FIGURE 3.

ICOS‐dependent modulation of cell surface ICOS‐L is a fast process that is impaired by fixation of ICOS⁺ cells. (A). M12 ICOS‐L GFP⁺ cells were incubated at 37°C for different times alone (closed symbols) or with ICOS⁺ D10 cells (open symbols), then stained in the cold for ICOS‐L and CD4. M12 ICOS‐L GFP⁺ cells (GFP⁺ CD4^– cells), were gated and analyzed for ICOS‐L and GFP fluorescence. Data shown as per cent ICOS‐L⁺ cells (left panel), or mean ICOS‐L fluorescence (middle panel), or mean GFP fluorescence (right panel). Asterisks indicate significant differences between conditions as determined by two‐way ANOVA (***: p < 0.001). (B). M12 ICOS‐L GFP⁺ cells were incubated at 37°C for different times alone (closed circles, solid line) or with ICOS⁺ D10 live cells (open circles, dashed lines), or with fixed ICOS⁺ D10 cells (squares, dashed line), then fluorescence analyzed and shown as in A). Symbols indicate significant differences (p < 0.001) in fluorescence of ICOS‐L GFP⁺ cells in the presence or absence of fixed ICOS⁺ cells (ϕϕϕ), or between ICOS‐L GFP⁺ cells plus or minus ICOS⁺ live cells (***), or between ICOS‐L GFP⁺ cells plus live or fixed ICOS⁺ cells (###), as determined by two‐way ANOVA