DOCK8 Drives Src-Dependent NK Cell Effector Function Conor J. Kearney, Stephin J. Vervoort, Kelly M. Ramsbottom, Andrew J. Freeman, Jessica Michie, Jane This information is current as Peake, Jean-Laurent Casanova, Capucine Picard, Stuart G. of September 23, 2021. Tangye, Cindy S. Ma, Ricky W. Johnstone, Katrina L. Randall and Jane Oliaro J Immunol 2017; 199:2118-2127; Prepublished online 9 August 2017; Downloaded from doi: 10.4049/jimmunol.1700751 http://www.jimmunol.org/content/199/6/2118 Supplementary http://www.jimmunol.org/content/suppl/2017/08/09/jimmunol.170075 http://www.jimmunol.org/ Material 1.DCSupplemental References This article cites 38 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/199/6/2118.full#ref-list-1 Why The JI? Submit online. by guest on September 23, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology DOCK8 Drives Src-Dependent NK Cell Effector Function Conor J. Kearney,*,† Stephin J. Vervoort,‡ Kelly M. Ramsbottom,* Andrew J. Freeman,* Jessica Michie,* Jane Peake,x Jean-Laurent Casanova,{,‖,#,** Capucine Picard,{,#,†† Stuart G. Tangye,‡‡,xx Cindy S. Ma,‡‡,xx Ricky W. Johnstone,†,‡ Katrina L. Randall,{{,‖‖ and Jane Oliaro*,† Mutations in the dedicator of cytokinesis 8 (DOCK8) gene cause an autosomal recessive form of hyper-IgE syndrome, characterized by chronic immunodeficiency with persistent microbial infection and increased incidence of malignancy. These manifestations suggest a defect in cytotoxic lymphocyte function and immune surveillance. However, how DOCK8 regulates NK cell–driven immune responses remains unclear. In this article, we demonstrate that DOCK8 regulates NK cell cytotoxicity and cytokine production in response to target cell engagement or receptor ligation. Genetic ablation of DOCK8 in human NK cells attenuated cytokine transcription and secretion through inhibition of Src family kinase activation, particularly Lck, downstream of target Downloaded from cell engagement or NKp30 ligation. PMA/Ionomycin treatment of DOCK8-deficient NK cells rescued cytokine production, indicating a defect proximal to receptor ligation. Importantly, NK cells from DOCK8-deficient patients had attenuated production of IFN-g and TNF-a upon NKp30 stimulation. Taken together, we reveal a novel molecular mechanism by which DOCK8 regulates NK cell–driven immunity. The Journal of Immunology, 2017, 199: 2118–2127. utosomal recessive hyper-IgE syndrome (AR-HIES) is a bacterial, and fungal infections; and recurrent pneumonia. Other http://www.jimmunol.org/ rare primary immunodeficiency disease characterized by clinical symptoms include eczema and an increased incidence of A elevated serum IgE levels; repeated cutaneous viral, severe allergies and asthma (1, 2). A major advance in the man- agement of patients with this rare disease was made in 2009 with *Immune Defence Laboratory, Cancer Immunology Division, Peter MacCallum Can- the discovery that mutations in the dedicator of cytokinesis 8 cer Centre, Melbourne, Victoria 3000, Australia; †Sir Peter MacCallum Department (DOCK8) gene, located on chromosome 9p, are responsible for of Oncology, The University of Melbourne, Parkville, Victoria 3052, Australia; most cases of AR-HIES (2, 3). ‡Gene Regulation Laboratory, Cancer Therapeutics Division, Peter MacCallum Can- cer Centre, Melbourne, Victoria 3000, Australia; xUniversity of Queensland and Lady DOCK8 is a member of the DOCK180-related family of guanine Cilento Children’s Hospital, Brisbane, Queensland 4006, Australia; {Laboratory of nucleotide exchange factors (GEFs), which promote the activity of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Imagine Rho GTPases such as Rac and Cdc42 (4), and are involved in by guest on September 23, 2021 Institute, Necker Medical School, University Paris Descartes, 75015 Paris, France; ‖Pediatric Hematology and Immunology Unit, Necker Hospital for Sick Children, diverse cellular processes including cell migration, differentiation, AP-HP, Paris, France; #St. Giles Laboratory of Human Genetics and Infectious Dis- and cell–cell interactions (4). Although DOCK8 is ubiquitously eases, Rockefeller Branch, The Rockefeller University, New York, NY 10065; **Howard Hughes Medical Institute, New York, NY 10065; ††Study Center for expressed, it is particularly enriched in lymphocytes, suggesting it Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, 75015 functions to promote immunity (5). Indeed, recent studies have Paris, France; ‡‡Immunology Division, Garvan Institute of Medical Research, xx found that DOCK8 is required for the survival and persistence of Darlinghurst, New South Wales 2010, Australia; St. Vincent’s Clinical School, Uni- + versity of New South Wales, New South Wales 2052, Australia; {{Department of CD8 T cells (6) and NKT cells (7), and for long-term Ab pro- Immunology and Infectious Disease, The John Curtin School of Medical Research, duction by B cells (5). DOCK8 also appears to be essential for Australian National University, Acton, Australian Capital Territory 2601, Australia; and ‖‖Australian National University Medical School, Australian National University, maintaining the structural integrity of T cells, serving to repress Acton, Australian Capital Territory 2601, Australia cell death during migration into skin (8), which is essential for the ORCIDs: 0000-0001-9003-5861 (J.P.); 0000-0001-8788-5056 (C.P.); 0000-0002- control of herpes virus skin infection (8, 9). 5360-5180 (S.G.T.); 0000-0001-9708-6020 (K.L.R.). Consistent with the clinical characteristics of AR-HIES, DOCK8 Received for publication May 24, 2017. Accepted for publication July 12, 2017. is also implicated in the regulation of the NK cell and CD8+ T cell This work was supported by the National Health and Medical Research Council of immune synapse. The immune synapse is a specialized structure Australia (Grant 1079318). that forms between the plasma membrane of two cells, facilitating J.O., K.L.R., and C.J.K. designed the study; C.J.K., S.J.V., K.M.R., A.J.F., and J.M. signaling or the triggering of killer cell effector function (10, 11). performed experiments, analysis, and interpretation of the data; C.S.M., S.G.T., J.P., C.P., J.-L.C., and R.W.J. provided clinical samples and contributed to writing the Formation of the immune synapse results in rapid signal trans- manuscript; C.J.K. and J.O. wrote the manuscript. duction events that promote optimal cell activation, but also The sequencing data presented in this article have been submitted to the Gene Expression triggers reorganization of the cell cytoskeleton. These changes Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE101467. facilitate the spatial rearrangements of molecules that contribute Address correspondence and reprint requests to Dr. Jane Oliaro, Immune Defence Lab- to the success and quality of the immune synapse (11–13). Upon oratory, Cancer Immunology Division, Peter MacCallum Cancer Centre, 305 Grattan immune synapse formation in T cells, protein kinase C u and Street, Melbourne, VIC 3000, Australia. E-mail address: [email protected] leukocyte-specific protein tyrosine kinase (Lck) rapidly polarize The online version of this article contains supplemental material. into a region proximal to the synapse contact site called the central Abbreviations used in this article: AR-HIES, autosomal recessive hyper-IgE syn- drome; DOCK8, dedicator of cytokinesis 8; GEF, guanine nucleotide exchange fac- SMAC region (11). These events trigger the TCR-mediated sig- tor; GSEA, gene set enrichment analysis; MTOC, microtubule-organizing center; naling cascades that culminate in T cell activation and acquisition NCR, natural cytotoxicity receptor; RNA-Seq, RNA sequencing; siRNA, small in- of effector functions. Furthermore, adhesion, costimulatory, and terfering RNA; WASp, Wiskott–Aldrich syndrome protein. conjugation-promoting molecules, such as LFA-1 and talin, mi- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 grate to a ring that surrounds the central SMAC region (called the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700751 The Journal of Immunology 2119 peripheral SMAC), which serves to improve the quality of the containing 5% BSA. After washing, proteins were detected using West immune synapse (10, 11). Coast SuperSignal. DOCK8 was initially identified as a regulator of the B cell im- Conjugation assay munological synapse, where it was found to be required for long-lived Ab production (5). Later, DOCK8 was found to also regulate the NK K562 and KHYG1 cells were labeled with CellTrace Violet and CFSE (Molecular Probes), respectively, for 20 min at 37˚C. Cells were washed twice, cell cytotoxic synapse (14, 15).