Producing T Cells

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 26, 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

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 26, 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 conjugated 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). Aldehyde dehydroge- DCs, regulation of cytoskeletal rearrangement as well as cytokine nase (ALDH) activity in individual cells was estimated using Aldefluor responses might play important roles in their development and staining kits (StemCell Technologies, Vancouver, BC, Canada), as de- functions. In this study, we demonstrate that Lnk/Sh2b3 is ex- scribed previously (33). Flow cytometry analysis was performed with Downloaded from a FACSCanto II (BD Biosciences). pressed in DCs and regulates the production and functions of DC lineage cells. In Lnk-deficient mice, the number of DCs in the Cell culture and ELISA spleen and peripheral LNs was increased. Lnk-deficient BM- CD11c+ BMDCs (1 3 105 cells) were replated in 100 ml medium in 96- derived DCs (BMDCs) proliferated better than did normal cells well plates and stimulated with various concentrations of GM-CSF. Cells in response to GM-CSF. In the mature DC fraction, GM-CSF– were pulse-labeled with [3H]thymidine (0.2 mCi/well) during 16 h of the 3 mediated induction of enzymes and surface molecules was en- 64-h culture period and incorporated [ H]thymidine was measured. Puri- http://www.jimmunol.org/ 4 hanced. Moreover, IL-15–mediated induction of IL-12Rb1 and fied MLN DCs (3 3 10 ) were stimulated for 3 d with 1 ng/ml GM-CSF, 1 IFN-g production were augmented in Lnk2/2 DCs. They sup- ng/ml IL-12 (R&D Systems, Minneapolis, MN), 1 ng/ml IL-15 (Pepro- + + Tech), or combined with both IL-12 and IL-15. Cultured cell supernatants ported IFN-g–producing CD4 T and CD8 T cells significantly were collected and evaluated for IFN-g with a mouse ELISA MAX more efficiently than did normal DCs. Thus, a lack of Lnk/Sh2b3- standard kit (BioLegend). mediated regulation in DCs might lead to inflammation or pro- Cell signaling tective immunity mediated by IFN-g–producing T cells. Purified splenic CD11c+ DCs were treated with lysis buffer (2% Triton X- 100, 150 mM NaCl, 50 mM Tris-HCl, 10 mM sodium fluoride, 1 mM Materials and Methods 3 sodium vanadate) and 1 protease inhibitor cocktail (Roche, Basel, by guest on September 26, 2021 Mice Switzerland). Total lysates or the supernatants precipitated with anti–Lnk/ Sh2b3 Ab were separated on 7.5% SDS-PAGE and transferred onto C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan) or CLEA polyvinylidene difluoride membranes. Anti–Lnk/Sh2b3 Abs were gener- Japan (Tokyo, Japan). C57BL/6 mice congenic for the Ly5 locus (CD45.1) 2 2 2 2 2 2 ated as described before (12). For signaling in BMDCs, cells purified and as well as Rag2 / , Lnk / , and Lnk / CD45.1 mice (13, 21) were 2 2 starved in medium without cytokines for 6–8 h were stimulated with 1 or maintained under specific pathogen-free conditions. Rag1 / OT-I and 2 2 10 ng/ml GM-CSF for 5 or 15 min and lysed. Total lysates derived from Rag1 / OT-II mice were purchased from Taconic and bred under specific 1.5 3 106 BMDCs were separated on a 5–20% gradient SDS-PAGE. The pathogen-free conditions. Experiments were performed with age- and sex- following Abs were obtained from Cell Signaling Technology (Danvers, matched mice at 6–14 wk of age. All mice were handled in accordance MA): anti-STAT5, anti–phospho-STAT5 (Tyr694), anti-ERK1/2, anti– with the Guidelines for Animal Experiments of the Research Institute, phospho-ERK1/2 (Thr202/Tyr204), and anti–phospho-JAK2 (Tyr1007/1008). National Center for Global Health and Medicine. For mature DCs, purified cells were stimulated with 0.1 ng/ml IL-12 or 1 Preparation of cells ng/ml IL-15 for 20 min. Cells were fixed and permeabilized with Phosflow buffer (BD Biosciences) and stained with anti–phospho-STAT4 or anti– BMDCs were generated using standard protocols. Femurs and tibias were phospho-STAT5. harvested