DOCK8 regulates fitness and function of regulatory T cells through modulation of IL-2 signaling
Akhilesh K. Singh, Ahmet Eken, David Hagin, Khushbu Komal, Gauri Bhise, Azima Shaji, Tanvi Arkatkar, Shaun W. Jackson, Estelle Bettelli, Troy R. Torgerson, Mohamed Oukka
Akhilesh K. Singh, Ahmet Eken, David Hagin, Khushbu Komal, Gauri Bhise, Azima Shaji, Tanvi Arkatkar, Shaun W. Jackson, Estelle Bettelli, Troy R. Torgerson, Mohamed Oukka
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Research Article Immunology

DOCK8 regulates fitness and function of regulatory T cells through modulation of IL-2 signaling

Abstract

Foxp3+ Tregs possess potent immunosuppressive activity, which is critical for maintaining immune homeostasis and self-tolerance. Defects in Treg development or function result in inadvertent immune activation and autoimmunity. Despite recent advances in Treg biology, we still do not completely understand the molecular and cellular mechanisms governing the development and suppressive function of these cells. Here, we have demonstrated an essential role of the dedicator of cytokinesis 8 (DOCK8), guanine nucleotide exchange factors required for cytoskeleton rearrangement, cell migration, and immune cell survival in controlling Treg fitness and their function. Treg-specific DOCK8 deletion led to spontaneous multiorgan inflammation in mice due to uncontrolled T cell activation and production of proinflammatory cytokines. In addition, we show that DOCK8-deficient Tregs are defective in competitive fitness and in vivo suppressive function. Furthermore, DOCK8 controls IL-2 signaling, crucial for maintenance and competitive fitness of Tregs, via a STAT5-dependent manner. Our study provides potentially novel insights into the essential function of DOCK8 in Tregs and immune regulation, and it explains the autoimmune manifestations associated with DOCK8 deficiency.

Authors

Akhilesh K. Singh, Ahmet Eken, David Hagin, Khushbu Komal, Gauri Bhise, Azima Shaji, Tanvi Arkatkar, Shaun W. Jackson, Estelle Bettelli, Troy R. Torgerson, Mohamed Oukka

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Figure 6

In vivo immune-suppressive functions are impaired in DOCK8-deficient Tregs.

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In vivo immune-suppressive functions are impaired in DOCK8-deficient Tre...
(A and B) DOCK8 is dispensable for the in vitro Treg-suppressive function. YFP–CD4+ naive T cells were sorted from control Foxp3Cre mice and labeled with cell tracer. In vitro suppression was performed with 5 × 105 irradiated splenocytes as APCs and titrated number of YFP+ Tregs sorted from Foxp3Cre and Foxp3CreDOCK8fl/fl mice as suppressor cells. Cultures were stimulated with anti-CD3 (1 μg/ml), and proliferation was measured by loss of cell tracer after 96-hour stimulation. Data are representative of 2 independent experiments. (B–H) DOCK8-deficient Tregs failed to suppress in vivo inflammation during EAE a mouse model to study CNS inflammation. (B) Schematic representation showing the DOCK8 deletion by tamoxifen injection and induction of EAE in 8- to 10-week-old control Foxp3CreERT2 mice and Foxp3CreERT2DOCK8fl/fl mice. (C) Control (Foxp3CreERT2) and Foxp3CreERT2DOCK8fl/fl mice were immunized with MOG35–55 in CFA and monitored for clinical signs of EAE. (D and E) Frequency and (F) absolute number of Tregs in the CNS of control and Foxp3CreERT2DOCK8fl/fl mice at the peak of disease. (G) The total CNS-infiltrating CD4+ T cells and (H) absolute number of total CNS infiltrating lymphocytes. Data are representative of 3 separate experiments with 5 mice per group. The data shown are the mean ± SD. Statistics were performed either with Prism software by using t test (2-tailed) (in C, E–H) and Mann-Whitney U test (in C). *P < 0.05, **P < 0.01. (I–K) DOCK8 flag tag mice showing the expression of DOCK8 on hematopoietic cells including Tregs, and its expression is upregulated during CNS inflammation. (I) Flow histogram showing that DOCK8 is specifically expressed on hematopoietic cells. (J and K) Control or DOCK8 flag tag mice were immunized with MOG35–55 in CFA, and their spleen and CNS were analyzed for DOCK8 expression at the peak of disease. (J) Upper panel shows DOCK8 expression in spleen at baseline (blue line) and at the peak of EAE (red line). Lower panel shows the DOCK8 expression at the peak of EAE (red line). (K) The MFI of DOCK8 on Foxp3–CD4+ T cells, Foxp3+CD4+ T cells, and CD8+ T cells in the spleen and CNS. Data represents 2–3 independent experiments with 3–6 mice per group. The data shown are the mean ± SD. Statistics were performed with Prism software by using 1-way ANOVA. **P < 0.01, ***P < 0.001.