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. 2018 Jan 2;128(1):415-426.
doi: 10.1172/JCI95837. Epub 2017 Dec 4.

γδTCR Recruits the Syk/PI3K Axis to Drive Proinflammatory Differentiation Program

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Free PMC article

γδTCR Recruits the Syk/PI3K Axis to Drive Proinflammatory Differentiation Program

Ryunosuke Muro et al. J Clin Invest. .
Free PMC article

Abstract

γδT cells produce inflammatory cytokines and have been implicated in the pathogenesis of cancer, infectious diseases, and autoimmunity. The T cell receptor (TCR) signal transduction that specifically regulates the development of IL-17-producing γδT (γδT17) cells largely remains unclear. Here, we showed that the receptor proximal tyrosine kinase Syk is essential for γδTCR signal transduction and development of γδT17 in the mouse thymus. Zap70, another tyrosine kinase essential for the development of αβT cells, failed to functionally substitute for Syk in the development of γδT17. Syk induced the activation of the PI3K/Akt pathway upon γδTCR stimulation. Mice deficient in PI3K signaling exhibited a complete loss of γδT17, without impaired development of IFN-γ-producing γδT cells. Moreover, γδT17-dependent skin inflammation was ameliorated in mice deficient in RhoH, an adaptor known to recruit Syk. Thus, we deciphered lineage-specific TCR signaling and identified the Syk/PI3K pathway as a critical determinant of proinflammatory γδT cell differentiation.

