Figure 3.

The effect of TIM-4 blockade with addition of exogenous TGF-β on inhibiting T helper 2 cell differentiation and inducing the conversion of CD4⁺CD25⁺Foxp3⁺ T regulatory cells. (A) TIM-4⁺ KCs were isolated from model mice and purified using fluorescence activated cell sorting, followed by addition of control mAb/TGF-β/TIM-4 mAb/TGF-β+TIM-4 mAb into medium for incubation. Following washing and resuspension of cells, they were stained with PEcy5-MHC II and PE-CD40 antibodies and acquired for FACS analysis. (B) Spleen CD4⁺CD25⁻ T cells were purified from recipient mice and labeled with CFSE, followed by co-culturing with KCs in condition described in A (control as untreated). The proliferation of T cells was then determined using a CFSE dilution gated on CD4⁺ populations. (C) The supernatants of the co-cultured system, including the cytokines IL-4, IL-6 and IL-13, were detected using enzyme-linked immunosorbent assay. (D) Splenic CD4⁺CD25⁻ T cells were purified from recipient mice and co-cultured with KCs in conditions as described in A. Following washing and re-suspension, the cells were stained with PE-Foxp3 and FITC-CD25 antibodies and underwent FACS analysis. Data are presented as the mean ± standard deviation. ^aP<0.05 vs. control and control mAb groups; ^bP<0.05 vs. TGF-β and TIM-4 mAb groups. TIM-4, T cell immunoglobulin-domain mucin-domain-4; TGF-β, transforming growth factor-β; KCs, kupffer cells; mAb, monoclonal antibodies; MHC II, major histocompatibility complex II; CD, cluster of differentiation; FACS, fluorescence-activated cell sorting; CFSE, carboxyfluorescein succinimidyl ester; IL, interleukin; Foxp3, forkhead box P3; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PEcy5, phycoerythrin-streptavidin.