Calcineurin sets the bandwidth for discrimination of signals during thymocyte development

Abstract

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the α β T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas highaffinity/avidity interactions induce death by apoptosis (negative selection) 1,2 . Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not Reprints and permissions information is available at www.nature.com/reprints.Correspondence and requests for materials should be addressed to G.R.C. (crabtree@stanford.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Contributions E.M.G., M.M.W. and G.R.C. generated the hypotheses, designed the experiments and wrote the manuscript. E.M.G. performed the experiments and generated the figures. K.C.-B. generated the NFATc3 conditional knockout mice, maintained this line in the NFATc2-null background and contributed to the experiments in Fig. 4. A.N.R. and L.H. contributed to pilot experiments and experiments shown in Fig. 2 and Supplementary Fig. 8. J.R.N. generated the Cnb1 conditional knockout mice, conducted pilot experiments and contributed to experimental rationale. L.M. contributed to experiments shown in Supplementary Fig. 5. B.I. provided the Raf-CAAX transgenic mice. The calcineurin/NFAT 3,4 and the Raf-MEK-ERK 5-7 pathways have been shown to be required for positive selection of thymocytes but not for their negative selection. Calcineurin B1 (Cnb1)-deficient CD4 + CD8 + double-positive thymocytes lack calcineurin activity, fail to dephosphorylate NFATc transcription factors and are not positively selected (Fig. 1a and ref. 3). Cnb1-deficient thymocytes have normal phosphorylation of JNK (c-Jun N-terminal kinase), p38, protein kinase C-θ, protein kinase D and glycogen synthase kinase 3α after crosslinking of the T-cell receptor (TCR) (Fig.1band ref. 3). Actin polymerization and Ca 2+ influx were also normal in these cells ( Supplementary Fig. 2a, b). Collectively, these results indicated that Cnb1-deficient thymocytes did not have widespread signalling defects downstream of the TCR. However, Cnb1-deficient thymocytes showed a specific and sever...

Aberrant Selection and Function of Invariant NKT Cells in the Absence of AP-1 Transcription Factor Fra-2

“…By analogy with T cell development (49), enhanced positive selection of lower affinity iNKT cells might also be caused by increased intracellular signaling. Intracellular signaling during iNKT cell selection and development could potentially be perturbed in the absence of Fra-2, likely via the dysregulation of AP-1 target genes evident from our microarray data; this could then affect the selection process.…”

Calcium signaling in lymphocytes

“…The RasGEF activity of RasGRP1 in the Golgi depends both on DAG and on increased intracellular Ca 2+ concentration [22]. RasGRP1 is barely expressed in thymocytes at the double-negative (CD4 − CD8 − , DN) to double-positive (CD4 + CD8 + , DP) stages [24]; instead, the calcineurin/ NFAT pathway was shown to modulate Ras activation in CD4 + CD8 + CD69 − DP thymocytes that have not yet undergone positive selection, altering the threshold for ERK activity and allowing weak TCR signals to induce positive selection [25]. B-Raf, MEK and ERK activation are all impaired in DP thymocytes from mice lacking the calcineurin regulatory subunit CnB1, suggesting that calcineurin/ NFAT-dependent transcription regulates the Raf/ Ras/ MAP kinase pathway at several levels, both upstream and downstream of Raf.…”

Sea squirts and immune tolerance

“…Moreover, over the last few years, multiple studies have demonstrated the regulation of signaling strength in the immune system. Studies in T cells and macrophages have revealed that calcineurin (which acts through an unknown mechanism), miR-181 and miR-155 (which regulate the translation of phosphatases, thereby affecting inhibitory signaling) all function to modify the signaling strength of ligand engagement on the cell surface (Gallo et al, 2007;Li et al, 2007;O'Connell et al, 2009). Obviously, the ability to manipulate signaling strength or response thresholds may be excellent targets for future clinical intervention, such as inducing tolerance.…”