Background Evidence suggests that human autoimmune diseases might be driven by pro-inflammatory Th1 cells whereas immuno-modulatory Th2 cells are rarely found. The mechanisms regulating Th2 cell differentiation from resting human T cells, however, are not completely characterised.
Objectives To delineate the mechanisms controlling Th2 differentiation, we defined optimal Th2 inducing conditions in human naive and memory CD4pos T cells in vitro and investigated activation of intracellular signalling pathways and their individual role in Th2 differentiation.
Methods A cell culture system was employed that permitted Th2 cell differentiation after short term priming. The phenotype of freshly isolated and primed T cells was determined by cytometric analysis of intracellular cytokines. Activation of intracellular signalling pathways was assessed by western blot analysis or kinase activity assays.
Results In the absence of TCR ligation, stimulation of CD28 induced Th2 differentiation from purified CD4pos peripheral blood memory but not from cord blood naive T cells. Co-stimulation via CD28 and the TCR, on the other hand, enhanced Th2 cell differentiation from naive T cells but suppressed it from memory T cells. Comparison of naive and memory T cell responses to CD28 stimulation, therefore, revealed critical signals facilitating Th2 cell differentiation. CD28 engagement initiated IL-4 gene transcription and activation of p38 MAP kinase in memory T cells but not in naive cells. By contrast, PI3-kinase and JNK/SAPK pathways were activated in both subsets. Whereas IL-4 had a synergistic effect with anti-CD28 on the generation of Th2 cells in memory T cells, it was not sufficient to complement CD28 signals in naive T cells. T cell differentiation in the presence of specific inhibitors to IL-4, the MAP kinases and PI3-kinase revealed that TCR independent, CD28 mediated Th2 differentiation is critically dependent on IL-4 stimulation and the activation of the MAP kinases p38 and ERK1/2. Whereas CD28 signals directly activated IL-4 and p38 in memory T cells, ERK phosphorylation required indirect stimulation by IL-2.
Conclusion The results indicate that generation of Th2 effectors requires coordinate signalling via the CD28 and IL-2 pathways. The mechanisms regulating Th2 cell differentiation might provide valuable tools for therapeutic modulation of chronic autoimmunity.
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