FIGURE 4.

Aquaporin 4 (AQP4) p61–80-specific T cells exhibit a proinflammatory bias. Peripheral blood mononuclear cells (PBMC) were stained with 5,6-Carboxylfluorescein diacetate succinimidyl ester (CFSE) and cultured for 10 days with AQP4 peptides (10μg/ml) or recombinant human (rh) AQP4 (5μg/ml). (A) CD4⁺CFSE^low proliferating T cells were analyzed for interleukin (IL)-17 and interferon (IFN)-γ production by intracellular staining after stimulation with phorbol 12-myristate 13-acetate/Ionomycin for 5 hours. (B) Frequencies of IL17⁺IFN-γ⁻, IL17⁺IFN-γ⁺, and IL17⁻IFN-γ⁺ were examined among proliferating p61–80-specific CD4⁺ T cells (n = 8 NMO and n = 5 healthy controls [HC]), p156–170-specific CD4⁺ T cells (n = 6 NMO and n = 3 HC), and rhAQP4-specific CD4⁺ T cells (n = 6 NMO and n = 5 HC). Frequencies of IL-17 and IFN-γ single positive T cells were used to calculate Th17/Th1 ratio. (C) PBMC were examined by fluorescence-activated cell sorting (FACS) for expression of regulatory T cells (Treg)markers including CD4, CD127, and CD25. (D) CFSE-labeled PBMC were cultured for 10 days with AQP4 p61–80 (10μg/ml) or rhAQP4 (5μg/ml). Proliferating CD4⁺ T cells (cell division index > 2) were examined by FACS for expression of CD25^high, defined as the top half of CD25⁺ cells, and Foxp3 (n = 8 NMO p61–80, n = 6 HC p61–80, n = 7 NMO rhAQP4, and n = 5 HC rhAQP4). Box and whisker plots include the median, distribution, and range. **p < 0.01 Mann–Whitney U test.