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 7

T/DC interactions are inhibited by CsA via suppression of TCR proximal signaling.

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T/DC interactions are inhibited by CsA via suppression of TCR proximal s...
Mice were injected in the footpad with gp33 or OVA-p pulsed DCs labeled with Deep Red Dye. After 18 to 20 hours, recipients were injected i.v. with green CMFDA-labeled P14 WT or LckS59A T cells. pLNs were collected at the indicated times, cleared, and imaged by 2-photon microscopy. (A) Example images (20 μm thick) at the indicated times are shown (DCs are red, P14 T cells are green). Arrows (white) indicate T/DC clusters. (B) The percentage of T/DC clusters as analyzed by Imaris software. Data were pooled from 3 or 4 experiments. (C) The proportion of small (3–4 cells), medium (5–9 cells), and large (10 or more cells) clusters in the total number of T cell clusters. The number of T cells per cluster as analyzed by Imaris software. Data were pooled from 2 to 4 independent experiments. (D–I) Mice were injected i.p. with CsA or PBS at the time of DC transfer and again the time of T cell transfer. pLNs were removed at 3 (D–F) or 6 (G–I) hours after T cell injection and imaged. Representative images are shown in (D) and (G). The percentage of T/DC clusters (E and H) and the number of T cells per cluster (F and I) were analyzed by Imaris software. Data were pooled from 3 to 4 independent experiments (E and H) or 3 independent experiments (F and I). (J–L) CsA was injected 1 hour before collecting pLNs. Representative images are shown in (J). The percentage of T/DC clusters (K) and the number of T cells per cluster (L) were determined with Imaris software. Data were pooled from 3 or 4 independent experiments (K) or 3 independent experiments (L). The graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 by 2-way ANOVA with Tukey’s multiple-comparison post hoc test.