Published February 27, 2019, doi:10.4049/jimmunol.1801157 The Journal of Immunology Sostdc1 Regulates NK Cell Maturation and Cytotoxicity Alberto J. Millan,* Sonny R. Elizaldi,* Eric M. Lee,* Jeffrey O. Aceves,* Deepa Murugesh,* Gabriela G. Loots,*,† and Jennifer O. Manilay* NK cells are innate-like lymphocytes that eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified roles for sclerostin domain–containing-1 (Sostdc1)inNK cell development and function. Sostdc1-knockout (Sostdc12/2) mice display a progressive accumulation of transitional NK cells (tNKs) (CD27+CD11b+) with age, indicating a partial developmental block. The NK cell Ly49 repertoire in Sostdc12/2 mice is also changed. Lower frequencies of Sostdc12/2 splenic tNKs express inhibitory Ly49G2 receptors, but higher frequencies express activating Ly49H and Ly49D receptors. However, the frequencies of Ly49I+,G2+,H+, and D+ populations were universally decreased at the most mature (CD272CD11b+) stage. We hypothesized that the Ly49 repertoire in Sostdc12/2 mice would correlate with NK killing ability and observed that Sostdc12/2 NK cells are hyporesponsive against MHC class I–deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-g expression to controls. Consistent with Sostdc1’s known role in Wnt signaling regulation, Tcf7 and Lef1 levels were higher in Sostdc12/2 NK cells. Expression of the NK development gene Id2 was decreased in Sostdc12/2 immature NK and tNK cells, but Eomes and Tbx21 expression was unaffected. Reciprocal bone marrow transplant experiments showed that Sostdc1 regulates NK cell maturation and expression of Ly49 receptors in a cell-extrinsic fashion from both nonhematopoietic and hematopoietic sources. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation and cytotoxicity, and identify potential NK cell niches. The Journal of Immunology, 2019, 202: 000–000. atural killer cells are innate lymphocytes that are im- mechanisms that drive and integrate these processes is still un- portant for early immune defense against tumors and clear. In particular, how the microenvironment regulates NK cell N virally infected cells. Since the initial discovery of maturation and function is still an area of ongoing investigation. NK cells in 1975, studies from many groups have identified NK Conventional NK cells develop in the bone marrow (BM) from cell receptors that are involved in self/nonself recognition, NK cell hematopoietic stem cells following a well-established sequence of precursors and stages of maturation, cytokines and transcription maturational stages, and egress to the peripheral organs to fully mature factors that are critical for NK cell development and function, and function (10–12). NK cell maturation (Fig. 1A) originates with and evidence for NK cell immune memory (1–9). Despite the immature NK (iNK) (CD27+CD11b2) cells, which progresses to over 40 years of NK cell history, the molecular and cellular the transitional NK (tNK) cell stage (CD27+CD11b+,alsoknownas DP [13], and not to be confused with tissue resident NK cells [14]), then to the final mature NK (mNK) cell stage (CD272CD11b+)(13, *Department of Molecular Cell Biology, School of Natural Sciences, University of 15–17). As NK cells progress through these stages, they lose prolif- † California, Merced, Merced, CA 95343; and Physical and Life Sciences Directorate, erative and cytokine-producing capability but gain cytotoxic ability Lawrence Livermore National Laboratories, Livermore, CA 94550 against target cells (12, 18, 19). Although the BM microenvironment ORCIDs: 0000-0002-0958-2399 (E.M.L.); 0000-0002-1431-5295 (J.O.M.). is critical for NK cell development, how the peripheral microenvi- Received for publication August 23, 2018. Accepted for publication February 6, 2019. ronment regulates NK cell maturation and cytotoxicity is incom- pletely understood and requires further investigation. This work was supported by University of California (UC) Merced faculty research funding, a UC Cancer Research Coordinating Committee grant, a Halcyon-Dixon Sclerostin domain–containing-1 (Sostdc1), also known as Wise, Trust award (to J.O.M.), and UC graduate student fellowships (to A.J.M.). G.G.L. Ectodin, Usag-1, and Sost-like, has been studied in the context of works under the auspices of the U.S. Department of Energy through the Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. tooth development, kidney disease, hair follicle formation, and 2/2 bone fracture (20–26). Sostdc1 can function as an antagonist of G.G.L. and D.M. provided the Sostdc1 mice. Study design: A.J.M. and J.O.M. Study conduct: A.J.M., S.R.E., E.M.L., J.O.A., and J.O.M. Data collection: A.J.M., both bone morphogenetic protein and canonical Wnt signaling S.R.E., E.M.L., and J.O.A. Data analysis: A.J.M., S.R.E., E.M.L., J.O.A., and J.O.M. pathways (21, 22, 24). Sostdc1 expression is highly expressed in Data interpretation: A.J.M. and J.O.M. Drafting manuscript: A.J.M. and J.O.M. Re- vising manuscript content: A.J.M. and J.O.M. Approving final version of manuscript: skin, brain, and intestine as well as in skeletal muscles, kidney, J.O.M. J.O.M. takes responsibility for the integrity of the data analysis. lungs, and vasculature (21, 23, 24). Most recently, we found it also Address correspondence and reprint requests to Dr. Jennifer O. Manilay, University to be expressed in the bone periosteum and mesenchymal stem of California, Merced, School of Natural Sciences, 5200 North Lake Road, Merced, cells to support bone formation and fracture remodeling (27). In CA 95343. E-mail address: [email protected] this study, we reveal Sostdc1’s cell-extrinsic roles in the regulation The online version of this article contains supplemental material. of NK cell maturation, Ly49 receptor expression, and cytotoxic Abbreviations used in this article: BM, bone marrow; DC, dendritic cell; Eomes, function in the BM and spleen. Eomesodermin; FCM, flow cytometry; iNK, immature NK; KO, knockout; LLNL, 2/2 Lawrence Livermore National Laboratories; b2m , b-2 microglobulin knockout; +/+ b2m , b-2 microglobulin sufficient; MHC-I, MHC class I; mNK, mature NK; poly(I:C), polyinosinic-polycytidylic acid; qPCR, quantitative PCR; Sostdc1, sclerostin Materials and Methods domain–containing-1; Tbx21, T-box protein 21; tNK, transitional NK; UC, University Mice of California; WT, wild-type. Sostdc12/2 mice have been described (23, 27) and were both bred and Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 transferred from Lawrence Livermore National Laboratories (LLNL) to www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801157 2 CELL-EXTRINSIC ROLES OF Sostdc1 ON NK CELLS University of California (UC) Merced to begin an independent breed- or isotype control mAb. Cells were stimulated with PMA and ionomycin at ing colony. C57B6/J (CD45.2+/5.2+) and B6.SJL-Ptprca Pepcb/BoyJ a final concentration of 100 ng/ml and 1000 ng/ml and incubated at 37˚C + + (CD45.1 /5.1 ) and B6.129P2-B2mtm1Unc/J b-2 microglobulin knock- and 5% CO2 conditions for 4 h (28). Protein transport inhibitors brefeldin 2/2 out (KO) (b2m ) mice were obtained from The Jackson Laboratory. A (BioLegend) and monensin (BioLegend) were added 1 h poststimulation Mice of 28–38 wk of age and of both sexes were used. No differences at a final concentration of 13. Cells were washed and cell surface stained between sexes nor mice from LLNL and UC Merced colonies have been for CD27, CD3, CD19, Gr1, NK1.1, CD11b, fixable viability dye, and Fc observed. All mice were housed in conventional housing with autoclaved block. Cells were then fixed and stained intracellularly with anti–IFN-g or feed. Mice were euthanized by carbon dioxide asphyxiation followed isotype mAb control using the BD Cytofix/Cytoperm Kit (BD Biosciences) by cervical dislocation. All animal procedures were approved by the UC according to the manufacturer’s suggested protocol. Cells were analyzed Merced and LLNL Institutional Animal Care and Use Committees. using FCM on the LSR II (Becton Dickinson). Flow cytometry NK cell proinflammatory cytokine assay Isolation of spleen and BM cells were performed and stained for flow Age- and sex-matched Sostdc12/2 and B6 control mice received 200 mgof cytometry (FCM) as described (27). Abs against CD161 (also known as poly(I:C) (Sigma-Aldrich) via injection into the i.p. cavity. Sixteen hours NK1.1, PK136), CD11b (M1/70), CD27 (LG.3A10), CD19 (6D5), CD3 later, splenic cells were harvested, processed to a single-cell suspension (2C11), Gr1-Ly6C/G (Gr1), Ly49G2 (4D11), Ly49I (YL1-90), Ly49H in RPMI1640 media supplemented with 10% FBS 3, 0.09 mM nones- (3D10), Ly49D (eBio4E5), CD45.2 (104), CD45.1 (A20), CD45 (30-F11), sential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml CD4 (GK1.5), CD8 (2.43), Ter119 (TER119), CD107a (1D4B), rat IgG2a penicillin, 100 mg of streptomycin, 0.025 mM BME, and 0.01 M HEPES k isotype control (RTK2758), IFN-g (XMG1.2), rat IgG1 k isotype control buffer, and RBCs were lysed with ammonium-chloride-potassium buffer. (RTK2071), GM-CSF (MP1-22E9), granzyme B (GB11), perforin A total of 5 3 106 splenic cells were additionally stimulated in 96-well (eBioOMAK-D) IL-12/IL-23 p40 (C15.6), TNF-a (TN3-19.12), and flat-bottom plates with 100 ng/ml of PMA and 1000 ng/ml of ionomycin BUV395 streptavidin, eFluor 780 fixable viability dye, eFluor 506 fixable and incubated at 37˚C and 5% CO2 conditions for 5 h. Brefeldin A viability dye were purchased from eBioscience, BioLegend, Miltenyi (BioLegend) was added 1 h poststimulation at a final concentration of Biotec, and BD Biosciences.