Surface CTLA-4 mediates the generation of functional CD4^hi Treg.

<p>Naïve CD4⁺CD25^- T cells were co-cultured with allogeneic CD40-activated B cells at a ratio of 10:1 in the presence of 10µg/ml CTLA-4 neutralizing mAb or its isotype control (IC). (A) Expressions of CD4, CD25 (up panel) and Foxp3, ICOS (bottom panel) in CD4⁺CD25^- and CD4^hi subsets from different treatment groups on day 6 post co-culture. (B) The absolute number of total CD4⁺ T cells (left panel) and CD4^hi Treg (right panel) from different treatment groups during 9 days. (C) Antigen-specific inhibition mediated by CD4^hi Treg from different treatment groups. CD4⁺CD25^– cells (5×10⁴, responder, R) and γ-irradiated allogeneic PBMC (5×10⁴, stimulator, S) were co-cultured while CD4^hi Treg from different groups were sorted on day 6 post co-culture and added into MLR system at different concentration. 3 days later, proliferation of responder was determined as described in “MLR assays”. Results shown here are representative of 4 independent experiments (n=4, *P<0.05, **P<0.01).</p

ICOS expressions in CD40-activated B cells and CD4⁺ T cell subsets.

<p>(A) Expressions of ICOS and ICOSL in CD40-activated B cells. (B-C) Naïve CD4⁺CD25^- T cells isolated from normal PBMC were co-cultured with allogeneic CD40-activated B cells at a ratio of 10:1 without exogenous cytokines for 6 days, the expressions of CD4, CD25 (B) and Foxp3, ICOS (C) in CD4⁺ T cell populations were determined by flow cytometry. The data represent for 20 independent experiments.</p

ICOS-ICOSL mediates the generation of CD4^hi Treg.

<p>Naïve CD4⁺CD25^- T cells were co-cultured with allogeneic CD40-activated B cells at a ratio of 10:1 for indicated time. ICOS-Ig and its relevant isotype controls (IC) were added at the final concentration of 5µg/ml or 50µg/ml since the initiation of co-culture and supplemented with replacement of medium every 3 days. The absolute number of total CD4⁺ T cells (A) and CD4^hi Treg (B) was examined. The percentage of PI⁺ cells in CD4⁺CD25^- and CD4^hi Treg on day 9 post co-culture (C) was examined as described in “cell death and proliferation assays”. Data for 4 different experiments are shown here and the 2-tailed unpaired Student t tests were used for comparing between group with ICOS-Ig and IC at indicated time. (n=4, *P<0.05, **P<0.01) (D) Exogenous IL-2 on proliferation and apoptosis of CD4^hi Treg. 50µg/ml ICOS-Ig was added into the co-culture with or without 250IU/ml recombinant human IL-2 and suppleted with replacement of medium every 3 days. The proliferation and apoptosis of CD4^hi Treg were determined by CFSE and PI staining correspondingly on day 9 post co-culture. Data represent for 3 independent experiments.</p

ICOS is not required for CD4^hi Treg effector function.

<p>CD4⁺CD25^– cells (5×10⁴, responder, R) and γ-irradiated allogeneic PBMC (5×10⁴, stimulator, S) were co-cultured as described in “MLR assays”. (A) CD4^hi Treg was sorted on day 9 from the co-culture supplemented with 50µg/ml ICOS-Ig or its relevant isotype controls (IC). The sorted cells were serially diluted and added into MLR as regulators. (B) Innocent CD4^hi Treg were used as regulators, and ICOS-Ig or IC was added respectively on the start of the MLR co-culture at the final concentration of 50µg/ml. On day 3 after co-culture, proliferation of responder was examined as described. The data represent for 4 independent experiments and the 2-tailed unpaired Student t tests were used for comparing between groups with ICOS-Ig and IC. (n=4, *P<0.05, **P<0.01).</p

Blockade of ICOS-ICOSL affects the expressions of CD4^hi Treg markers.

