VIP limits T-cell proliferation and Th1 polarization of T cells in vitro. (A-B) pDCs isolated from B6 and VIP-KO marrow by FACS were activated with R848 and CpG for 24 hours, followed by coculturing with syngeneic T cells for 72 hours at a pDC:T-cell ratio of 1:10. CD4⁺ and CD8⁺ T-cell proliferation was analyzed by CFSE (carboxyfluorescein diacetate succinimidyl ester) dilution using flow cytometry. Representative histograms for CFSE dilution and mean values of the fraction of proliferated cells and the proliferation index. Two independent experiments that included 3 biological replicates per group with a total of n = 14 technical replicates in the VIP-KO pDC group, n = 10 in the wild-type pDC group, and n = 14 in the no pDC group. (A) Proliferation for activated CD4⁺ T cells. (B) Proliferation for activated CD8⁺ T cells. (C-D) Representative flow plots of IFN-γ expression on CD4⁺ T cells (C) and CD8⁺ T cells (D) analyzed by flow cytometry after intracellular staining. (E-F) Representative flow plots of IFN-γ and TNF-α coexpression on CD4⁺ T cells and CD8⁺ T cells based on 2 independent experiments that each included 2 biological replicates with a total of 8 technical replicates for each group. Statistics: 2-tail unpaired Student t test, *P < .05, **P < .01, ***P < .001, ****P < .0001.