Figure 2.
CD28 expressed by human T cells mediate rapid B7 acquisition upon ligand binding. (A) Scheme of Treg and Tconv isolation from human PBMCs. Flow cytometry histograms show surface expressions of CD4, CD25, and CD127 and intracellular expression of Foxp3 of purified Treg and Tconv cells. (B) Effects of CD28 blockade and CTLA4 blockade on the abilities of human Tregs to acquire CD80 and CD86 from Raji APCs. Left: A cartoon depicting a Treg:Raji (CD80−/−CD86−/−CD80-GFP+) or Treg:Raji (CD80−/−CD86−/−CD86-GFP+) coculture assay, after which GFP signal in Tregs was measured. Right: Representative flow cytometry histograms showing the amounts of CD80-GFP or CD86-GFP on Tregs after a 15-min incubation with Raji under the indicated conditions. Bar graphs on the immediate right show the normalized MFI values of acquired GFP on Tregs, calculated by setting the MFI of the “−, −, −, −” condition as 0 and the MFI of “+, +, −, −” condition as 100. (C) Same as in B except replacing human Tregs with Tconvs. (D) Effects of CD28 deficiency or blockade on the abilities of Jurkat to acquire CD80, CD86, and MHCII. Upper: A cartoon depicting the assay setup. Lower: Representative flow cytometry histograms showing the relative levels of acquired CD80, CD86, or MHCII on the indicated Jurkat cells after a 15-min incubation with either Raji (CD80−/−CD86−/−CD80-GFP+) or Raji (CD80−/−CD86−/−CD86-GFP+) cells. Bar graphs immediate right show the normalized MFI values of acquired CD80-GFP, CD86-GFP, or HLA-DR on Jurkat cells, calculated by setting the MFI of “−, −, −” condition as 0 and the MFI of “+, +, −” condition as 100. Error bars are SD from three independent coculture experiments performed on three different days, data in B and C were generated using PBMCs from three independent age-matched donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test.

CD28 expressed by human T cells mediate rapid B7 acquisition upon ligand binding. (A) Scheme of Treg and Tconv isolation from human PBMCs. Flow cytometry histograms show surface expressions of CD4, CD25, and CD127 and intracellular expression of Foxp3 of purified Treg and Tconv cells. (B) Effects of CD28 blockade and CTLA4 blockade on the abilities of human Tregs to acquire CD80 and CD86 from Raji APCs. Left: A cartoon depicting a Treg:Raji (CD80−/−CD86−/−CD80-GFP+) or Treg:Raji (CD80−/−CD86−/−CD86-GFP+) coculture assay, after which GFP signal in Tregs was measured. Right: Representative flow cytometry histograms showing the amounts of CD80-GFP or CD86-GFP on Tregs after a 15-min incubation with Raji under the indicated conditions. Bar graphs on the immediate right show the normalized MFI values of acquired GFP on Tregs, calculated by setting the MFI of the “−, −, −, −” condition as 0 and the MFI of “+, +, −, −” condition as 100. (C) Same as in B except replacing human Tregs with Tconvs. (D) Effects of CD28 deficiency or blockade on the abilities of Jurkat to acquire CD80, CD86, and MHCII. Upper: A cartoon depicting the assay setup. Lower: Representative flow cytometry histograms showing the relative levels of acquired CD80, CD86, or MHCII on the indicated Jurkat cells after a 15-min incubation with either Raji (CD80−/−CD86−/−CD80-GFP+) or Raji (CD80−/−CD86−/−CD86-GFP+) cells. Bar graphs immediate right show the normalized MFI values of acquired CD80-GFP, CD86-GFP, or HLA-DR on Jurkat cells, calculated by setting the MFI of “−, −, −” condition as 0 and the MFI of “+, +, −” condition as 100. Error bars are SD from three independent coculture experiments performed on three different days, data in B and C were generated 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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