and single-cell suspensions were prepared from flushed BM cells. Cells were then cultured in RPMI 1640 supplemented with 10% FBS, 50 Differentiation of T cells by coculturing with DCs mM 2-ME, 1% nonessential amino acids (Life Technologies, Carlsbad, Purified naive T cells (5 3 104) and MLN DCs (2 3 104) were cocultivated CA), antibiotics, and either 10 ng/ml recombinant murine GM-CSF for 3 d on plate-bound anti-CD3ε (1 mg/ml) (eBioscience). Designated (PeproTech, London, U.K.) or 100 ng/ml recombinant human Flt3L recombinant cytokines (listed below) were added to the cultures: TGF-b1 (PeproTech). Medium was removed and replaced with fresh medium (0.5 ng/ml) (R&D Systems), IL-15 (0.1, 0.25, or 1 ng/ml), and GM-CSF containing GM-CSF on days 2 and 4. For cultures supplemented with (0.1, 0.25 or 1 ng/ml). Three days after the initial stimulation, cells were Flt3L, medium was not changed. At day 6 or 7, BMDCs were harvested restimulated for 4 h with PMA (50 ng/ml) plus ionomycin (500 ng/ml). and used for experiments. The cells were fixed and permeabilized with the Foxp3 fixation/ For DC isolation, mesenteric LNs (MLNs) or spleens were collected and permeabilization kit (eBioscience) and were used for detection of nu- digested with 100 U/ml collagenase type D (Roche Applied Science, clear Foxp3 and IFN-g cytokines. Cultured cell supernatants were col- Mannheim, Germany) for 30 min at 37˚C. DCs were purified by positive lected and evaluated for IL-12/23 p40 and IFN-g with a mouse ELISA immunomagnetic selection or by sorting with the FACSAria (BD Bio- MAX standard kit (BioLegend). For DC-free cultures, 5 3 105 naive CD4+ sciences, Franklin Lakes, NJ). Immunomagnetic selection was performed T cells were cultured for 3 d with plate-bound anti-CD3ε (2 mg/ml) using biotinylated anti–CD11c Ab (N418) (BioLegend, San Diego, CA) (eBioscience) and soluble anti-CD28 (2 mg/ml) (eBioscience) in the and anti-biotin–conjugated beads and the MACS system (Miltenyi Biotec, presence or absence of TGF-b1, IL-12 (PeproTech), IFN-g (PeproTech), or Gladbach, Germany) according to the manufacturers’ recommendations. retinoic acid (RA) (Sigma-Aldrich, St. Louis, MO). Purification yielded up to 80% CD11c+ cells. DC sorting with the FAC- 2 2 + SAria was performed to purify the CD3 CD19 CD11c population. Pu- Quantitative RT-PCR analysis rification yielded up to 95% CD11c+ cells. For T cell isolation, spleens were collected and disrupted through a 70-mm cell strainer. Naive T cells Total RNA was extracted from purified DCs with the RNeasy kit (Qiagen, were purified by using the FACSAria to sort for the CD4+CD62L+CD44lo Hilden, Germany). cDNA was synthesized with Superscript III (Life population. Technologies) according to the manufacturer’s instructions. TaqMan real- The Journal of Immunology 3 time quantitative RT-PCR was used to detect IL-12R mRNA. TaqMan sisting of acetone/dibutylphthalate (1:1) was applied to the stripped skin probes for IL-12Rb1(Il12rb1) (Mm00434189_m1), IL-12Rb2(Il12rb2) region. Mice were killed and axillary LNs were collected for analysis 48 h (Mm00434200_m1) and GAPDH (Mm99999915_g1) were obtained from later. Life Technologies. BrdU incorporation assay in vivo BM transplantation Mice were i.p. injected with 200 ml 10 mg/ml BrdU solution (Sigma- Femoral BM cells were washed with PBS and counted. C57BL/6 (CD45.1) Aldrich). Incorporation of BrdU into cells in BM and spleen was ana- nucleated BM cells (5 3 106/recipient mouse) and CD45.2 Lnk2/2 BM lyzed 2 or 24 h after injection by using a BrdU flow kit (BD Biosciences). cells (2.5-5 3 105) were i.v. transfused into lethally irradiated (9.5 Gy) C57BL/6 (CD45.1) mice. Six weeks later, PBLs were collected from the Statistical analysis recipient mice and chimerism was confirmed by the expression of CD45.1 Differences between groups were analyzed for statistical significance with or CD45.2. a Student t test and considered significant when p values were ,0.05. Immunohistochemistry 2/2 Results Freshly dissected spleens from WT C57BL/6J mice or Lnk