Differential costimulation of peptide-stimulated proliferation of CD28−CD45RAhi cells by CD137L expressed on autologous fibroblasts. Replicate cultures of sorted CD28−CD45RO− CD8+ T cells were stimulated with autologous fibroblasts that had been transduced with individual costimulatory ligands and pulsed with dilutions of viral peptide (from 40 to 0.4 μg/mL) or unpulsed, or autologous monocytes pulsed with viral peptide or unpulsed. T cells were harvested at 10 days and the number of antigen-specific cells per well was calculated from the total cell count per well multiplied by the proportion of multimer-positive cells determined by flow cytometry. (A) Fibroblasts transfected with test construct stained with isotype control antibody (open histograms) or test antibody (filled histograms). (B) Fibroblasts transfected with control construct stained with isotype control antibody (open histograms) or test antibody (filled histograms). (C) Donor 28. (D) Donor 26. These experiments were performed twice on each donor, and the results in both cases confirm that on 41BBL transfection was able to support T-cell proliferation. At each peptide concentration, the number of antigen-specific cells per well stimulated by CD137L-transfected fibroblasts was significantly greater (P < .05 by Student t test) than stimulation with CD275-transfected fibroblasts or CD80/CD86-transfected fibroblasts. Error bars represent the standard deviation of the cell count from 3 independent wells at each peptide concentration multiplied by the proportion of antigen specific cells as determined by peptide-specific MHC class I multimers and flow cytometer.
