Figure 5. VPAC2 KO Tregs exhibit reduced in vitro suppressive activity.

The Treg suppressive activity of CD4⁺CD25⁺ Tregs from WT vs VPAC2 KO spleens and lymph nodes over WT Teff cell proliferation was determined by [³H]-thymidine incorporation assay. (A) Purity of isolated Tregs prior to culture was determined by flow cytometry. Representative plots of CD4⁺CD25⁺ cells and Foxp3⁺ cells (gated on CD4⁺CD25⁺ cells) are shown. (B) Tregs and Teffs at different ratios (Treg:Teff = 0:1, 1:1, 1:2, 1:4, 1:8) were co-cultured with WT APCs in complete RPMI 1640 medium with IL-2 (50U/ml) and MOG (20μg/mL). Proliferation was measured 48 hours after culture by thymidine incorporation assay (see Materials and methods). Results shown are representative of three independent experiments of n = 6 mice/group. In addition, cultures were performed in triplicate. *P<.05 (Student's t-test).