mice were 2/2 embedded in 4% carboxymethyl cellulose and frozen in n-hexane chilled DCs numbers were increased in lymphoid organs in Lnk in dry ice. Cryostat sections of frozen tissues were fixed with acetone, air- mice dried, and stained with FITC-conjugated anti-CD3ε and PE-conjugated anti–CD11c Abs. Confocal microscopic analysis was performed with an Polymorphisms in the LNK/SH2B3 gene locus are reportedly as- Olympus FV-500 confocal microscope. sociated with several autoimmune diseases (27–30). Because DCs greatly affect immune responses and Lnk/Sh2b3 is expressed in DC migration assay various hematopoietic cells and progenitors, we investigated the Downloaded from Mice were anesthetized and hair was removed from each chest. A total expression of Lnk/Sh2b3 in DCs. We purified mature CD11c+ volume of 400 ml 2% FITC isomer-I (Sigma-Aldrich) in a vehicle con- http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Increased numbers of DCs and their precursors in mice lacking Lnk/Sh2b3. (A) Lnk/Sh2b3 protein expression in DCs. Lysates prepared from purified splenic DCs or from BMDCs were subjected to immunoprecipitation and immunoblotting using anti–Lnk/Sh2b3 Ab. The expression level did not change during maturation induced by stimulation FIGURE 2. GM-CSF–dependent growth and signaling responses were with LPS (1 mg/ml) for 24 h. (B) The absolute numbers of CD11c+ DCs in enhanced in Lnk2/2 BMDCs. (A) Relative cell number of BMDCs gen- spleens (n = 7) and axillary LNs (n = 5) obtained from WT (open symbols) erated from WT and Lnk2/2 mice by cultivating the same numbers of BM or Lnk-deficient (KO; filled symbols) mice. (C) BM cells were analyzed for cells with 10 ng/ml GM-CSF for 4 d (n = 4) or with 100 ng/ml Flt3L for the expression of c-Kit lineage markers (CD3ε, TER119, B220, CD19, 7d(n = 3). (B) BM cells from WT (open symbols) or Lnk2/2 (KO; filled NK1.1, CD11c, I-Ab, Gr-1, CD11b, Sca-1, and IL-7Ra)(left panels). symbols) mice were cultured in the presence of GM-CSF for 6 d, har- Then, CMPs (Lin2Sca-12IL-7Ra2c-Kit+CD34+CD16/322) and GMPs vested, and CD11c+ DC lineage cells were purified using anti-CD11c (Lin2Sca-12IL-7Ra2c-Kit+CD34+CD16/32+) were separated by the ex- magnetic beads. Purified CD11c+ cells were reseeded in the presence of pression pattern of CD34 and CD16/32 (right panels). Numbers represent various concentrations of GM-CSF and their growth responses were the percentages of cells falling in each box. (D) Flow cytometric analysis assessed by [3H]thymidine incorporation. Data are means 6 SD of three of Lin2Sca-12IL-7Ra2 BM cells for MDPs (c-Kit+Flt3+)(left) and CDPs independent experiments. (C) Purified CD11c+ BMDCs were starved (c-KitintFlt3+M-CSFR+)(right). Numbers represent the percentages of overnight and then stimulated with GM-CSF at the indicated times and cells falling into each box. (E) The absolute numbers of CMPs, GMPs, concentrations. Cells were lysed and the lysates were subjected to immu- MDPs, and CDPs per femur obtained from WT (open symbols) or KO noblotting using anti–phosphorylated JAK2 (pJAK2), anti–phosphorylated (filled symbols) mice (n = 5–10). Horizontal bars in the plots represent STAT5 (pSTAT5), anti-phosphorylated ERK1/2 (pERK1/2), anti-STAT5, mean values of indicated groups. *p , 0.05, **p , 0.01. and anti-ERK1/2. *p , 0.05. 4 Lnk/Sh2b3 REGULATES DC FUNCTION

DCs from spleens (.95% purity) of WT or Lnk2/2 mice, and the DC precursors in BM of Lnk2/2 mice cell lysates were subjected to immunoprecipitation and immuno- To better understand how DC numbers increased in the absence of blotting using anti–Lnk/Sh2b3 Abs. The 68-kDa band corre- Lnk/Sh2b3, we examined DC precursors in BM. We studied the sponding to the Lnk/Sh2b3 protein was detected in lysates from 2/2 expression of Lnk/Sh2b3 in DC precursors by the use of the Lnk- WT splenic DCs, but not from the Lnk splenic DCs (Fig. 1A, Venus knockin mice, in which a part of the Lnk gene was replaced left). To rule out the possibility of contamination of a small by a modified GFP Venus and the