<p>Naïve CD4⁺CD25^- T cells were co-cultured with allogeneic CD40-activated B cells at a ratio of 10:1 with the supplement of 50µg/ml ICOS-Ig or its relevant isotype controls (IC) for 9 days. (A) Expressions of CD4, CD25 (up panel) and Foxp3 (bottom panel) in CD4⁺CD25^- T cells and CD4^hi Treg on day 9 post co-culture. (B) Expressions of T cell activation markers CD27, CD44, CD45RO (up panel), Treg-related markers GITR, surface CTLA-4, whole-cell CTLA-4 (middle panel), intracellular inhibitory cytokines IL-10 and TGF-β (bottom panel) in CD4⁺CD25^- T cells and CD4^hi Treg on day 9 post co-culture. Data represent for 4 different experiments are shown.</p

ICOS regulates the dynamic transfer of CTLA-4 between cell surface and cytoplasm.

<p>(A) The surface expressions of CD107a (left panel) and CD63 (right panel) on CD4^hi Treg were determined by FACS analysis after 9 days of co-culture with allogenic CD40-activated B cells at a ratio of 10:1 with 50ug/ml of ICOS-Ig or its relevant isotype controls (IC). (B-C) Inhibition of endocytosis restored the surface CTLA-4 expression and impaired suppressive capacity of CD4^hi Treg caused by ICOS-ICOSL blockade. The surface expression of CLTA-4 in CD4^hi Treg (B) were determined by FACS analysis after 9 days of co-culture with allogenic CD40-activated B cells at a ratio of 10:1 with 50µg/ml of ICOS-Ig or its relevant isotype controls (IC). 30µM of E64 and 100uM of pepstatin A (EP) solved in DMSO were added in the culture for inhibiting endocytosis. Antigen-specific inhibition mediated by CD4^hi Treg sorted on day 6 post co-culture from different treatment group (C) were determined as described in “MLR assays”. Results shown are representative of four independent experiments (n=4, *P<0.05, n.s. no significant difference).</p

The blockade of TLR5 reduced CD4^hiCD25⁺ regulatory T cells proliferation by inducing S phase arrest.

<p>(A) Flow cytometric analysis of the CFSE signal in CD4^hiCD25⁺ regulatory T cells generated with no treatment (dotted line), with isotype-matched mAb (dashed line), and with anti-TLR5 blocking mAb (solid line). Filled histogram is the CFSE signal on Day 0 (left panel). Statistical analysis of the MFI of the CFSE in CD4^hiCD25⁺ regulatory T cells. Data show Mean+SEM, n  = 6. (right panel). (B) Cell cycle analysis of CD4^hiCD25⁺ regulatory T cells generated with no treatment (left), with isotype-matched mAb (middle), and with anti-TLR5 blocking mAb (right). Numbers indicate the percentage of CD4^hiCD25⁺ regulatory T cells in S phase (left panel). Statistical analysis of percentage of CD4^hiCD25⁺ regulatory T cells in S phase. Data show Mean+SEM, n  = 6 (right panel). All data shown are representative from three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, one way ANOVA with Tukey’s pairwise comparisons.</p

LR5 blockade reduced the generation of CD4^hiCD25⁺ regulatory T cells and was independent of apoptosis.

