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Recruitment of calcineurin to the TCR positively regulates T cell activation

Abstract

Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (LckS59). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70Y493), as well as some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (Lck that cannot be phosphorylated) or LckS59E (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.

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Figure 1: Components of TCR-induced pathways are phosphorylated differentially by CsA and FK506.
Figure 2: Knockdown of calcineurin expression in T cells recapitulates the effects of CsA and FK506.
Figure 3: Cytosolic calcineurin is recruited to TCR microclusters.
Figure 4: Calcineurin-containing microclusters also contain phosphorylated TCR.
Figure 5: Association of calcineurin with signaling components requires Lck activity and intact ZAP-70.
Figure 6: Phosphorylated LckS59 is a substrate of calcineurin in vivo and in vitro.
Figure 7: Substitutions of Lck Ser59 affect TCR-proximal signaling.
Figure 8: Calcineurin's effect on inhibitory phosphorylated LckS59 promotes TCR-induced LFA-1-mediated cell adhesion.

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Acknowledgements

We thank R. Germain (NIAID) for the DCEK cells and Amphotropic Phoenix packaging cell lines; B. Dong for technical assistance; and S. Koyasu for suggesting that we investigate whether CsA has an effect on the activation of p38 via the alternative pathway. Supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, NIH and the Overseas Associateship funded by the Indian Department of Biotechnology, Ministry of Science and Technology (L.I.S.).

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D.D. and J.D.A. conceived of the project, designed experiments, analyzed data and wrote the manuscript; D.D., P.R.M. and L.I.S. performed the experiments; and V.A.B., I.A. and L.E.S. performed the imaging experiments and analyzed the imaging data.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Recruitment of calcineurin is TCR specific and requires Lck.

Immunoblot analysis of (a) Jurkat and (b) human CD4+ T cells left unactivated or activated with OKT3 for the indicated times in the presence of medium or CsA (above lanes) followed by probing for phosphorylated protiens (right margin). Immunoblot analysis of Immunoprecipitates of TCR-ζ from Jurkat and Lck-deficient JCam1.6 cells (c) activated for different times (aove lanes) followed by reprobing with anti-TCR-ζ to ensure equal loading. Immunoprecipitation and immunoblotting with anti-TCR of Jurkat cells (d) activated with either PMA and ionomycin (P+I) or anti-CD3 (OKT3) for the indicated times. Data are representative of 3 independent experiments with similar results.

Supplementary Figure 2 Calcineurin dephosphorylates the inhibitory pLckS59.

Immunoblot analysis of (a) Lymph node cells from AND TCR-transgenic mice activated with MCC-pulsed APC’s (I-Ek expressing DCEK) for the indicated times (above lanes) in the presence of medium (-) and CsA or FK506 (+). Immunoprecipitation with anti-TCR-ζ and in vitro phosphatase assay of TCR complexes from Jurkat cells (b) activated with OKT3 for the different times (above lanes) in the presence of medium (-) or CsA (+), using human recombinant calcineurin (rhCN) for 30 min at 30°C. Western blot analysis of Lck negative JCam1.6 cells (c) retrovirally transduced with cDNA encoding Lck, LckS59A and LckS59E. Data are representative of 3 independent experiments. ELISA for IL-2 secretion after 12 h in Lck-negative JCam1.6 cells (d) stably expressing Lck, LckS59A, or LckS59E and activated with anti-CD3 + anti-CD28 or PMA + ionomycin (P+I). The data represent 2 independent experiments (mean ± SD).

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Supplementary Figure 3 Effect of inhibitors on cell adhesion and IL-2 secretion,

Cell quantification (trypan blue exclusion) of Jurkat cells (a) activated with OKT3 for 30 min and dropped on ICAM-1-coated wells in the presence of medium (-), CsA (+) or cyclohexamide (+) (bottom rows). Percentage binding calculated for each with respect to the activated sample, taken as 100%. The data are representative of 3 independent experiments (mean ± SEM; *** P value < 0.005; paired t-test). Cytokine analysis using ELISA in Jurkat cells (b) activated anti-CD3 and anti-CD28 for 8 h. The data are representative of 3 independent experiments (mean ± SEM). Analysis of IL-2 using ELISA in Jurkat cells at 8 h (c) and human CD4+ T cells at 16 h (d) activated with anti-TCR Ab or PMA+Ionomycin (P+I) in the presence of the drugs CsA, FK506, PP1, VIVIT and VEET (bottom lanes). The data represent the mean ± SD from 2 independent experiments (* P value < 0.05, ** P value < 0.01, paired t-test).

Source data

Supplementary Figure 4 LFA-1 expression on cells treated with drugs.

Flow cytometry of (a) Jurkat, (b) human CD4+ T cells and (c) Lck-negative J Cam1.6 cells expressing Lck, LckS59A and LckS59E left unactivated or activated with anti-CD3 for 30 min with mediun (-), FK506 or CsA (+). The black solid line represents isotype control.

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Supplementary Data Set 1

Uncropped immunoblots. (PDF 22404 kb)

Supplementary Data Set 2

Original images. (PDF 2403 kb)

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Dutta, D., Barr, V., Akpan, I. et al. Recruitment of calcineurin to the TCR positively regulates T cell activation. Nat Immunol 18, 196–204 (2017). https://doi.org/10.1038/ni.3640

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