Figure 9. Overexpression of NOX2 rescues the suppressive activity of old CD8 Tregs.

CD8 Tregs were induced from older individuals and transfected with a control or NOX2 expression vector. (A and B) Surface expression of NOX2 was evaluated 24 hours after transfection by flow cytometry. (A) One representative image and (B) results (mean ± SD) from 3 independent experiments are shown. (C) CD8 Tregs transfected with NOX2 expression vector or a control vector were analyzed for their suppressive function by mixing them with naive CD4 T cells and quantifying stimulation-induced pZAP70. Frequencies of pZAP70⁺ CD4 T cells (mean ± SD) from 10 independent experiments are shown. (D and E) Old CD8 Tregs were transfected with a control or NOX2 expression vector and tested for in vivo–suppressive activity by injecting them into NSG mice that had been reconstituted with naive CD4 T cells and monocytes. Nine days after transfer, expansion of CD4 T cells was quantified by enumerating the frequency and total numbers of human CD4 T cells in the murine spleen. Results are shown as mean ± SD from 6 independent experiments. (F–H) Old transfected CD8 Tregs were examined for in vivo function, as above. CD4 T cells were labeled with CFSE prior to the transfer. Proliferation of CD4 T cells was assessed by flow cytometric analysis of CFSE dilution in splenocytes harvested after 9 days. A representative image is shown, and results from 6 independent experiments are summarized as (mean ± SD) division index and proliferation index. Unpaired 2-tailed Student’s t test was used for comparisons.