Fig. 5.
3H-7β/α-OHC were preferentially converted into 3H-7β/α, 27-OHC in mouse Th17 but minimally in Th1 T cells. (A) Conversion of 3H-7β-OHC or 7α-OHC by mouse Th17 and Th1 cells in culture (purified total CD4+ T cells activated under Th17 or Th1 conditions for 3 d). Conditioned media were fractionated using HPLC as described in SI Appendix. Radio activities of 50 μL 1-min fractions were determined. Data shown were from a representative experiment of three independent experiments. (B) Conversion of 3H-7β-OHC by cultured Th17 cells differentiated from WT and Cyp27a1 KO naïve CD4+ T cells. Conditioned media were purified as above. Fractions containing the converted products were pooled and further purified by another HPLC; 30-s fractions were collected to resolve converted species, which resulted in peaks A and B. MS analysis revealed that peak B was consistent with being 7β, 27-OHC. (C) HPLC fractionation of conditioned medium from 3H-7β-OHC–spiked culture of untransfected COS7 cells or COS7 cells transiently expressing human CYP27A1 protein. CPM, counts per minute.