Keywords: Cell Biology; Cytokines; Immunology; Signal transduction; T cell development.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Syk plays a dominant role in γδTCR signaling and γδT cell development.
(A and B) Flow cytometric analysis of CD3ε and TCRδ expression in thymocytes from the indicated mice at E15.5 (WT, n = 16; Zap70–/–, n = 10; Sykb–/–, n = 8; and Zap70–/– Sykb–/–, n = 2) and on day 0 (WT, n = 19; Zap70–/–, n = 4; Sykb–/–, n = 7; and Zap70–/– Sykb–/–, n = 8). The total number of thymocytes is indicated above each flow cytometric plot (A). Graphs indicate the total number of γδT cells per mouse (B). (C and D) TCR-induced ERK phosphorylation in thymic γδT cells from the indicated mice on day 0 (Zap70–/–, n = 3; Sykb–/–, n = 4; and Zap70–/– Sykb–/–, n = 3). Histograms indicate p-ERK levels after a 2-minute stimulation (C). MFI relative to the nonstimulated control (D). (E) Histograms show CD5 expression in thymic γδT cells from the indicated mice at E15.5 (WT, n = 13; Zap70–/–, n = 10; Sykb–/–, n = 9; and Zap70–/– Sykb–/–, n = 2) and on day 0 (WT, n = 17; Zap70–/–, n = 3; Sykb–/–, n = 7; and Zap70–/– Sykb–/–, n = 5). Graphs indicate the MFI relative to WT mice. All data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by 1-way ANOVA (B and E) and 2-way ANOVA (D). Data represent the combined results of 3 independent experiments (A, B, and E) or a single experiment (C and D). Max, maximum.
Figure 2
Figure 2. Syk is required for γδT17 development.
(A) Intracellular staining for IL-17A production after stimulation with PMA and ionomycin in total or Vγ4+ γδT thymic cells from the indicated mice on day 0. SSC-A, side scatter area.(B) Total IL-17–producing and Vγ4+ γδT thymic cell numbers per mouse on day 0 (WT, n = 23; Zap70–/–, n = 4; Sykb–/–, n = 7; and Zap70–/– Sykb–/–, n = 5). (C) Representative profiles for cell-surface TCRδ and intracellular RORγt expression in thymic γδT cells (n = 4–5). Graphs indicate the frequency of RORγt cells in total and Vγ4+ γδT cells. (D) Number of Vγ4, Vγ1, Vγ5, and Vγ6 (17D1+Vγ5) cells per mouse at E15.5 (WT, n = 16; Zap70–/–, n = 10; Sykb–/–, n = 8; and Zap70–/– Sykb–/–, n = 2) and on day 0 (WT, n = 19; Zap70–/–, n = 4; Sykb–/–, n = 7; and Zap70–/– Sykb–/–, n = 8). (E) Total IL-17–producing and Vγ4+ γδT cell numbers from the thymus, spleen, and lungs of the indicated fetal liver chimeric mice. The mice were analyzed 8 weeks after the reconstitution. Data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by 1-way ANOVA (BD) and unpaired t test (E). Data represent the combined results of 3 independent experiments (AD) or 2 independent experiments (E).
Figure 3
Figure 3. Zap70 fails to functionally substitute Syk in γδT17 development.
(A) Scheme of the reconstitution of fetal liver T progenitor cells in FTOC. Gr-1TER119 fetal liver cells from WT or Syk-deficient mice at E15.5 were infected with retroviruses expressing EGFP alone or Syk or Zap70 along with EGFP. The infected cells were reconstituted in dGUO-treated WT fetal thymic lobes, and the reconstituted thymic lobes were further cultured for 9 to 14 days. (B) Expression of Syk and Zap70 in EGFP+CD3ε+TCRδ+ cells from FTOC on day 9. (C) Expression of CD3ε and TCRδ in EGFP+ cells on day 9. (D) Expression of CD5 in EGFP+ γδT cells on day 9. (E and F) Intracellular staining for IL-17A and IFN-γ production in EGFP+ γδT cells on day 14. (G) Frequency of the total EGFP+ γδT cells shown in C (n = 6–8). (H) Relative MFI of CD5 expression in the γδT cells shown in D (n = 6–8). (J) Frequency of the IL-17A+ γδT cells shown in E (n = 7–9). (I) Frequency of the IFN-γ+ γδT cells shown in F (n = 7–9). Graphs indicate the data for individual thymic lobes (circles) and the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA (GJ). Data represent at least 2 independent experiments.
Figure 4
Figure 4. The PI3K pathway controls γδT17 development.
