DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs
Erin Janssen, Sudha Kumari, Mira Tohme, Sumana Ullas, Victor Barrera, Jeroen M.J. Tas, Marcela Castillo-Rama, Roderick T. Bronson, Shariq M. Usmani, Darrell J. Irvine, Thorsten R. Mempel, Raif S. Geha
Erin Janssen, Sudha Kumari, Mira Tohme, Sumana Ullas, Victor Barrera, Jeroen M.J. Tas, Marcela Castillo-Rama, Roderick T. Bronson, Shariq M. Usmani, Darrell J. Irvine, Thorsten R. Mempel, Raif S. Geha
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Research Article Immunology

DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

Abstract

Patients deficient in the guanine nucleotide exchange factor DOCK8 have decreased numbers and impaired in vitro function of Tregs and make autoantibodies, but they seldom develop autoimmunity. We show that, similarly, Dock8–/– mice have decreased numbers and impaired in vitro function of Tregs but do not develop autoimmunity. In contrast, mice with selective DOCK8 deficiency in Tregs develop lymphoproliferation, autoantibodies, and gastrointestinal inflammation, despite a normal percentage and in vitro function of Tregs, suggesting that deficient T effector cell function might protect DOCK8-deficient patients from autoimmunity. We demonstrate that DOCK8 associates with STAT5 and is important for IL-2–driven STAT5 phosphorylation in Tregs. DOCK8 localizes within the lamellar actin ring of the Treg immune synapse (IS). Dock8–/– Tregs have abnormal TCR-driven actin dynamics, decreased adhesiveness, an altered gene expression profile, an unstable IS with decreased recruitment of signaling molecules, and impaired transendocytosis of the costimulatory molecule CD86. These data suggest that DOCK8 enforces immunological tolerance by promoting IL-2 signaling, TCR-driven actin dynamics, and the IS in Tregs.

Authors

Erin Janssen, Sudha Kumari, Mira Tohme, Sumana Ullas, Victor Barrera, Jeroen M.J. Tas, Marcela Castillo-Rama, Roderick T. Bronson, Shariq M. Usmani, Darrell J. Irvine, Thorsten R. Mempel, Raif S. Geha

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Figure 4

Tregs from mice with a Treg-specific DOCK8 deficiency have impaired fitness.

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Tregs from mice with a Treg-specific DOCK8 deficiency have impaired fitn...
(A and B) Proliferation measured by Cell Trace Violet dilution (A) and IL-2 secretion in culture supernatants (B) by CD4+CD25– Teffs isolated from the spleens of Foxp3YFP–Cre/Dock8flox/flox and Foxp3YFP–Cre control mice cultured for 3 days with anti-CD3+anti-CD28–coated beads. (C) Percentage of YFP+ Tregs of CD4+ T cells and percentage of CD44–CD62Lhi rTregs and CD44+CD62Llo aTregs among total Tregs in spleens of 30-week-old Foxp3YFP–Cre/Dock8flox/flox mice and Foxp3YFP–Cre controls. (D) Percentage of YFP+ cells of total CD4+ cells in the intraepithelial lymphocytes (IEL) and LP of the stomachs and colons and in the skin from Foxp3YFP–Cre/Dock8flox/flox mice and Foxp3YFP–Cre controls. (E) qPCR analysis of Foxp3, Il2ra, Tgfb, and Il10 mRNA levels in FACS-sorted CD4+CD25+YFP+ Tregs from Foxp3YFP–Cre/Dock8flox/flox mice and Foxp3YFP–Cre controls. Results are expressed as fold increase relative to the ratio of the mRNA of interest/b2microglobulin in Foxp3YFP–Cre controls. (F) Suppression of the proliferation of CD4+CD25– Teffs stimulated with mitomycin C–treated APCs and soluble anti-CD3 mAb at a Teff/Treg ratio of 1:1. Teff proliferation was measured by FACS analysis of Cell Trace Violet dilution. (G) YFP+/YFP– cell ratio in the spleens and MLNs of Foxp3YFP–Cre/+/Dock8flox/flox females and Foxp3YFP–Cre/+ controls normalized to a ratio of 1.0 in the controls. (H) Quantitative analysis of surface CD25 and intracellular FOXP3 protein expression by splenic CD4+YFP+ cells from Foxp3YFP–Cre/+/Dock8flox/flox females and Foxp3YFP–Cre/+ controls. Results in A, B, and F are representative of 3 independent experiments. Symbols represent individual mice, and error bars represent mean and SEM. t test, NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.