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. Author manuscript; available in PMC: 2016 Nov 2.

Published in final edited form as: Nat Immunol. 2016 May 2;17(7):825–833. doi: 10.1038/ni.3463

Importance of a novel ICOS signaling motif in T_FH cell development. (a) Evolutionary conservation of the proximal (IProx), PI3K-binding YxxM and distal motifs in the cytoplasmic tail of ICOS. (b,d) Flow cytometry of cells from host B6 mice 7 d after adoptive transfer of Icos^–/– SMARTA CD4⁺ T cells transduced with retrovirus encoding empty vector (EV) or wild-type ICOS (WT) or mIProx, YF or TL mutant ICOS and infected with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent CXCR5⁺SLAM^lo T_FH cells (b) or CXCR5⁺PD1^hi GC T_FH cells (d). Cumulative data for b (c) or d (e) from three independent experiments. (f, h) Flow cytometry of cells from host CD4-Cre × Bcl6^fl/fl mice 10 d after adoptive transfer of Icos^–/– SMARTA CD4⁺ T cells transduced as in (b), and were immunized with KLH-gp61 absorbed to alum. Numbers adjacent to outlined areas indicate percent CD95⁺GL7⁺ GC B cells (f) and CD138⁺IgD^– plasma cells (h). Cumulative data for f (g) or h (i) from two independent experiments. Each data point represents a single mouse. (j) Quantification of anti-KLH-gp61 IgG from sera of immunized mice analyzed with ELISA and presented as absorbance at 450 nm. The endpoint titer (k) and area under curve (AUC; l) were calculated. Shown are mean ± SEM; ANOVA with post-hoc Tukey's corrections. *P < 0.01; **P < 0.001; ***P < 0.0001.

Importance of a novel ICOS signaling motif in T_FH cell development. (a) Evolutionary conservation of the proximal (IProx), PI3K-binding YxxM and distal motifs in the cytoplasmic tail of ICOS. (b,d) Flow cytometry of cells from host B6 mice 7 d after adoptive transfer of Icos^–/– SMARTA CD4⁺ T cells transduced with retrovirus encoding empty vector (EV) or wild-type ICOS (WT) or mIProx, YF or TL mutant ICOS and infected with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent CXCR5⁺SLAM^lo T_FH cells (b) or CXCR5⁺PD1^hi GC T_FH cells (d). Cumulative data for b (c) or d (e) from three independent experiments. (f, h) Flow cytometry of cells from host CD4-Cre × Bcl6^fl/fl mice 10 d after adoptive transfer of Icos^–/– SMARTA CD4⁺ T cells transduced as in (b), and were immunized with KLH-gp61 absorbed to alum. Numbers adjacent to outlined areas indicate percent CD95⁺GL7⁺ GC B cells (f) and CD138⁺IgD^– plasma cells (h). Cumulative data for f (g) or h (i) from two independent experiments. Each data point represents a single mouse. (j) Quantification of anti-KLH-gp61 IgG from sera of immunized mice analyzed with ELISA and presented as absorbance at 450 nm. The endpoint titer (k) and area under curve (AUC; l) were calculated. Shown are mean ± SEM; ANOVA with post-hoc Tukey's corrections. *P < 0.01; **P < 0.001; ***P < 0.0001.