Lnk/Sh2b3 Controls the Production and Function of Dendritic Cells and Regulates the Induction of IFN- −γ Producing T Cells This information is current as Taizo Mori, Yukiko Iwasaki, Yoichi Seki, Masanori Iseki, of September 28, 2021. Hiroko Katayama, Kazuhiko Yamamoto, Kiyoshi Takatsu and Satoshi Takaki J Immunol published online 14 July 2014 http://www.jimmunol.org/content/early/2014/07/13/jimmun ol.1303243 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2014/07/14/jimmunol.130324 Material 3.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published July 14, 2014, doi:10.4049/jimmunol.1303243 The Journal of Immunology Lnk/Sh2b3 Controls the Production and Function of Dendritic Cells and Regulates the Induction of IFN-g–Producing T Cells Taizo Mori,*,1 Yukiko Iwasaki,*,†,1 Yoichi Seki,* Masanori Iseki,* Hiroko Katayama,* Kazuhiko Yamamoto,† Kiyoshi Takatsu,‡,x and Satoshi Takaki* Dendritic cells (DCs) are proficient APCs that play crucial roles in the immune responses to various Ags and pathogens and polarize Th cell immune responses. Lnk/SH2B adaptor protein 3 (Sh2b3) is an intracellular adaptor protein that regulates B lymphopoiesis, megakaryopoiesis, and expansion of hematopoietic stem cells by constraining cytokine signals. Recent genome-wide association studies have revealed a link between polymorphism in this adaptor protein and autoimmune diseases, including type 1 diabetes and celiac disease. We found that Lnk/Sh2b3 was also expressed in DCs and investigated its role in the production and function of DC Downloaded from lineage cells. In Lnk2/2 mice, DC numbers were increased in the spleen and lymph nodes, and growth responses of bone marrow– derived DCs to GM-CSF were augmented. Mature DCs from Lnk2/2 mice were hypersensitive and showed enhanced responses to IL-15 and GM-CSF. Compared to normal DCs, Lnk2/2 DCs had enhanced abilities to support the differentiation of IFN-g– producing Th1 cells from naive CD4+ T cells. This was due to their elevated expression of IL-12Rb1 and increased production of IFN-g. Lnk2/2 DCs supported the appearance of IFN-g–producing T cells even under conditions in which normal DCs supported induction of regulatory T cells. These results indicated that Lnk/Sh2b3 plays a regulatory role in the expansion of DCs and might http://www.jimmunol.org/ influence inflammatory immune responses in peripheral lymphoid tissues. The Journal of Immunology, 2014, 193: 000–000. endritic cells (DCs), which were originally named after of c-Kit/CD117 (2, 3), as well as common DC precursors (CDPs) their characteristic morphology, are sparsely but widely defined with a Lin2IL-7Ra2c-KitintFlt3/CD135+ M-CSFR/CD115+ D distributed cells of hematopoietic origin. They are pro- immunophenotype (4), have been shown to be DC progenitors in BM. fessional APCs and have crucial functions in the initiation of innate Both cell types are components of granulocyte–macrophage progen- and adaptive immunity in infection and inflammation and in the itors (GMPs). Commitment to the DC lineage occurs at the MDP induction of tolerance under steady-state conditions (1). The stage, and MDPs give rise to monocytes and to CDPs that exclusively by guest on September 28, 2021 number of DCs in the periphery is maintained by the continuous produce plasmacytoid DCs (pDCs) and pre-DCs, a circulating DC- generation of precursors in the bone marrow (BM) as well as by restricted progenitor that gives rise exclusively to conventional DCs local expansion of resident DCs and their apoptosis. Macrophage (cDCs) in both lymphoid and nonlymphoid tissue DCs (2–10). DC progenitors (MDPs), originally defined as lineage (Lin)2 cells Lnk, recently designated as SH2B adaptor protein 3 (Sh2b3), expressing a CX3CR1 promotor–driven GFP transgene and low levels belongs to an adaptor protein family that includes SH2-B (Sh2b1) and APS (Sh2b2). They share the presence of a homologous N- terminal domain with putative proline-rich protein interaction *Department of Immune Regulation, Research Institute, National Center for Global Health and Medicine, Chiba 272-8516, Japan; †Department of Allergy and Rheuma- motifs, followed by the pleckstrin homology and Src homology 2 tology, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan; (SH2) domains and a conserved C-terminal tyrosine phosphory- ‡ Department of Immunobiology and Pharmacological Genetics, Graduate School of lation site. Lnk/Sh2b3 negatively regulates cytokine and growth Medicine and Pharmaceutical Science for Research, University of Toyama, Toyama 2/2 930-0194, Japan; and xPrefectural Institute for Pharmaceutical Research, Toyama factor signals involved in lymphohematopoiesis (11–14). Lnk 939-0363, Japan mice are characterized by overproduction of B cells and expansion 1T.M. and Y.I. contributed equally to this work. of hematopoietic stem cells (HSCs), as well as overactive mega- Received for publication December 9, 2013. Accepted for publication June 5, 2014. karyocytopoiesis and erythropoiesis, owing to the absence of This work was supported by Japan Society for the Promotion of Science Grants-in- negative regulation of stem cell factor, thrombopoietin, and 2 2 Aid for Scientific Research 22590446 and 25293097 (to S.T.), as well as by National erythropoietin signaling pathways (13–19). Analysis of Lnk / Center for Global Health and Medicine Grants 22-114, 22-205, and 25-107 (to S.T.). HSCs has shown that Lnk/Sh2b3 controls thrombopoietin-induced Address correspondence and reprint requests to Dr. Satoshi Takaki, Department of self-renewal, quiescence, and proliferation of HSCs (20, 21). Ac- Immune Regulation, Research Institute, National Center for Global Health and 2/2 Medicine, 1-7-1 Konodai, Ichikawa, Chiba 272-8516, Japan. E-mail address: cordingly, aged Lnk mice manifest some characteristics of my- [email protected] eloproliferative disease (22). In humans, mutations in the LNK/ The online version of this article contains supplemental material. SH2B3 gene have been found in a portion of myeloproliferative Abbreviations used in this article: ALDH, aldehyde dehydrogenase; BM, bone mar- disease patients (23–25). Additionally, Lnk/Sh2b3 regulates cyto- row; BMDC, bone marrow–derived DC; CD, celiac disease; cDC, conventional DC; skeletal rearrangement. Lnk2/2 megakaryocytes cultivated on CDP, common DC precursor; CMP, common myeloid progenitor; DC, dendritic cell; GMP, granulocyte–macrophage progenitor; HSC, hematopoietic stem cell; Lin, lin- VCAM-1 (a ligand for a4b1 and a4b7 integrins) showed altered cell eage; LN, lymph node; MDP, macrophage DC progenitor; MHC-II, MHC class II; shapes and proplatelet formation compared with wild-type (WT) MLN, mesenteric lymph node; pDC, plasmacytoid DC; RA, retinoic acid; SH2, Src cells (19). We have reported that Lnk/Sh2b3 promotes stabilization homology 2; Sh2b3, SH2B adaptor protein 3; Treg, regulatory T cell; WT, wild-type. of the developed thrombus, mainly through integrin aIIbb3-medi- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 ated actin cytoskeletal reorganization (26). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303243 2 Lnk/Sh2b3 REGULATES DC FUNCTION Recent genome-wide association studies have demonstrated the Flow cytometry presence of a nonsynonymous single nucleotide polymorphism in Cells were incubated with anti–CD16/32 mAb (2.4G2, BD Biosciences) to LNK/SH2B3 as a risk factor for several autoimmune diseases, prevent nonspecific binding of Abs via FcR interactions without CD16/32 including type 1 diabetes and celiac disease (CD) (27–30). CD staining. In general, 1 3 106 cells were incubated on ice for 20 min with is a common intestinal inflammatory disorder resulting from FITC-, PE-, PE-Cy7–, allophycocyanin-, allophycocyanin-Cy7–, and intolerance to gluten (31), and increased production of IL-15 biotin-conjugated mAbs for cell surface staining. The following conju- gated Abs were purchased from eBioscience (San Diego, CA): CD3ε (145- by intestinal epithelial cells has been reported in CD patients. 2C11), CD11b (M1/70), CD16/32 (93) , CD19 (1D3), CD34 (RAM34), Activation by IL-15 and the killing of intestinal epithelial cells CD40 (1C10), CD86 (GL1), CD115 (M-CSFR) (AFS98), CD103 (2E8), expressing stress- and inflammation-induced nonclassical MHC CD127 (IL-7Ra) (A7R34), Foxp3 (FJK-165), MHC class II (MHC-II; M5/ class I molecules has been suggested as an etiologic event (32). 114.15.2), NK1.1 (PK136), Sca-1 (D7), SIRP-1a (PB4), and TER119 (TER119). The following conjugated Abs were purchased from Bio- The functions of Lnk/Sh2b3 that enhance the risk for autoimmune Legend: B220 (RA3-6B2), CCR9 (242503), CD4 (RM4-5), CD8a (53- inflammation, however, have been largely unrevealed. 6.7), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL14), DCs show high motility and morphological diversity. They CD207 (4C7), Gr-1 (RB6-8C5), IFN-g (XMG1.2), Ly-6C (HK1.4), capture Ags in the periphery and migrate to lymph nodes (LNs). PDCA-1 (927), and Siglec-H (551). The following conjugated Abs were They form protruding dendrites and extending lamellipodia in purchased from BD Biosciences: c-Kit (2B8), CD11c (HL3), CD135 (Flt3), CD212 (IL-12Rb1) (114), phospho-STAT4 (38/p-Stat4), phospho- response to various stimuli. Considering those characteristics of STAT5 (47/Stat5 [pY694]), and I-Ab (AF6-120.1).