Lnk gene expression was fraction of B cells that highly express Lnk/Sh2b3 in the spleen monitored by Venus expression on flow cytometry (35). The Lnk (13), we prepared BMDCs by culturing BM cells in the presence expression levels were gradually upregulated as DC precursors of GM-CSF, and the lysates were subjected to immunoblotting. matured from Lin2Sca-12IL-7Ra2c-Kit+CD34+CD16/322 com- We confirmed the expression of Lnk/Sh2b3 protein in BMDCs, mon myeloid progenitors (CMPs) in BM, and they further in- and the expression level was not changed during LPS-induced creased in cDCs in spleen (Supplemental Fig. 2A). We evaluated maturation (Fig. 1A, right). the number of Lin2Sca-12IL-7Ra2c-Kit+CD34+CD16/32+ GMPs We next assessed DC production in Lnk2/2 mice. The total live + as well as CMPs, from which GMPs branch. The percentage of cells and absolute number of CD11c DCs were increased in the CMPs in Lnk2/2 BM was higher than that in WT BM. There were spleens and peripheral LNs of Lnk-deficient mice compared with more CMPs in BM of Lnk2/2 mice compared with that of WT normal mice (Fig. 1B, Supplemental Fig. 1A, 1B). The expres- mice. There was also an increased tendency in GMPs, although sion levels of costimulatory molecules such as CD40 and CD86 + +/+ 2/2 this did not achieve statistical significance. (Fig. 1C, 1E). GMPs on CD11c DCs were similar in Lnk and Lnk mice 2 2 2 + + 2 2 differentiate into Lin Sca-1 IL-7Ra c-Kit Flt3 MDPs and

/ Downloaded from (Supplemental Fig. 1C). Lnk mice showed mild splenomegaly Lin2Sca-12IL-7Ra2c-KitintFlt3+M-CSFR+ CDPs that give rise to because of enhanced lymphohematopoiesis (13, 15). To examine 2/2 cDCs and pDCs (4). We evaluated MDP and CDP fractions and whether increased DCs in the spleen of Lnk mice accumulated found that the number of MDPs increased in Lnk2/2 mice (Fig. within specific regions, we undertook an immunohistochemical + 1D, 1E). Thus, DC lineage precursor cells in BM tended to in- analysis. Splenic CD11c DCs resided diffusely in the marginal crease in the Lnk-deficient strain compared with normal DC de- zone border (34), and the pattern was not largely disturbed in 2/2 velopment. Lnk mice (Supplemental Fig. 1D). We asked whether the en- http://www.jimmunol.org/ larged size of the spleen or the increased number of lymphocytes Enhanced proliferation of BMDCs upon stimulation with in the absence of Lnk/Sh2b3 might affect the survival or pro- GM-CSF duction of DC lineage cells. We generated lymphocyte-deficient GM-CSF and Flt3L support development of DCs in the steady-state Lnk2/2Rag22/2 mice and confirmed that the mice also had in- as well as the inflammatory state. We examined the proliferation of creased numbers of DCs in the spleen (Supplemental Fig. 1E). BMDCs with GM-CSF as well as Flt3L. We found that more Thus, the increased number of DCs was independent of lymphoid CD11c+ DCs were generated from Lnk-deficient BM cells than lineage cells. from WT BM (Fig. 2A). The difference in the cumulative pro- by guest on September 26, 2021

FIGURE 3. Primary DCs showed increased responsiveness to GM-CSF. (A) Aldehyde dehydrogenase activity was induced by GM-CSF in mature DCs from spleen and MLNs. Freshly prepared splenic and MLN cells were incubated with Aldefluor and stained for CD11c expression. Shown are representative plots of CD11c+ cells from spleen (top panels) and MLNs (bottom panels). Numbers adjacent to the gates indicate the percentages of cells in each population. (B) The numbers of CD11c+Aldefluor+ cells in spleen (n = 7) or MLNs (n = 6) from WT (open symbols) or Lnk2/2 (KO; filled symbols) mice. (C) Splenic CD11c+ DCs were isolated and incubated with various concentrations of GM-CSF for 12 h. Aldefluor+ cells were assessed by flow cytometry as in (A) to determine the percentage in culture. Data are means 6 SD of six independent experiments. (D) The expression of CD8a and CD103 in the CD11c+ cell population from spleen and MLNs. (E) The percentages of CD11c+CD8a+CD103+ cells in spleen and MLNs (n = 5) (WT, open symbols; KO, filled