SLAT regulates Th1 and Th2 inflammatory responses by controlling Ca2+/NFAT signaling
Stéphane Bécart, Céline Charvet, Ann J. Canonigo Balancio, Carl De Trez, Yoshihiko Tanaka, Wei Duan, Carl Ware, Michael Croft, Amnon Altman
Stéphane Bécart, Céline Charvet, Ann J. Canonigo Balancio, Carl De Trez, Yoshihiko Tanaka, Wei Duan, Carl Ware, Michael Croft, Amnon Altman
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Research Article Immunology

SLAT regulates Th1 and Th2 inflammatory responses by controlling Ca2+/NFAT signaling

Abstract

SWAP-70–like adapter of T cells (SLAT) is a novel guanine nucleotide exchange factor for Rho GTPases that is upregulated in Th2 cells, but whose physiological function is unclear. We show that SLAT–/– mice displayed a developmental defect at one of the earliest stages of thymocyte differentiation, the double-negative 1 (DN1) stage, leading to decreased peripheral T cell numbers. SLAT–/– peripheral CD4+ T cells demonstrated impaired TCR/CD28-induced proliferation and IL-2 production, which was rescued by the addition of exogenous IL-2. Importantly, SLAT–/– mice were grossly impaired in their ability to mount not only Th2, but also Th1-mediated lung inflammatory responses, as evidenced by reduced airway neutrophilia and eosinophilia, respectively. Levels of Th1 and Th2 cytokine in the lungs were also markedly reduced, paralleling the reduction in pulmonary inflammation. This defect in mounting Th1/Th2 responses, which was also evident in vitro, was traced to a severe reduction in Ca2+ mobilization from ER stores, which consequently led to defective TCR/CD28-induced translocation of nuclear factor of activated T cells 1/2 (NFATc1/2). Thus, SLAT is required for thymic DN1 cell expansion, T cell activation, and Th1 and Th2 inflammatory responses.

Authors

Stéphane Bécart, Céline Charvet, Ann J. Canonigo Balancio, Carl De Trez, Yoshihiko Tanaka, Wei Duan, Carl Ware, Michael Croft, Amnon Altman

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Figure 8

Impaired TCR/CD28-induced signaling events in SLAT–/– mice.

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Impaired TCR/CD28-induced signaling events in SLAT–/– mice. ...
(A) WT and SLAT–/– total T cells were stimulated with crosslinked anti-CD3 plus anti-CD28 (20 μg/ml each) mAbs for the indicated times. Cell lysates were separated by SDS-PAGE and immunoblotted with the indicated Abs. SAPK, stress-activated protein kinase. (B and C) WT and SLAT–/– T cells were stimulated with anti-CD3 (10 μg/ml) plus anti-CD28 (2.5 μg/ml) mAbs and recombinant IL-2 (100 U/ml) for 48 hours, rested overnight, and restimulated with anti-CD3/CD28 mAbs or ionomycin (Iono) for 2 hours. Cytoplasmic (C) and nuclear (N) fractions were purified and immunoblotted with anti-NFATc1 Ab (B) or anti-NFATc2 Ab (C). Fractions were also immunoblotted with an anti-SLAT Ab, or with anti-tubulin and anti-lamin B Abs to confirm purity of the cytoplasmic and nuclear fractions, respectively. (D–F) Defective Ca2+ signaling in SLAT–/– T cells. Changes in [Ca2+]i were analyzed by flow cytometry in indo-1–loaded T cells from WT and SLAT–/– mice. TCR/CD28 costimulation was performed using a crosslinking goat anti-hamster (gαh) Ig Ab in the absence (D) or presence (E) of 1 mM EGTA, followed by the addition of 1 μM thapsigargin (Thapsi) (D) or 2 mM CaCl2 (E). (F) [Ca2+]i changes in response to thapsigargin, followed by EGTA treatment. Data are expressed as a histogram displaying FL5/FL4 ratio versus time (s). (G) A graph showing mean levels of IP3 (± SD) generated in primary WT or SLAT–/– T cells after CD3/CD28 crosslinking for the indicated times. (H and I) WT and SLAT–/– CD4+ T cells were stimulated under neutral conditions with anti-CD3 (1 μg/ml) plus anti-CD28 (2.5 μg/ml) in the presence or absence of the indicated ionomycin concentrations for 6 days (1st stim.). After 24 hours of restimulation with anti-CD3/CD28 Abs with or without ionomycin, production of IL-4 (H) or IFN-γ (I) was measured by ELISA. Results are expressed as mean ± SD. Statistical differences were determined as in Figure 2. #P < 0.001, WT versus SLAT–/– mice.