Blockade of either CD28 or CTLA4 inhibits B7 depletion from APCs by human Tregs. (A) Schematics of a Treg:Raji coculture assay examining Treg-mediated depletion of B7 ligands from APCs. GFP fluorescence in Raji (CD80−/−CD86−/−CD80-GFP+) or Raji (CD80−/−CD86−/−CD86-GFP+) cells was measured by flow cytometry after 9 h coculture with human Tregs. (B) Representative flow cytometry histograms and quantification graph of CD80-GFP amounts in Raji (CD80−/−CD86−/−CD80-GFP+) after incubation with Tregs under the indicated conditions. Normalized MFIs of CD80-GFP were calculated by setting the MFI of Raji (CD80−/−CD86−/−) as 0 and the MFI of “−, −, −, −” as 100. (C) Same as B except measuring CD86-GFP in Raji (CD80−/−CD86−/−CD86-GFP+) cells. Error bars are SD from three independent coculture experiments using PBMCs from three independent age-matched donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test.
Figure 9.

Blockade of either CD28 or CTLA4 inhibits B7 depletion from APCs by human Tregs. (A) Schematics of a Treg:Raji coculture assay examining Treg-mediated depletion of B7 ligands from APCs. GFP fluorescence in Raji (CD80−/−CD86−/−CD80-GFP+) or Raji (CD80−/−CD86−/−CD86-GFP+) cells was measured by flow cytometry after 9 h coculture with human Tregs. (B) Representative flow cytometry histograms and quantification graph of CD80-GFP amounts in Raji (CD80−/−CD86−/−CD80-GFP+) after incubation with Tregs under the indicated conditions. Normalized MFIs of CD80-GFP were calculated by setting the MFI of Raji (CD80−/−CD86−/−) as 0 and the MFI of “−, −, −, −” as 100. (C) Same as B except measuring CD86-GFP in Raji (CD80−/−CD86−/−CD86-GFP+) cells. Error bars are SD from three independent coculture experiments using PBMCs from three independent age-matched donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test.

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