symbols). (F) Splenic cells were cultured in the presence or absence of 1 ng/ml GM-CSF for 18 h. Cells were stained and representative plots for the expression of CD8a and CD103 in live CD11c+ cells are shown (n = 3). Horizontal bars in (B)and(E) represent means of indicated groups. *p , 0.05, **p , 0.01. The Journal of Immunology 5 liferation curve became evident after day 7, when 60–70% of were largely normal in the skin of Lnk2/2 mice (Supplemental cultured BM cells became CD11c+. This result could reflect the Fig. 3C). These results suggested that Ag capture and migration increased proliferative capacity of DC precursor cells or altered ability was not significantly compromised in Lnk2/2 DCs. growth responses to cytokines. To evaluate the cytokine respon- Next, we examined whether responses to GM-CSF were also siveness of committed BMDCs, we harvested and purified affected in mature DCs by Lnk deficiency. GM-CSF reportedly CD11c+ BMDCs after 7 d of cultivation. The cells were reseeded induces ALDH activity essential for RA production in mature in the presence of various concentrations of GM-CSF, and we DCs. ALDH activity can be monitored by use of Aldefluor dye investigated their proliferation by measuring thymidine uptake. (33). The number of Aldefluor-derived fluorescence-positive Lnk2/2 CD11c+ BMDCs showed an augmented proliferative re- (Aldefluor+) DCs, that is, freshly isolated DCs carrying ALDH sponse to GM-CSF (Fig. 2B). The phosphorylation of JAK2 activity, was increased in MLNs and slightly elevated in spleens (necessary for all of the biological functions after activation of from animals lacking Lnk/Sh2b3 (Fig. 3A, 3B). We examined the GM-CSFR) (36) was enhanced and prolonged in BMDCs in the induction of ALDH activity in splenic DCs following cultivation absence of Lnk/Sh2b3 (Fig. 2C). Phosphorylation of STAT5 in- with GM-CSF. Positive Aldefluor staining of splenic DCs was duced by JAK2, as well as phosphorylation of ERK1/2, was also more strongly induced by GM-CSF in cells from Lnk2/2 animals augmented and prolonged in Lnk2/2 CD11c+ BMDCs. Thus, in than those from WT DCs (Fig. 3C). developing DCs, responses induced by GM-CSF were augmented In spleen, cDCs are divided into two populations by their ex- and resulted in enhanced proliferation in the absence of Lnk/ pression of CD11b and CD8a, namely CD11b+CD8a2 and Sh2b3. We assessed increased proliferation of DC progenitors CD11b2CD8a+ DCs. The ratio of CD11b+CD8a2 to CD11b2 in vivo by BrdU incorporation assay. Most DC progenitors in BM CD8a+ DCs was not skewed by Lnk deficiency. However, the size Downloaded from rapidly incorporated BrdU, and BrdU+ fractions in splenic pre-cDCs of the CD103+ fraction in CD8a+ DCs as well as the absolute and cDCs were increased in Lnk2/2 mice compared with WT mice number were significantly increased in Lnk2/2 spleens (Fig. 3D, within 1 d after bolus injection of BrdU (Supplemental Fig. 2B). 3E). CD103+ DCs, which usually reside in nonlymphoid tissues, are known to develop and expand in response to GM-CSF and Peripheral mature DCs showed enhanced responsiveness to have the ability to activate CD8+ T cells into IFN-g–producing GM-CSF in the absence of Lnk/Sh2b3 cells (39, 40). Overnight cultivation of splenocytes demonstrated http://www.jimmunol.org/ We inquired whether any of the functions of DCs were altered in the that expression of CD103 on peripheral CD8a+ DCs was main- absence of Lnk/Sh2b3, and we first examined Ag capture and tained by the presence of GM-CSF (Fig. 3F). These results sug- migration of DCs by painting FITC on skin and measured DCs gested that GM-CSF–induced responses in DCs were exaggerated carrying FITC in draining LNs (37). Although the ratio of FITC- in Lnk2/2 mice. + 2/2 bearing CD11c DCs seemed to be slightly reduced in Lnk 2/2 mice, reflecting the increased total DC number (Supplemental Lnk DCs were hyperresponsive to IL-15 and supported Fig. 3A), the absolute numbers of migrating FITC+MHC-II+ more IFN-g–producing Th1 cells DCs in draining LNs were comparable in Lnk+/+ and Lnk2/2 mice DCs play important roles in the priming and differentiation of (Supplemental Fig. 3B). Dermal MHC-IIhigh DC subpopulations effector T cells in immune responses. We next examined whether by guest on September 26, 2021 determined by the expression of CD207, CD11b, and CD103 (38) dysfunction of Lnk/Sh2b3 in DCs might affect their ability to

