Single cell analysis of cytokine expression kinetics by human CD4(+) T-cell clones during activation or tolerance induction

Exposure to optimal peptide antigen concentrations induces human CD4(+) T-c ell clones to proliferate and secrete various cytokines. Higher (>10-fold o ptimal) antigen concentrations cause long-term proliferative unresponsivene ss, which can be reversed by exogenous interleukin-2 (IL-2). We call this c ondition 'tolerance'. We used intracellular cytokine staining and flow cyto metric analysis to investigate the kinetics of interferon-gamma, tumour nec rosis factor-alpha, IL-4 and IL-5 production during the initial phase of to lerance induction. Single cell analysis of interferon-gamma and IL-4 or IL- 5 coexpression showed functional heterogeneity of cloned human CD4(+) T cel ls. Superstimulation with phorbol 12-myristate 13-acetate and ionomycin (PI ) revealed enhanced responsiveness shortly after tolerizing treatment, foll owed by reduced responsiveness. Both tolerized and activated T cells had si milarly reduced cytokine responses when further stimulated with antigen dur ing the following 48 hr, with limited enhancement following additional stim ulation with PI. We conclude that cytokine induction is normally followed b y a refractory phase, but that the expression of cytokines is enhanced in t he initial phase of tolerance induction.