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Keywords:

  • T cell polarization;
  • Th1 cells;
  • Th2 cells;
  • differential Ca2+-signals;
  • lipid rafts;
  • ion channels;
  • NFAT;
  • cytokine profiles;
  • cell death sensitivity

Abstract

T helper lymphocytes become polarized upon antigen and cytokine stimuli received after their maturation in the thymus. Since the balance of Th1 and Th2 responses is critical in healthy and pathological immune responses, understanding the molecular base of T cell polarization still remained an important question. Using our Th0/Th1/Th2 hybridoma model system, we performed a comparative study on polarized Th1 and Th2 cells in terms of their membrane raft expression/composition, their TCR mediated activation signaling, and sensitivity to activation-induced cell death (AICD) using flow and image cytometric methods. We show here that the TCR stimulation induced more intense and sustained Ca2+-response in Th1 cells compared to Th2 ones correlates well with a shorter nuclear residence time of the Ca2+-dependent NFAT transcription factor in Th2 cells. In addition, NFAT translocation directly depended on lipid raft integrity/membrane cholesterol level. Expression pattern of raftophilic accessory proteins (CD4, CD59, and CD48) and lipids (GM1, cholesterol) were also different in the Th1 and Th2 hybridomas, similarly to differentiated spleen Th cells. The activation-induced, remarkably clustered and polarized membrane distribution of TCR/CD3 complex in Th1, but not in Th2 cells, together with an increased raft localization of Kv1.3 ion channels regulating the Ca2+-response, are consistent with the above properties of NFAT. Finally, the polarized Th cells, especially Th1, were more sensitive to AICD than their unpolarized Th0 precursor. These results suggest that the membrane microdomain organization—Ca2+-signaling—NFAT activation axis is an important determinant of polarized Th cell effector function and fate. © 2012 International Society for Advancement of Cytometry