FIGURE 4. Cocultivation of naive CD4+ T cells with Lnk2/2 DCs in the presence of GM-CSF or IL- 15 facilitated the induction of IFN-g–producing Th1 cells. (A) CD45.2+ naive CD4+ T cells were cultured with DCs isolated from CD45.1+ WT or Lnk2/2 (KO) MLNs in the presence of plate-coated anti- CD3ε alone or in combination with GM-CSF (1 ng/ ml) or IL-15 (1 ng/ml) for 3 d. Representative plots and histograms gated on CD45.2+ T cells and the percentages of Foxp3+ or IFN-g+ cells are shown. Numbers in histograms are the median fluorescence intensities (MFI) of IFN-g staining obtained by coculturing with WT or Lnk2/2 (KO) DCs. Similar results were obtained from two additional experiments. (B) IFN-g+ CD4+ T cells induced by increasing doses of GM-CSF or IL-15 in the presence of WT DCs (WT; open circles) or Lnk2/2 DCs (KO; filled circles). (C) Relative MFI ratio of IFN-g staining in CD4+ T cells. (D and E) The amounts of IFN-g (D) and IL-12/23 p40 (E) in the coculture supernatants were analyzed by ELISA. Data present the means 6 SD of three independent experiments (B–E). *p , 0.05, **p , 0.01. 6 Lnk/Sh2b3 REGULATES DC FUNCTION support differentiation of Th cells from naive CD4+ T cells. We isolated CD11chi DCs from MLNs of Lnk2/2 or WT mice and cultured them with naive splenic CD4+ T cells purified from WT mice allotypically distinguished from DCs by the expression of CD45.1 or CD45.2. After 3 d of cultivation, expression of IFN-g and Foxp3 in T cells was analyzed by intracellular staining. In the presence of GM-CSF, cocultivating with Lnk2/2 DCs generated more IFN-g–producing T cells compared with cocultivation with WT DCs (Fig. 4A–D). Involvement of IL-15 in the pathogenesis of CD has been suggested (32). IL-15 induces several responses critical for T cell priming in DCs (41, 42). In the presence of IL- 15, Lnk2/2 DCs supported more IFN-g–producing cells compared with normal DCs (Fig. 4A–D). The induction of IFN-g–producing CD4+ T cells was increased in a dose-dependent manner for both GM-CSF and IL-15 (Fig. 4B, 4C). Those two cytokines had an additive effect for the induction of IFN-g+CD4+ T cells under these coculture conditions (data not shown). IL-12, produced from DCs and a strong inducer of Th1 responses, was not increased in the supernatants (Fig. 4E). Downloaded from

Lnk2/2 DCs upregulated IL-12Rb expression and produced more IFN-g We tried to clarify the molecular mechanisms by which Lnk2/2 DCs enhanced their capacity to support IFN-g+CD4+ T cells.

We screened cytokines that were involved in Th1 cell induction http://www.jimmunol.org/ when DCs were cultured alone. Lnk2/2 DCs tended to produce more IFN-g compared with normal DCs (Fig. 5A). Consistent with the amounts of IL-12 in coculture supernatants described above (Fig. 4E), IL-12 production from Lnk2/2 DCs and WT DCs were comparable (data not shown). IFN-g production was significantly increased by the stimulation of IL-15 from Lnk2/2 DCs but not from normal DCs (Fig. 5A). IL-12 is known to induce IFN-g production in DCs. We found that IL-12–induced IFN-g production was exaggerated in the absence of Lnk/Sh2b3 by guest on September 26, 2021 (Fig. 5A). Addition of IL-15 together with IL-12 strongly induced IFN-g production by normal DCs, and it completely masked the difference between Lnk2/2 and normal DCs. IL-15 is required for IL-12Rb1 expression by DCs (41). Thus, the 2 2 results indicated that the increased responsiveness of Lnk2/2 FIGURE 5. Enhanced responsiveness of Lnk / DCs to IL-15 resulted DCs to IL-15 might be responsible for their increased IFN-g in upregulation of IL-12Rb1 expression and increased secretion of IFN-g. A 2/2 production, particularly