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

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

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 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

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