) Representative histogram showing CD25 expression on CD4⁺Foxp3⁺ Tregs from WT (filled plot) and KO (unfilled plot) mice. (C) CD4⁺ cells were purified from spleens of WT, Het, and KO mice and serum starved for 20 min. IL-2 was then added in complete culture medium, and the cells were incubated for the designated time points. Cells were then fixed and stained for p-STAT5 by flow cytometry. CD4⁺CD25⁺ cells were gated, and the unstimulated plot (filled plot) for each genotype is overlaid with the stimulated plot (unfilled plot) (this is representative of three separate experiments, n = 6 mice per group). (D) Line graph representing the histograms shown in (C) showing the kinetics of p-STAT5; there is a statistical significance at 2 min between WT and KO groups. *p < 0.05, **p < 0.01.

Suppressive capability of Tregs from PTPN22-deficient mice. CD4⁺CD25⁺ Tregs were isolated from splenocytes from WT, Het, and KO mice using magnetic separation. CFSE-labeled Teff (CD4⁺CD25⁻) T cells (3 × 10⁴) were cultured in the presence of 1 × 10⁵ irradiated splenocytes and soluble anti-CD3 Ab (1 μg/ml). Tregs were titrated into the cultures at the ratios indicated. The cells were cultured for 72 h, and then proliferation was assessed by flow cytometry. This experiment is representative of three separate experiments. The 96-h time point shows similar results from two separate experiments (data not shown).