if mediated by autocrine IL-12. DCs ( ) DCs isolated from MLNs of WT or Lnk (KO) mice were cultured freshly isolated from MLNs of Lnk2/2 mice expressed more IL- with GM-CSF, IL-12, IL-15, or a combination of IL-12 and IL-15 for 2 d. 2 2 Cell supernatants were evaluated for IFN-g by ELISA. (B) DCs purified 12Rb1 (Fig. 5B). After starvation in culture, Lnk / DCs ten- from MLNs by cell sorting were lysed immediately (Fresh) or incubated ded to show increased phosphorylation of STAT5, although this for 24 h with or without IL-15. The expression of Il12rb1 was measured by did not achieve statistical significance (Fig. 5C, 5D). They quantitative PCR. (C) DCs from MLNs of WT (CD45.1+; filled gray) or expressed more IL-12Rb1 in response to IL-15 (Fig. 5B). Ac- Lnk2/2 (CD45.2+; black line) mice were stimulated by IL-15 or IL-12 in cordingly, IL-12–mediated STAT4 phosphorylation was ob- the same tubes and analyzed for phosphorylation of STAT5 (pSTAT5) or 2 2 served in a higher proportion of Lnk / DCsthaninnormal STAT4 (pSTAT4). Numbers in histograms are the median fluorescence 2 2 DCs (Fig. 5C, 5D). intensity (MFI) obtained from WT or Lnk / (KO) cells. Similar results were obtained from two additional experiments. (D) Relative MFI ratio of 2/2 Dysregulated functions of Lnk DCs were cell intrinsic pSTAT5 and pSTAT4 staining in unstimulated or stimulated cells. (E and F) + To confirm that the altered characteristics of Lnk2/2 DCs were cell BM chimeric mice were prepared by transferring CD45.1 WT BM cells mixed with CD45.2+ Lnk2/2 (KO) BM cells into lethally irradiated intrinsic and not a consequence of Lnk deficiency in the micro- 2 2 CD45.1+ recipient B6 mice. CD45.1+ WT and CD45.2+ Lnk / DCs were environment of the lymphoid organ, we generated BM chimeric isolated from MLNs of the chimeric mice and the responses to IL-12 and mice in which nearly half of the myeloid lineage cells were de- IL-15 were evaluated for IFN-g production (E) or the expression of Il12rb1 2/2 + rived from Lnk CD45.2 precursors and the remainder were (F)asin(A) and (B). Data are the means 6 SD of three independent 2/2 derived from normal CD45.1+ precursors. We found that Lnk - experiments (A, B, and D–F). *p , 0.05, **p , 0.01. derived CD45.2+CD8a+CD103+ cDCs are increased even in the spleen of BM chimeric mice whereas the percentage of CD4+ cDCs, CD8a+ cDCs, and pDCs is not largely changed in spleen chimeric mice and IFN-g production was examined (Fig. 5E). In and MLNs between CD45.1+ and CD45.2+ (Supplemental Fig. Lnk2/2 DCs, more Il12rb1 transcripts accumulated in the pres- 2C). This result indicated that the intrinsic effect of Lnk defi- ence of low concentrations of IL-15 than in normal DCs that had ciency induced CD8a+CD103+ cDCs development. Lnk2/2 been simultaneously isolated from the same chimeric mice (Fig. CD45.2+ DCs and normal CD45.1+ DCs were purified from 5F). Thus, responses mediated by IL-15 in DCs were enhanced The Journal of Immunology 7 in the absence of Lnk/Sh2b3, resulting in an increased proportion (Fig. 6C). Thus, the ability of DCs to support Th1 cells or Tregs of Il12rb1-expressing DCs. The enhanced responses of Lnk2/2 was altered by Lnk deficiency in response to inflammatory DCs were due to cell-intrinsic changes and did not depend on cytokines GM-CSF and IL-15. Consequently, the deficiency led environmental changes. to enhanced expression of IL-12Rb1aswellasaugmented production of both RA and IFN-g. Lnk2/2 DCs supported the appearance of IFN-g+CD4+ T cells even in the presence of TGF-b Discussion In the presence of TGF-b, naive CD4+ T cells differentiate into DC homeostasis is dependent on the rate at which DC progenitors Foxp3+ peripheral regulatory T cells (Tregs). RA facilitates TGF- enter the blood from BM, cell division, and apoptosis regulated by b–induced Treg production; however, it also has coadjuvant cytokines (43, 44). Lnk/Sh2b3 expressed in DCs constrained GM- properties that induce Th1 immunity in the presence of IL-15 (42). CSF signaling and controlled the number of DCs