Figure 6.
CTLA4 mediates cis-endocytosis of CD80 acquired via trogocytosis. (A) Effects of filler cells on CTLA4-mediated depletion of Treg trogocytosed Myc-CD80. Human Tregs were precultured with Myc-CD80–expressing Raji (CD80−/−CD86−/−Myc-CD80+) in the presence of SEB, isolated by FACS, and incubated alone or with 30-fold excess of CD28−/− Jurkat filler cells for 0 or 3 h, in the presence or absence of ipilimumab and/or 28.3scFv before flow cytometry measurement of Myc-CD80 and HLA-DR amounts. Bar graph summarizes data from three technical replicates of Tregs isolated from a single human donor. (B) Effect of TCR stimulation on CTLA4-mediated cis depletion of Treg trogocytosed Myc-CD80. Tregs were precultured with Myc-CD80–expressing Raji (CD80−/−CD86−/−Myc-CD80+) in the presence or absence of SEB, isolated by FACS, and incubated with 30-fold excess of CD28−/− Jurkat filler cells for 0 or 3 h, in the presence or absence of ipilimumab and/or 28.3scFv before flow cytometry measurement of Myc-CD80 and HLA-DR. Bar graph summarizes data from three technical replicates of Tregs isolated from a single human donor. (C) Flow cytometry histograms showing Myc-CD80 and endogenous CD80 expression on indicated Raji cells measured by anti-CD80 staining and anti-Myc staining, respectively. (D) Flow cytometry histograms of anti-Myc staining of indicated Jurkat cells in the presence or absence of ipilimumab. (E) Flow cytometry histograms showing the GFP signal (total expression of CTLA4), anti-CTLA4 staining (surface expression of CTLA4), and anti-CD28 staining (surface expression of CD28) on indicated Jurkat cells. (F) Ipilimumab effects on the cell surface expression of trogocytosed Myc-CD80 in Jurkat expressing CTLA4WT or CTLA4ΔICD. Flow cytometry histograms show anti-Myc staining of indicated Jurkat after 1 h incubation in the presence of excess filler cells at 37°C or on ice, with or without ipilimumab. In A and B, normalized MFIs were calculated by setting the absolute 0 as 0 and the highest MFI value among all the conditions as 100 in each replicate. Error bars: SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test.

CTLA4 mediates cis-endocytosis of CD80 acquired via trogocytosis. (A) Effects of filler cells on CTLA4-mediated depletion of Treg trogocytosed Myc-CD80. Human Tregs were precultured with Myc-CD80–expressing Raji (CD80−/−CD86−/−Myc-CD80+) in the presence of SEB, isolated by FACS, and incubated alone or with 30-fold excess of CD28−/− Jurkat filler cells for 0 or 3 h, in the presence or absence of ipilimumab and/or 28.3scFv before flow cytometry measurement of Myc-CD80 and HLA-DR amounts. Bar graph summarizes data from three technical replicates of Tregs isolated from a single human donor. (B) Effect of TCR stimulation on CTLA4-mediated cis depletion of Treg trogocytosed Myc-CD80. Tregs were precultured with Myc-CD80–expressing Raji (CD80−/−CD86−/−Myc-CD80+) in the presence or absence of SEB, isolated by FACS, and incubated with 30-fold excess of CD28−/− Jurkat filler cells for 0 or 3 h, in the presence or absence of ipilimumab and/or 28.3scFv before flow cytometry measurement of Myc-CD80 and HLA-DR. Bar graph summarizes data from three technical replicates of Tregs isolated from a single human donor. (C) Flow cytometry histograms showing Myc-CD80 and endogenous CD80 expression on indicated Raji cells measured by anti-CD80 staining and anti-Myc staining, respectively. (D) Flow cytometry histograms of anti-Myc staining of indicated Jurkat cells in the presence or absence of ipilimumab. (E) Flow cytometry histograms showing the GFP signal (total expression of CTLA4), anti-CTLA4 staining (surface expression of CTLA4), and anti-CD28 staining (surface expression of CD28) on indicated Jurkat cells. (F) Ipilimumab effects on the cell surface expression of trogocytosed Myc-CD80 in Jurkat expressing CTLA4WT or CTLA4ΔICD. Flow cytometry histograms show anti-Myc staining of indicated Jurkat after 1 h incubation in the presence of excess filler cells at 37°C or on ice, with or without ipilimumab. In A and B, normalized MFIs were calculated by setting the absolute 0 as 0 and the highest MFI value among all the conditions as 100 in each replicate. Error bars: SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test.

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