To provide an independent measure of infiltrated ocular lymphocytes, we next performed flow cytometry analyses of these cells. Consistent with our findings on tissue immunostaining, CD3
+ lymphocytes were noted in both wild-type and Nrf2 knockout mice fed the HF diet (
Fig. 5A). Among those cells there were two subpopulations, CD3
+ CD4
− cells and CD3
+ CD4
+ cells (8.8% and 6.3%, respectively, in HF
Nrf2 −/− mice). However, those two groups of cells were either diminished or absent in eyes from
Nrf2 −/− mice on the normal diet (1.1% and 0.4%, respectively). CD4
+ FoxP3
+ Tregs were identified in both wild-type and Nrf2 knockout mice on the HF diet (
Fig. 5B), and the percentage of Tregs in the total CD3
+ population was higher in wild-type mice. In contrast, the number of IL-17-producing lymphocytes was significantly higher in
Nrf2 −/− mice (
Fig. 5C, 10.5% ± 0.9 vs. 4.1% ± 0.6, mean ± SEM,
P < 0.01, Student's
t-test). Further analyses revealed that the majority of IL-17 producers were γδ T cells (
Fig. 5F); and there was a significant increase of IL-17
+ and TCRγδ
+ cells in HF-fed
Nrf2 −/− mice (
Fig. 5D) (5.6 ± 1.5 vs. 1.9 ± 0.1, mean ± SEM,
P < 0.05, Student's
t-test). The number of Th17 cells (CD4
+ IL-17
+) was minimal in the eyes from either wild-type or
Nrf2 −/− mice (
Fig. 5E). Thus, γδ T cells are the major source of IL-17 production in the degenerating retina/RPE. We further characterized the subsets of γδ T cells in the RPE/choroid in HF diet–fed
Nrf2 −/− mice. As shown in
Figure 6, the profile was different between spleen and RPE/choroid, and Vγ6 was the major subset in the eye.