Calcineurin inhibitors suppress acute graft-versus-host disease via NFAT-independent inhibition of T cell receptor signaling
Shizuka Otsuka, … , Roberto Weigert, Jonathan D. Ashwell
Shizuka Otsuka, … , Roberto Weigert, Jonathan D. Ashwell
Published April 6, 2021
Citation Information: J Clin Invest. 2021;131(11):e147683. https://doi.org/10.1172/JCI147683.
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Research Article Immunology

Calcineurin inhibitors suppress acute graft-versus-host disease via NFAT-independent inhibition of T cell receptor signaling

Abstract

Inhibitors of calcineurin phosphatase activity (CNIs) such as cyclosporin A (CsA) are widely used to treat tissue transplant rejection and acute graft-versus-host disease (aGVHD), for which inhibition of gene expression dependent on nuclear factor of activated T cells (NFAT) is the mechanistic paradigm. We recently reported that CNIs inhibit TCR-proximal signaling by preventing calcineurin-mediated dephosphorylation of LckS59, an inhibitory modification, raising the possibility of another mechanism by which CNIs suppress immune responses. Here we used T cells from mice that express LckS59A, which cannot accept a phosphate at residue 59, to initiate aGVHD. Although CsA inhibited NFAT-dependent gene upregulation in allo-aggressive T cells expressing either LckWT or LckS59A, it was ineffective in treating disease when the T cells expressed LckS59A. Two important NFAT-independent T cell functions were found to be CsA-resistant in LckS59A T cells: upregulation of the cytolytic protein perforin in tissue-infiltrating CD8+ T cells and antigen-specific T/DC adhesion and clustering in lymph nodes. These results demonstrate that effective treatment of aGVHD by CsA requires NFAT-independent inhibition of TCR signaling. Given that NFATs are widely expressed and off-target effects are a major limitation in CNI use, it is possible that targeting TCR-associated calcineurin directly may provide effective therapies with less toxicity.

Authors

Shizuka Otsuka, Nicolas Melis, Matthias M. Gaida, Debjani Dutta, Roberto Weigert, Jonathan D. Ashwell

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Figure 4

CsA-mediated immunosuppression in aGVHD requires inhibition of TCR signaling.

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CsA-mediated immunosuppression in aGVHD requires inhibition of TCR signa...
For aGVHD induction, a combination of donor T cell–depleted BM cells and LN cells from B6 WT or B6 LckS59A mice were injected into irradiated BAF1 mice. CsA was given daily. WT BM cells were given alone as a negative control. (A and B) Mice receiving allogenic WT (n = 7), WT+CsA (n = 8), LckS59A (n = 11), LckS59A+CsA (n = 10), and control WT BM (n = 6) were scored for aGVHD. (C) The mortality during the course of aGVHD. (D) Samples of liver at 30 days after transplant were stained with H&E. Livers of recipient mice receiving WT and LckS59A cells showed aGVHD signs with portal inflammation (a), bile duct lymphocytes and bile duct epithelium (b), and endothelialitis (c). CsA-treated recipient mice receiving WT donor cells had no significant aGVHD signs in the liver, whereas aGVHD signs such as portal inflammation, focal bile duct lymphocytes, and focal endothelialitis were observed in mice receiving LckS59A cells. Magnification is ×100. Scale bars: 500 μm. (E) Histopathology scores of mice receiving WT alone (n = 6), WT+CsA (n = 7), LckS59A alone (n = 8), or LckS59A+CsA (n = 8) BM+lymph node cells or control BM alone (n = 4) at day 30. (F) Liver-infiltrating cells were collected from recipient mice receiving WT alone (n = 10), WT+CsA (n = 8), LckS59A alone (n = 11), or LckS59A+CsA (n = 11) BM+lymph node cells or control BM alone (n = 5) at day 7 to 8. The graphs show the mean ± SEM. *P < 0.05, **P < 0.01, 2-tailed Student’s t test. (G) Representative histograms of CD69 expression on freshly isolated intrahepatic CD4+ and CD8+ T cells from recipient mice at day 7. The graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001, Student’s t test. (H) IL-2 and IFN-γ expression by restimulated liver-infiltrating cells from day 7 recipients that had received WT (n = 9), WT+CsA (n = 8), LckS59A (n = 10), or LckS59A+CsA (n = 11). Representative contour plots of cytokine expression in CD4+ and CD8+ T cells are shown. The graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001, Student’s t test.