We previously demonstrated that altered peptide ligands (APL) can partially activate T cells, resulting in multiple distinct functional phenotypes, including the induction of anergy. Such APL stimulate a unique pattern of T cell receptor (TCR) phospho-zeta species, and lack associated ZAP-70 kinase activity. While these data suggested that selective signaling pathways downstream of the TCR/CD3 molecules are activated upon APL stimulation, they did not directly demonstrate this. Thus, we pursued intracellular signaling events successfully stimulated by APL. Because our previous studies showed that cyclosporin A (CsA) completely inhibited anergy induction, we assessed whether TCR ligation by APL cause a rise in cytosolic calcium (Ca+2). Our results show that these ligands can induce Ca+2 transients, in contrast to data generated using analogue peptides in other antigen systems. These opposing results may reflect differences in the intracellular signaling pathways utilized by different APL, or may be due to the exquisite sensitivity of the assay used here. Importantly, the APL-stimulated Ca+2 induction is both initiated and sustained at lower levels than that stimulated by a strong agonist signal, but resembles that stimulated by a weaker agonist stimulus. Alone, the less than optimal Ca+2 induction does not cause anergy, because ionomycin treatment together with the APL does not result in a proliferative signal. Instead, we propose that a combination of this and other signaling pathways induces T cell anergy. Overall, these data support the concept of differential signaling in T cells, as a direct consequence of the phosphotyrosine status of the TCR/CD3 molecules.
We have demonstrated Th2 clonal anergy as a consequence of partial T cell activation by immunogenic peptide and chemically fixed APC, as well as by altered peptide ligand and live antigen-presenting cells (APC). Either stimulation resulted in a profound inability of the T cells to proliferate upon restimulation with antigen and functional APC, a similar phenomenon to that found with Th1 cells. The anergic state was long lasting and was restricted to proliferation, since the T cells retained the ability to produce cytokines upon restimulation, albeit at slightly reduced levels. Th2 anergy induction was inhibited by cyclosporine A, but not by provision of exogenous costimulation or growth factors. The data presented unify Th1 and Th2 cells with regard to anergy and suggest that the fundamental control during anergy for both subsets is prevention of clonal expansion, thus blocking amplification of the immune response.