ABSTRACT
The expression of interleukin-2 (IL-2) is a key event in T helper (Th) lymphocyte activation, controlling both, the expansion and differentiation of effector Th cells as well as the activation of regulatory T cells. We demonstrate that the strength of TCR stimulation is translated into the frequency of memory Th cells expressing IL-2 but not into the amount of IL-2 per cell. This molecular switch decision for IL-2 expression per cell is located downstream of the cytosolic Ca2+ level. Here we show that in a single activated Th cell, NFATc2 activation is digital but NF-kappaB activation is graded after graded T cell receptor (TCR) signaling. Subsequently, NFATc2 translocates into the nucleus in an all-or-none fashion per cell, transforming the strength of TCR-stimulation into the number of nuclei positive for NFATc2 and IL-2 transcription. Thus, the described NFATc2 switch regulates the number of Th cells actively participating in an immune response.
Subject(s)
Interleukin-2/metabolism , NFATC Transcription Factors/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Helper-Inducer/metabolism , Active Transport, Cell Nucleus/drug effects , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , Calcineurin/pharmacology , Calcium/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cells, Cultured , Flow Cytometry , Humans , Interferon-gamma/metabolism , Interleukin-2/genetics , Ionomycin/pharmacology , Lectins, C-Type , Lymphocyte Activation/drug effects , Models, Theoretical , NF-kappa B/metabolism , NFATC Transcription Factors/genetics , Phosphorylation , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Helper-Inducer/drug effectsABSTRACT
Dephosphorylation of NFAT by the Ca(2+)-calmodulin-dependent Ser/Thr protein phosphatase calcineurin is a bottleneck of T cell receptor-dependent activation of T cells. In dimeric complexes with immunophilins, the immunosuppressants cyclosporine A (CsA) and tacrolimus (FK506) block this process by inhibition of the enzymatic activity of calcineurin. We have identified the pyrazolopyrimidine compound NCI3 as a novel inhibitor of calcineurin-NFAT signaling. Similar to CsA and FK506, NCI3 inhibits dephosphorylation and nuclear translocation of NFAT, IL-2 production and proliferation of stimulated human primary T cells with IC(50) values from 2 to 4.5 microM. However, contrary to CsA and FK506, NCI3 neither blocks calcineurin;s phosphatase activity nor requires immunophilins for inhibiting NFAT activation. Our data suggest that NCI3 binds to calcineurin and causes an allosteric change interfering with NFAT dephosphorylation in vivo but not in vitro. NCI3 acts not only on the endogenous calcineurin but also on a C-terminally truncated, constitutively active version of calcineurin. The novel inhibitor described herein will be useful in better defining the cellular regulation of calcineurin activation and may serve as a lead for the development of a new type of immunosuppressants acting not by direct inhibition of the calcineurin phosphatase activity.
