FIGURE 5.

FoxP3+ T regulatory cells (T_reg) are reduced in dTg HEL/TCR mice but accumulate in the retina in late stages of disease and have greater immunosuppressive activity than 3A9 TCR T_reg. (A,B) Analysis of T regulatory cell (T_reg) numbers in dTg HEL/TCR mice during EAU. Absolute numbers of T_reg in the submandibular eye DLN and retina were quantified by flow cytometry using FoxP3 and CD25 expression in CD4+ T cells. At onset of disease (P21) T_reg numbers were extremely low (2 × 10³) in the dTg DLN compared to those of 3A9 mice. No T_reg were detected in 3A9 retinas and effectively nil in the retinas of dTg mice at P21. As EAU developed, there was an increase in retinal T_reg which reached its maximum (4 × 10³/retina) by P45 (n = 6–8/age group). Data were analyzed using one-way ANOVA and Tukey’s Multiple Comparison post hoc Test with *p < 0.05, **p < 0.01, ***p < 0.001 on a 95% level of confidence. Cell numbers are the average of 3–4 pairs of retina or DLN, i.e., cell count/pair. 1 × 10⁵ total events were recorded. Total numbers provided were extrapolated based on total cell counts, determined using a Coulter cell counter prior to sample processing. (C) T_eff cell proliferation is significantly reduced in the presence of increasing concentrations of AgX T_reg compared to 3A9 T_reg (i.e., T_reg isolated from mice with EAU aged between P50 and P70; n = 3/genotype): dTg T_reg show greater suppressive ability when [T_reg:T_eff] ratio is [>2:1]. Data were analyzed using two-way ANOVA with *p < 0.05, ****p < 0.0001 on a 95% level of confidence.