Io of 10:1 induced a population of CD4hiCD25+ regulatory T cells [28]. The CD4hiCD25+ T cells were alloantigen specific CD45RO+CCR72CD62L+ memory T cells and expressed FOXP3, IFN-c, CTLA-4, and GITR [28,29]. Suppressive MLR experiment demonstrated that these cells could suppress T cell Trations of IL-1a and IL-1b in the IL-1ra proliferation in a cell-cell contact dependent manner which was partially dependent on the surface CTLA-4, indicating that these cells are iTregs [28]. In this experiment, we investigated the role of TLR5-related signals in the generation and function of human CD4hiCD25+ regulatory T cells induced by allogeneic CD40activated B cells and have unveiled a novel function of TLR5related signaling in iTregs. Our results indicated that TLR5related signaling enhances the proliferation but not the suppressive function of human CD4hiCD25+ regulatory T cells induced by allogeneic CD40-activated B cells.suspended cells are routinely CD19 positive. These B cells were cryopreserved in 10 DMSO medium for future use.?Isolation of Naive CD4+CD252CD45RO2 T Cells and Induction of CD4hiCD25+ Regulatory T CellsThe CD4hiCD25+ regulatory T cells were induced by the coculture of the CD4+CD252CD45RO2 T cells with the allogeneic CD40-activated B cells at a T-cell: B-cell ratio of 10:1 for 6 days as ?described previously [28] unless otherwise specified. Human naive CD4+CD252CD45RO2 T cells were isolated from healthy donors PBMC by CD4+ T cell enrichment using the human CD4 T Cells Enrichment Cocktails (StemCell Technologies, ?Canada), followed by negative selection using a human naive CD4+ T Cell Isolation Kit and LD Column (Miltenyi Biotec, Germany) according to manufacturer’s instructions.TLR5 Blockade and Chemical Inhibition of ERK1/2 Phosphorylation10 mg/ml of anti-TLR5 mAb, and its relevant isotype control (Invivogen, CA) were used for the blockade of TLR5. 20 mM of PD98059 and its solvent control DMSO (Merck, Germany) were used for chemical inhibition of ERK1/2 phosphorylation. Antibodies and PD98059 were added to CD4+CD252CD45RO2 T cells one hour before co-culturing with allogeneic CD40activated B cells and were replenished when cell culture medium was changed.Flow Locytic AECOPD; {P,0.01 vs. the Neutrophilic AECOPD; `P,0.05 vs. the Paucigranulocytic Cytometric AssaysAll fluorescence-conjugated antibodies were from BD-Biosciences unless otherwise specified: CD4-pacific blue (Biolegend, CA), CD25-APC-Cy7, CTLA-4-PE, GITR-PE, TLR5-PE (Imgenex, CA), human Foxp3 staining kit (clone: PCH101) (eBiosciences, CA), p-p44/42 (Thr202/Tyr204)-AlexaFluor-488 (Cell Signaling, MA). Annexin V/propidium iodide (Gibco-BRL, Life Technologies, CA) was used for measuring apoptosis. Propidium iodide (Gibco-BRL, Life Technologies, CA) was used for cell cycle ?analysis. For measuring cell proliferation, naive CD4+CD252CD45RO2 T cells were stained with CFSE before co-culturing with allogeneic CD40-activated B cells. Cells were analyzed using FACS LSRII (BD Biosciences, CA) and results were analyzed using FlowJo v8.8.2 (Tree Star, OR). Cell cycle analysis results were analyzed using ModFit (Verity Software House, ME).Materials and Methods Ethics StatementWritten consent for the use of buffy coat for research purposes was obtained from the donors by the Hong Kong 23977191 Red Cross Blood Transfusion Services at the time of blood donation. The use of buffy coat for this experiment was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB Reference Number: UW 07-390).Mixed Lymphocyte Reaction (MLR) AssaysCD4hiCD25+ regulatory T ce.Io of 10:1 induced a population of CD4hiCD25+ regulatory T cells [28]. The CD4hiCD25+ T cells were alloantigen specific CD45RO+CCR72CD62L+ memory T cells and expressed FOXP3, IFN-c, CTLA-4, and GITR [28,29]. Suppressive MLR experiment demonstrated that these cells could suppress T cell proliferation in a cell-cell contact dependent manner which was partially dependent on the surface CTLA-4, indicating that these cells are iTregs [28]. In this experiment, we investigated the role of TLR5-related signals in the generation and function of human CD4hiCD25+ regulatory T cells induced by allogeneic CD40activated B cells and have unveiled a novel function of TLR5related signaling in iTregs. Our results indicated that TLR5related signaling enhances the proliferation but not the suppressive function of human CD4hiCD25+ regulatory T cells induced by allogeneic CD40-activated B cells.suspended cells are routinely CD19 positive. These B cells were cryopreserved in 10 DMSO medium for future use.?Isolation of Naive CD4+CD252CD45RO2 T Cells and Induction of CD4hiCD25+ Regulatory T CellsThe CD4hiCD25+ regulatory T cells were induced by the coculture of the CD4+CD252CD45RO2 T cells with the allogeneic CD40-activated B cells at a T-cell: B-cell ratio of 10:1 for 6 days as ?described previously [28] unless otherwise specified. Human naive CD4+CD252CD45RO2 T cells were isolated from healthy donors PBMC by CD4+ T cell enrichment using the human CD4 T Cells Enrichment Cocktails (StemCell Technologies, ?Canada), followed by negative selection using a human naive CD4+ T Cell Isolation Kit and LD Column (Miltenyi Biotec, Germany) according to manufacturer’s instructions.TLR5 Blockade and Chemical Inhibition of ERK1/2 Phosphorylation10 mg/ml of anti-TLR5 mAb, and its relevant isotype control (Invivogen, CA) were used for the blockade of TLR5. 20 mM of PD98059 and its solvent control DMSO (Merck, Germany) were used for chemical inhibition of ERK1/2 phosphorylation. Antibodies and PD98059 were added to CD4+CD252CD45RO2 T cells one hour before co-culturing with allogeneic CD40activated B cells and were replenished when cell culture medium was changed.Flow Cytometric AssaysAll fluorescence-conjugated antibodies were from BD-Biosciences unless otherwise specified: CD4-pacific blue (Biolegend, CA), CD25-APC-Cy7, CTLA-4-PE, GITR-PE, TLR5-PE (Imgenex, CA), human Foxp3 staining kit (clone: PCH101) (eBiosciences, CA), p-p44/42 (Thr202/Tyr204)-AlexaFluor-488 (Cell Signaling, MA). Annexin V/propidium iodide (Gibco-BRL, Life Technologies, CA) was used for measuring apoptosis. Propidium iodide (Gibco-BRL, Life Technologies, CA) was used for cell cycle ?analysis. For measuring cell proliferation, naive CD4+CD252CD45RO2 T cells were stained with CFSE before co-culturing with allogeneic CD40-activated B cells. Cells were analyzed using FACS LSRII (BD Biosciences, CA) and results were analyzed using FlowJo v8.8.2 (Tree Star, OR). Cell cycle analysis results were analyzed using ModFit (Verity Software House, ME).Materials and Methods Ethics StatementWritten consent for the use of buffy coat for research purposes was obtained from the donors by the Hong Kong 23977191 Red Cross Blood Transfusion Services at the time of blood donation. The use of buffy coat for this experiment was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB Reference Number: UW 07-390).Mixed Lymphocyte Reaction (MLR) AssaysCD4hiCD25+ regulatory T ce.