Despite large-scale T cell activation, only a minor subset of T cells responding in vitro to Actinobacillus actinomycetemcomitans differentiate into effector T cells

Recent studies in our laboratory have demonstrated that Actinobacillus actinomycetemcomitans has a potent T cell stimulatory effect, activating more than half of all T cells, However, since the fate of these activated T cells was not known, the present study sought to determine whether all of these T cells differentiate into effector cells. To that end, the intracellular expression of T cell cytokines (IL-2, IFN-gamma, IL-4 and IL-10) in response to A. actinomycetemcomitans was determined by flow cytometry. Results demonstrated a time-dependent increase in the expression of the cytokines, most reaching peak levels at 24-48 h. At 48 h, the proportion of T cells expressing each of the cytokines were as follows: IL-2 (1.7% +/- 0.3), IFN-gamma (1.8% +/- 0.5), IL-4 (1.0% +/- 0.2) and IL-10 (1.5% +/- 0.5). These data indicated that only 2-5% of all T cells stimulated with A. actinomycetemcomitans expressed any T cell cytokines. The finding of large-scale T cell activation in the absence of cytokine expression suggests that the activation of T cells in response to A. actinomycetemcamitans is incomplete. To investigate this phenomenon, peripheral blood mononuclear cells (PBMC) were cultured with A. actinomycetemcomitans for 24 h followed by sorting of the activated (CD69(+)) cells by immunomagnetic separation and restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Results demonstrated that nearly 90% of the T cells were unresponsive to further restimulation. A possible explanation for this unresponsiveness is the induction of clonal anergy among the responding T cells. To determine possible preferential effects of the stimulation on specific cytokines, the expression of each cytokine among T cells responding to A. actinomycetemcomitans was compared to the maximum levels achieved by PMA + ionomycin stimulation. Results showed that number of IL-2(+) and IFN-gamma(+) T cells observed in response to A. actinomycetemcomitans were between 2% and 7% of those seen in response to PMA + ionomycin. Conversely, the proportions of T cells expressing IL-4 or IL-10 were between 35% and 90% of those following stimulation with PMA + ionomycin. Hence, A. actinomycetemcamitans appears to more preferentially induce T cells expressing IL-4 and IL-10. Collectively, these data suggest that the in vitro stimulation of T cells with A. actinomycetemcomitans leads to partial activation, i.e. only a minor subset of T cells responding to A. actinomycetemcomitans differentiate into effector cells, while a significant proportion become unresponsive to restimulation, suggesting clonal anergy.