A Ca2+ concentration of 1.5 mM, as present in IMDM but not in RPMI, is critical for maximal response of Th cells to PMA/ionomycin

A Ca 2+ concentration of 1.5 mM, as present in IMDM but not in RPMI, is critical for maximal response of Th cells to PMA/ ionomycin In the original activation of T lympho-cytes, distinct cytokine genes and genes for other functionally relevant proteins are imprinted for reexpression upon secondary activation by antigen [1, 2]. To measure this imprinting independently of a particular antigen, T cells can be stimulated with PMA and ionomycin, mimicking TCR stimulation [3]. PMA directly activates protein kinase C and hence leads to the nuclear translocation of NF-κB via phosphorylation of its antagonist IκB [4–6]. Ionomycin induces increased cytoplasmic Ca 2+ concentrations that activate calcineurin [7, 8]. Calcineurin dephosphorylates NFAT and thus enables its translocation to the nucleus [9–11]. NFAT, NF-κB, and AP-1, which are also activated by PMA/ionomycin, induce the transcription of imprinted genes [1, 12, 13]. The nuclear translocation of NFAT occurs in an all-or-none fashion, due to the multiple dephosphorylation events required, a process mediated by the Ca 2+-dependent phosphatase calcineurin [11]. Thus, when a population of T cells is restimulated, calcineurin activity determines the frequency of cytokine-producing cells in that population, rather than the amount of cytokine expressed per cell [14, 15]. When addressing the functional potential of Th cells in murine T cell transfer-induced colitis [16], we compared the frequencies of cytokine-producing mucosal Th cells, isolated ex vivo and restimu-lated with PMA and ionomycin in RPMI (Roswell Park Memorial Institute 1640) or in IMDM (Iscove's modified Dulbecco's medium). In IMDM, the frequencies of Th cells expressing IFN-γ, IL-17, IL-10, IL-22, or TNF were consistently higher than in RPMI. The difference in frequencies ranged from 1.4-fold for IFN-γ to more than threefold for TNF (Fig. 1 and Supporting Information Fig. 1). This effect was also observed for Th cells from spleen and mesenteric LNs of colitic mice (data not shown).