Figure 5.

Flow cytometric analysis of Th-1 cytokine production. (a,b) Donor antigen-specific IFN-γ production by CD4+ T cells: comparison of subgroups according to functional B-cell phenotype on ELISPOT. CD8-depleted PBMCs were stimulated with donor antigen under same conditions as in ELISPOT, then assayed by flow cytometry by using a cytokine capture system. White bars: Samples (n = 8) from patients with ELISPOT pattern showing evidence of B-dependent antidonor IFN-γ production (with or without evidence of regulation). Black bars: Samples (n = 3) from patients with ELISPOT pattern showing only suppression of antidonor IFN-γ production by B cells with NO evidence of B-dependent responses. (a) Shows the percentage of CD4+ cells expressing only IFN-γ (IFN-γ + IL-10−) or coexpressing with IL-10 (IFN-γ + IL-10+). (b) Shows the comparison of the percentage of total cells expressing IFN-γ/% total cells expressing IL-10. (c–e) Polyclonal stimulation with anti-CD3/anti-CD46 monoclonal antibodies: comparison of subgroups according to functional B-cell phenotype on ELISPOT. White bars: Samples (n = 4) in which antidonor-specific ELISPOT showed only suppression of antidonor IFN-γ production by B cells with NO evidence of B-dependent responses. Black bars: Samples (n = 8) in which antidonor-specific ELISPOT showed evidence of a regulated B-dependent antidonor response. Gray bars: Samples (n = 4) in which antidonor-specific ELISPOT showed evidence of an unregulated B-dependent antidonor response. (c) Percentage of CD4+ cells staining for IFN-γ alone (IFN-γ + IL-10−) compared with cells staining for both (IFNγ + IL-10+). (d) Median fluorescence intensity of staining for IFN-γ or IL-10 in the single-positive (IFNγ + IL-10−) or double-positive (IFNγ + IL-10+) CD4+ populations as indicated. (e) Ratio of mean fluorescence intensity of IFN-γ staining to IL-10 staining in the double-positive (IFN-γ + IL-10+) population in (d). *P < 0.05 by Mann-Whitney U test.