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. 2010 Dec 29;5(12):e14452. doi: 10.1371/journal.pone.0014452

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Tapirdamaz et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A. CFSE-labeled PBMC from an LTx-recipient were stimulated for 6 days with allogeneic splenocyte-derived CD40-B cells or with splenocytes from the same donor. Dot-plots and CFSE-dilution graphs of CD3⁺ T cells show increased T-cell proliferation upon stimulation with CD40-B cells compared to splenocytes. Percentages in dot plots depict % dividing (CFSE^low) T cells. Percentages in histograms are calculated PF. B: Paired comparisons of PF of LTx-recipient CD3⁺, CD4⁺ and CD8⁺ T cells upon stimulation with donor-derived CD40-B cells, autologous patient CD40-B cells, splenocytes or without stimulus. Sixty CFSE-labelled PBMC-samples of 6 LTx-recipients, obtained either before, or at different time points after transplantation, were stimulated with donor-derived or autologous CD40-B cells or with splenocytes from the same donor for 6 days. Bars represent median values. C: Donor-specific T-cell responses obtained by subtraction of PF reacting to autologous CD40-B cells from PF responding to donor-derived CD40-B cells, or subtraction of PF in the absence of allo-antigen (negative controls) from PF responding to donor splenocytes. Bars represent median values. D: Paired comparisons of the coefficients of variation of triplicate measurements of LTx-recipient CD3⁺ PF in 108 PBMC-samples of 6 LTx-recipients upon stimulation with donor-derived CD40-B cells or donor-derived splenocytes. Bars represent median values. E. Flowcytometry dotplot showing CFSE-dilution versus intracellular IFN-γ of CD3+ T cells stimulated with autologous or allogeneic CD40-B cells, and re-stimulated at day 5 for 24 hours with CD40-B cells of the same donor, the last 15 hours in the presence of Brefeldin A. Only T cells that divided (CFSE^low) in response to allogeneic CD40-B cells produce IFN-γ. F. Comparison of CFSE-MLR with IFN-γ ELISPOT. Four different CFSE-labeled PBMC samples from LTx-recipients were stimulated for 5 days with allogeneic splenocyte-derived CD40-B cells, and re-stimulated for 24 hours with CD40-B cells of the same donor to detect intracellular IFN-γ expression. In parallel, samples of the same PBMC were analyzed for IFN-γ production upon stimulation with the same CD40-B cells in IFN-γ ELISPOT. IFN-γ-producing cells in ELISPOT are depicted as percentage of PBMC, while the percentages of IFN-γ producing cells in CFSE-MLR are depicted as percentages of CD3+ T cells.