(A and B) TCR-induced Akt phosphorylation in thymic γδT cells from Zap70–/– or Sykb–/– mice. Histograms show staining profiles of p-Akt in cells from WT (black lines) and mutant (red lines) mice, overlaid with nonstimulated profiles (shaded) after a 1-minute stimulation (A). MFI relative to nonstimulated controls (B). Thymocytes from adult Zap70–/– mice (n = 3) and neonatal Sykb–/– mice (n = 4) were used. (CG) E15.5 fetal thymus from WT mice was cultured with vehicle alone (DMSO, 0.01%), IC87114 (1 μM), or SF1670 (2.5 μM) for 7 days (n = 5–11). (C) Flow cytometric profiles for CD3ε and TCRδ expression and absolute number of γδT cells. (D) Intracellular staining profiles for IL-17A production in γδT cells and absolute number of IL-17A–producing γδT cells (per lobe). (E) Intracellular staining profiles for RORγt expression in γδT cells and frequency of RORγt+ γδT cells. (F) Intracellular staining profiles for IFN-γ production in γδT cells and absolute number of IFN-γ–producing γδT cells (per lobe). (G) mRNA expression of Rorc, Sox13, and Sox4 in isolated γδT cells. Gene expression was normalized to β-actin (Actb) mRNA. (H) Number of Vγ4+ and Vγ6+ γδT cells. Data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired t test (B and G) and 1-way ANOVA (CF and H). Data represent 2 independent experiments (A and B) or a single experiment (G), or the combined results of 2 independent experiments (CF and H).
Figure 5
Figure 5. Impaired development of γδT17 cells in PI3K-deficient mice.
(A) Flow cytometric profiles for CD3ε and TCRδ in total thymocytes from 0-day-old WT and Pik3cd–/–Pik3cg–/– mice. The total number of thymocytes is shown above each flow cytometric plot. Graph indicates the total number of γδT cells per mouse (n = 4–6). (B) Flow cytometric analysis of CD5 expression in thymic γδT cells (n = 4–6). (C and D) TCR-induced ERK (C) and Akt (D) phosphorylation in thymic Vγ4+ γδT cells from 1-day-old WT and Pik3cd–/– Pik3cg–/– mice. Graphs indicate the MFI relative to the nonstimulated control. (E) Intracellular staining for IL-17A and IFN-γ production in neonatal thymic γδT cells from 0-day-old WT and Pik3cd–/– Pik3cg–/– mice after stimulation with PMA and ionomycin. The number of IL-17A+ and IFN-γ+ γδT cells per mouse is shown (n = 3–6). (F) Number of Vγ4+ and Vγ6+ γδT cells (per mouse) in the indicated mice (n = 4–6). All data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-way ANOVA (C and D) and unpaired t test (A, E, and F). Data represent a single experiment using more than 7 neonatal mice per group.
Figure 6
Figure 6. Syk mediates the Lat-independent TCR signal to the PI3K/Akt pathway.
(A) Flow cytometric profiles for CD3ε and TCRδ in total thymocytes from 5-week-old WT and Lat–/– mice. The total number of thymocytes is shown above each flow cytometric plot (n = 3). (B) Flow cytometric analysis of CD5 expression in thymic γδT cells (n = 3). (C) TCR-induced ERK phosphorylation in thymic γδT cells. Graph indicates the MFI relative to the nonstimulated control (n = 3). (D) TCR-induced Akt phosphorylation in thymic γδT cells pretreated or not with IC87114 (10 μM). Graph shows the MFI relative to the nonstimulated control (n = 3). (E) Flow cytometric analysis of Zap70 and Syk expression in thymic γδT cells from 5-week-old WT and Lat–/– mice (n = 3). (F) TCR-induced Akt phosphorylation in Lat–/– γδT cells pretreated or not with BAY61-3606 (10 μM). Graph shows the MFI relative to the nonstimulated control (n = 3). (G) Intracellular staining for IL-17A production in neonatal thymic γδT cells from WT mice (n = 3) and Lat–/– mice (n = 5) after stimulation with PMA and ionomycin. The number of IL-17A+ γδT cells (per mouse) is shown. All data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-way ANOVA (C, D, and F) and unpaired t test (G). Data represent 2 independent experiments (A, B, D, F, and G) or a single experiment (C and E).
Figure 7
Figure 7. RhoH mediates the γδTCR signaling required for γδT17 development.
(A and B) TCR-induced ERK and Akt phosphorylation in thymic γδT cells from WT (n = 3) and Rhoh–/– mice (n = 3). (C) Representative CD5 expression profiles in thymic γδT cells (n = 3). (D) Number of cells in the indicated thymic γδT subsets from WT and Rhoh–/– mice at E15.5 (WT, n = 5; Rhoh–/–, n = 5) and on day 0 (WT, n = 8; Rhoh–/–, n = 9). (E and F) Staining for Vγ4 and IL-17A in thymic (day 0; WT, n = 4; Rhoh–/–, n = 4) and splenic (6-week-old; WT, n = 8; Rhoh–/–, n = 8) γδT cells after stimulation with PMA and ionomycin. Graph shows the quantification of IL-17A+ γδT cells (per mouse). (GI) WT and Rhoh–/– mice were treated daily for 7 days with IMQ cream or control cream on the ear (n = 3). Kinetics of IMQ-induced ear swelling (G), representative H&E staining of ear sections on day 7 (H), and flow cytometric analysis of IL-17A+ cells in γδT cells from ear-draining lymph nodes on day 7 (I). Scale bar: 100 μm. All data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-way ANOVA (A, B, and G)and unpaired t test (D and F). Data represent more than 2 independent experiments (AF) or a single experiment (GI).

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