Lls generated with or without the blockade of TLR5 were sorted using FACSAria after 9 days of coculture. The sorted CD4hiCD25+ regulatory T cells were titrated and co-cultured with 56104 responder CD4+CD252 T cells from the same donor of the CD4hiCD25+ regulatory T cells and 56104 c-irradiated target PBMC from the donor of the CD40-activated B cells as stimulator for 3 days. 3H-thymidine was added to the coculture in the last 18 hours and the proliferation was analyzed by 3 H-thymidine incorporation assay as we described previously [28].Generation of CD40-activated B CellsPBMC were isolated from buffy coat of healthy adult donors from the Hong Kong Red Cross Blood Transfusion Services. CD40-acitvated B cells were generated from PBMC via CD40 stimulation using lethally irradiated (96Gy) Ermore, perfusates exclude the influence of other organs. It should be NIH3T3 cells transfected with the human CD40 ligand (t-CD40-L cells) as stimulator in B cell medium as we described previously [30,31]. Briefly, isolated PBMC were co-cultured with the lethally irradiated (96Gy) t-CD40-L cells in IMDM (GIBCO-BRL, Life Technologies, CA) supplement with the 2ng/ml of IL-4 (R D systems, MN), 5.561027 M of cyclosporine A (Sigma-Aldrich, MO), 50 mg/ml of transferrin (Sigma-Aldrich, MO), 5 mg/ml of insulin (Sigma-Aldrich, MO), 15 mg/ml of gentamycin (GIBCO-BRL, Life Technologies, CA), and 10 of heat-inactivated human AB serum (Innovative Research, MI) at 37uC in 5 CO2. Cells were sub-cultured to new 6-well plates of t-CD40-L cells every 3 to 4 days. After 14 days of co-culture, more than 95 of the viableStatistical AnalysisGraphs and statistical analysis were performed using Prism 5.0 for Windows software (GraphPad Software, CA). One-way ANOVA with Tukey’s pairwise comparisons 1315463 was used for comparing the percentage of regulatory T cells, apoptotic T cells, and percentage of CD4hiCD25+ regulatory T cells in S phase. p value of ,0.05 was considered to be significant.TLR5 Enhances Induced Treg ProliferationResults TLR5-related Signals Enhance the Generation of CD4hiCD25+ Regulatory T Cells Independent of Cell ApoptosisWe first investigated the TLR5 expression in the CD4hiCD25+ regulatory T cells. A population of CD4hiCD25+ regulatory T cells and a population of CD4+CD252 T cells without any regulatory ?function could be identified in the co-culture of naive CD4+CD252CD45RO2 T cells with allogeneic CD40-activated B cells for 6 days (Title Loaded From File Figure 1A). As shown in Figure 1 B-E, CD4hiCD25+ regulatory T cells exhibited an up-regulated surface (Figure 1B and C) and total TLR5 protein expression (Figure 1D and E). Interestingly, in CD4+CD252 T cells, surface TLR5 ?expression level was lower than that of naive CD4+CD252CD45RO2 T cells while total TLR5 expression was the same (Figure 1 B-E). The up-regulated TLR5 expression in CD4hiCD25+ regulatory T cells prompted us to investigate the effect of TLR5-related signals on the generation and function of CD4hiCD25+ regulatory T cells. It was found that the blockade of TLR5 using anti-TLR5 blocking antibodies decreased CD4hiCD25+ regulatory T cells generation (Figure 1F and G). Frequency of CD4hiCD25+regulatory T cells decreased from 61 of total CD4+ T cells to about 36 after 6 days of co-culture (p,0.001) (Figure 1G), indicating that TLR5 signaling was involved in CD4hiCD25+ regulatory T cells generation. Since TLR5 was reported to be antiapoptotic [32], and could promote the survival of cells and mice subjected to lethal irradiation [33,34], we further studied whether the reduced CD4hiCD25+ reg.Lls generated with or without the blockade of TLR5 were sorted using FACSAria after 9 days of coculture. The sorted CD4hiCD25+ regulatory T cells were titrated and co-cultured with 56104 responder CD4+CD252 T cells from the same donor of the CD4hiCD25+ regulatory T cells and 56104 c-irradiated target PBMC from the donor of the CD40-activated B cells as stimulator for 3 days. 3H-thymidine was added to the coculture in the last 18 hours and the proliferation was analyzed by 3 H-thymidine incorporation assay as we described previously [28].Generation of CD40-activated B CellsPBMC were isolated from buffy coat of healthy adult donors from the Hong Kong Red Cross Blood Transfusion Services. CD40-acitvated B cells were generated from PBMC via CD40 stimulation using lethally irradiated (96Gy) NIH3T3 cells transfected with the human CD40 ligand (t-CD40-L cells) as stimulator in B cell medium as we described previously [30,31]. Briefly, isolated PBMC were co-cultured with the lethally irradiated (96Gy) t-CD40-L cells in IMDM (GIBCO-BRL, Life Technologies, CA) supplement with the 2ng/ml of IL-4 (R D systems, MN), 5.561027 M of cyclosporine A (Sigma-Aldrich, MO), 50 mg/ml of transferrin (Sigma-Aldrich, MO), 5 mg/ml of insulin (Sigma-Aldrich, MO), 15 mg/ml of gentamycin (GIBCO-BRL, Life Technologies, CA), and 10 of heat-inactivated human AB serum (Innovative Research, MI) at 37uC in 5 CO2. Cells were sub-cultured to new 6-well plates of t-CD40-L cells every 3 to 4 days. After 14 days of co-culture, more than 95 of the viableStatistical AnalysisGraphs and statistical analysis were performed using Prism 5.0 for Windows software (GraphPad Software, CA). One-way ANOVA with Tukey’s pairwise comparisons 1315463 was used for comparing the percentage of regulatory T cells, apoptotic T cells, and percentage of CD4hiCD25+ regulatory T cells in S phase. p value of ,0.05 was considered to be significant.TLR5 Enhances Induced Treg ProliferationResults TLR5-related Signals Enhance the Generation of CD4hiCD25+ Regulatory T Cells Independent of Cell ApoptosisWe first investigated the TLR5 expression in the CD4hiCD25+ regulatory T cells. A population of CD4hiCD25+ regulatory T cells and a population of CD4+CD252 T cells without any regulatory ?function could be identified in the co-culture of naive CD4+CD252CD45RO2 T cells with allogeneic CD40-activated B cells for 6 days (Figure 1A). As shown in Figure 1 B-E, CD4hiCD25+ regulatory T cells exhibited an up-regulated surface (Figure 1B and C) and total TLR5 protein expression (Figure 1D and E). Interestingly, in CD4+CD252 T cells, surface TLR5 ?expression level was lower than that of naive CD4+CD252CD45RO2 T cells while total TLR5 expression was the same (Figure 1 B-E). The up-regulated TLR5 expression in CD4hiCD25+ regulatory T cells prompted us to investigate the effect of TLR5-related signals on the generation and function of CD4hiCD25+ regulatory T cells. It was found that the blockade of TLR5 using anti-TLR5 blocking antibodies decreased CD4hiCD25+ regulatory T cells generation (Figure 1F and G). Frequency of CD4hiCD25+regulatory T cells decreased from 61 of total CD4+ T cells to about 36 after 6 days of co-culture (p,0.001) (Figure 1G), indicating that TLR5 signaling was involved in CD4hiCD25+ regulatory T cells generation. Since TLR5 was reported to be antiapoptotic [32], and could promote the survival of cells and mice subjected to lethal irradiation [33,34], we further studied whether the reduced CD4hiCD25+ reg.