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 alphabeta 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 high-affinity/avidity interactions induce death by apoptosis (negative selection). 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 in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.

Figure 1. Specific and severe defect in Raf–MEK–ERK activation in Cnb1-deficient thymocytes

a, Expression of CD4 and CD8 on Cnb1-deficient and control thymocytes. The numbers in the corners of the panels represent the percentage of cells in each quadrant. b, Immunoblot analysis of phosphorylated and total proteins in Cnb1-deficient and control double-positive thymocytes after CD3ε crosslinking. GSK, glycogen synthase kinase; PKC, protein kinase C; PKD, protein kinase D. c, Immunoblot analysis of phosphorylated ERK1/2 in Cnb1-deficient and control double-positive thymocytes after CD3ε crosslinking. d, Immunoblot analysis of Egr1 induction in double-positive thymocytes from Cnb1-deficient and control littermates. Brg1 shows equal loading. e, Immunoblot analysis of phosphorylated MEK1/2 in Cnb1-deficient and control double-positive thymocytes after CD3ε crosslinking. f, Raf-B kinase activity in Cnb1-deficient and control double-positive thymocytes after CD3ε crosslinking. IP, immunoprecipitation; IB, immunoblotting.

Figure 2. Developmental but not direct requirement for calcineurin/NFAT activity for proper activation of ERK

a, ERK1/2 phosphorylation in double-positive cells from untreated and CsA-treated mice in the presence of CsA (200 ng ml⁻¹) or UO126 (10 µM) after CD3ε crosslinking. b, Erg1 induction in double-positive cells from untreated or CsA-treated mice and in double-positive cells stimulated in the presence of CsA (200 ng ml⁻¹) or UO126 (10 µM) after CD3ε crosslinking. Brg1 shows equal loading. c, ERK1/2 phosphorylation in double-positive CD69-negative Cnb1-deficient and control thymocytes after CD3ε crosslinking for 2 min (solid lines). Grey areas, unstimulated. d, BrdU incorporation and ERK1/2 phosphorylation in double-positive CD69-negative thymocytes from Cnb1-deficient mice and control littermates injected once with BrdU after CD3ε crosslinking for 2 min. The numbers in the corners of the panels represent the percentage of cells in each quadrant. e, ERK1/2 phosphorylation in NFATc2/NFATc3 double knockout (DKO) and control double-positive CD69-negative thymocytes after CD3ε crosslinking for 2 min (solid lines). Grey lines, unstimulated. The numbers in graphs c and e represent the percentage of cells in the indicated interval.

Figure 3. Reconstitution of Raf–MEK–ERK signalling partly rescues positive selection in the absence of calcineurin activity

a, Expression of CD4 and CD8 in Raf-CAAX transgenic and control mice treated with CsA or left untreated. b, Analysis of CD69 and TCR-β expression on thymocytes from Raf-CAAX transgenic and control mice treated with CsA or left untreated. c, Analysis of CD24 and Qa2 expression on thymocytes from Raf-CAAX transgenic and control mice treated or not with CsA. d, Absolute numbers of TCR^high CD4 and CD8 single-positive cells (n≥8, each square represents an individual mouse; bar equals mean value). P values refer to a one-tailed t-test. Open symbols, CsA, Raf-CAAX; filled symbols, CsA. e, Absolute numbers of thymocytes for mice of indicated genotype (n≥8; error bars show s.d.). Open bars, control; filled bars, CsA-treated. The numbers in the corners of the panels represent the percentage of cells in each quadrant.

Figure 4. Transition to the ‘high ERK competence’ state is required to respond functionally to positively selecting ligands

a, Phospho-ERK1/2 in OT-I double-positive thymocytes from mice stimulated with SIINFEKL or RTYTYEKL. White symbols, untreated; grey symbols, CsA-treated. b, Egr1 upregulation in OT-I double-positive thymocytes stimulated with SIINFEKL or RTYTYEKL. c, CD69 upregulation in OT-I double-positive thymocytes after 4 h of stimulation. Δ indicates the percentage difference in CD69 thymocytes stimulated by APCs only or by peptide-pulsed APCs. Solid lines, APCs plus peptide; grey areas, APCs only. d, CD4 and CD8 expression in thymocytes from mice of the indicated genotypes. e, Percentage of CD4 single-positive expressing Vβ chains reactive or not to endogenous superantigens (squares show results for individual mice; bars show medians; n≥3). White symbols, untreated control; light grey symbols, Bim-deficient; dark grey symbols, Bim-deficient and CsA-treated. f, Percentages (numbers shown in each panel refer to the percentage of cells in the indicated interval) and absolute numbers of HY⁺ CD8 single-positive CD24^low cells in mice of the indicated genotypes (squares show results for individual mice, bars show means; n≥4). White symbols, untreated; grey symbols, CsA-treated.