<p>(A) Flow cytometric analysis of the percentage of CD4^hiCD25⁺ regulatory T cells generated on Day 6 (right panel) from naïve CD4⁺CD25⁻CD45RO⁻ T cells (left panel). (B) Flow cytometric analysis of the expression of surface TLR5 in freshly isolated naïve CD4⁺CD25⁻CD45RO⁻ T cells (dotted line), and CD4⁺CD25⁻ (dashed line) and CD4^hiCD25⁺ regulatory T cells (solid line) after 6 days of co-culture of naïve CD4⁺CD25⁻CD45RO⁻ T cells with allogeneic CD40-activated B cells. Filled histogram indicates the staining obtained from isotype-matched mAb controls. (C) Mean fluorescence intensity (MFI) of the expression of surface TLR5. Data show Mean+SEM, n  = 6. (D) Flow cytometric analysis of total TLR5 in freshly isolated naïve CD4⁺CD25⁻CD45RO⁻ T cells (dotted line), CD4⁺CD25⁻ (dashed line), and CD4^hiCD25⁺ regulatory T cells (solid line) after 6 days of co-culture of naïve CD4⁺CD25⁻CD45RO⁻ T cells with allogeneic CD40-activated B cells. Filled histogram indicates the staining obtained from isotype-matched mAb control. (E) Mean fluorescence intensity (MFI) of the expression of total TLR5. Data show Mean+SEM, n  = 6. (F) Flow cytometric analysis of the generation of CD4^hiCD25⁺ regulatory T cells with no treatment (left panel), with isotype-matched mAb (middle panel), and with anti-TLR5 blocking mAb (right panel) during the co-culture. (G) Mean percentage of CD4^hiCD25⁺ regulatory T cells generated with no treatment, with isotype-matched mAb, and with anti-TLR5 blocking mAb. Data shown Mean+SEM, n  = 6. (H) Flow cytometric analysis of the percentage of apoptotic CD4^hiCD25⁺ regulatory T cells (upper panel) or CD4⁺CD25⁻ T cells (lower panel) after 6 days of co-culture of naïve CD4⁺CD25⁻CD45RO⁻ T cells with allogeneic CD40-activated B cells. All results shown are representative of three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, one way ANOVA with Tukey’s pairwise comparisons.</p

Reduced phosphorylated ERK1/2 might contribute to S phase arrest in CD4^hiCD25⁺ regulatory T cells.

<p>(A) Flow cytometric analysis of the expression of phosphorylated ERK1/2 in CD4^hiCD25⁺ regulatory T cells generated with no treatment (dotted line), isotype-matched mAb (dashed line), and with anti-TLR5 blocking mAb (solid line). Filled histogram is the staining obtained from isotype-matched mAb control for staining antibody (left panel). Statistical analysis of the MFI of p-ERK1/2 in CD4^hiCD25⁺ regulatory T cells. Data show Mean+SEM, n  = 10. All data shown are representative from five independent experiments (right panel). (B) Statistical analysis of the percentage of CD4^hiCD25⁺ regulatory T cells generated on Day 6 with or without the inhibition of ERK1/2 phosphorylation by PD98059. DMSO treated group is the control for PD98059. Data show Mean+SEM, n  = 6. All results shown are from 3 independent experiments (left panel). Cell cycle analysis of CD4^hiCD25⁺ regulatory T cells generated on Day 6 with or without the inhibition of ERK1/2 phosphorylation by PD98059. DMSO treated group is the control for PD98059. Numbers indicate the percentage of CD4^hiCD25⁺ regulatory T cells in S phase. All results shown are from 3 independent experiments (middle panel). Mean percentage of CD4^hiCD25⁺ regulatory T cells in S phase with the inhibition of ERK1/2 phosphorylation by PD98059. Data show Mean+SEM, n  = 6. All results shown are from 3 independent experiments (right panel). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, one way ANOVA with Tukey’s pairwise comparisons.</p

TLR5-related signals did not affect the function of CD4^hiCD25⁺ regulatory T cells.

<p>(A). Flow cytometric and statistical analysis of the expression of surface CTLA-4 (upper left panel), intracellular CTLA-4 (lower left panel), surface GITR (upper right panel), and FOXP3 (lower right panel) of CD4^hiCD25⁺ regulatory T cells generated with no treatment (dotted line), with isotype-matched mAb (dashed line), and with anti-TLR5 blocking mAb (solid line) after 6 days of co-culture of naïve CD4⁺CD25⁻CD45RO⁻ T cells and allogeneic CD40-activated B cells and filled histogram indicates staining obtained from the isotype-matched mAb for staining antibodies. Data were shown in Mean+SEM, n  = 6. (B) ³H-thymidine incorporation of CD4^hiCD25⁺ regulatory T cells in suppressive MLR at different regulatory T cells: responsders ratio. Data show Mean+SEM, n  = 6. All data shown are from 3 independent experiments. NS, not significant, one way ANOVA with Tukey’s pairwise comparisons.</p