that differenti- Because Lnk2/2 DCs showed better responsiveness to GM-CSF in ated from progenitor cells. The number of CD11cintCD45RAlo 2 2 2 the induction of ALDH activity required for RA production and CD43(Ly48)intSIRP-aintCD4 CD8 MHC-II splenic pre-cDCs 2 2 showed better responsiveness to IL-15 in Th1 T cell induction, we (45) was not increased significantly in Lnk / mice (data not asked whether induction of Foxp3+ cells might be altered by co- shown). Pre-cDCs reportedly expand primarily in the BM and cultivation with Lnk2/2 DCs. As reported, addition of TGF-b to differentiate into cDCs in lymphoid tissues via cell division culture medium resulted in induction of Foxp3+ T cells (Fig. 6A). (8). Interestingly, Lnk deficiency did not affect proliferation in Although IFN-g+ cells barely differentiated in cocultivation with the periphery. The transition from pre-cDCs to cDCs might be + normal DCs, some IFN-g cells differentiated during coculture prompt, or alternatively the cell division of pre-cDCs might not be Downloaded from with Lnk2/2 DCs (Fig. 6A). Addition of IL-15 increased IFN-g+ strongly affected by DC precursor trophic cytokines. cell production in a dose-dependent manner, and induction of Our previous studies indicated that Lnk/Sh2b3 restricted activa- IFN-g+Foxp3+ cellsoccurredonlyinthepresenceofLnk2/2 tion of ERK1/2 in thrombopoietin-dependent signaling pathways DCs (Fig. 6A, 6B). GM-CSF alone showed only marginal effects. of megakaryocytes (19) and in Kit ligand–activated MC9 mast However, the combination of GM-CSF and IL-15 supported ap- cell lines (14). Tong et al. showed that Lnk/Sh2b3 blocked + + + parent differentiation of IFN-g cells and IFN-g Foxp3 cells in erythropoietin-mediated erythropoiesis by negatively regulating http://www.jimmunol.org/ coculture with Lnk2/2 DCs but not with normal DCs (Fig. 6A). JAK2, STAT5, Akt, and ERK1/2 activation (17). In DCs, we As shown above, Lnk2/2 DCs produced more IFN-g but not demonstrated enhanced activation of GM-CSF–mediated signaling IL-12 compared with normal DCs in response to IL-15. To pathways, JAK2, STAT5, and ERK1/2 MAPK in the absence of confirm the effects of IFN-g overproduced by Lnk2/2 DCs in Lnk/Sh2b3. We also found that ALDH activity induced by GM-CSF Treg differentiation, we directly added IFN-g or IL-12 to CD4+ in DCs was enhanced by Lnk deficiency. ALDH is the enzyme that T cells in the presence of TGF-b,orTGF-b in combination with catalyzes retinaldehyde to RA and plays an important role in the RA. As reported (42), IL-12 supported the appearance of IFN-g+ pathway of RA synthesis from vitamin A (retinol). Splenic DCs Foxp32 cells in the presence of TGF-b and RA (Fig. 6C), al- treated with GM-CSF facilitate TGF-b–dependent differentiation of though the reduction of Foxp3+ cells was not evident under our naive T cells to Foxp3+ T cells (Fig. 6A, WT DCs) (33). However, by guest on September 26, 2021 culture conditions. In the presence of TGF-b, IL-12 induced DC responses to IL-15 in addition to those against GM-CSF were IFN-g production both in Foxp32 and Foxp3+ cells. Essentially also augmented by the absence of Lnk/Sh2b3. The responses to 2 2 the same results were observed following the addition of IFN-g those cytokines made Lnk / DCs strongly support the appearance

FIGURE 6. Lnk2/2 DCs induced the IFN-g+ Th1 phenotype even in the presence of TGF-b. (A)CD45.2+ naive CD4+ T cells were cultured with DCs isolated from MLNs of WT or Lnk2/2 (KO) mice on plates coated with anti-CD3ε in combination with TGF-b (0.5 ng/ml), GM-CSF (1 ng/ml), and IL-15 (1 ng/ml) as indicated for 3 d. Representative plots gated on CD45.2+ T cells show the percentages of Foxp3+,IFN-g+, and Foxp3+IFN-g+ cells. Similar results were obtained from two additional experiments. (B) IFN-g+ T cells were induced by increasing doses of IL-15 in the presence of Lnk2/2 DCs (d)but not WT DCs (s) in combination with TGF-b (0.5 ng/ml) and GM-CSF (1 ng/ml). Data are the means 6 SD of three independent experiments. (C)NaiveCD4+ T cells were cultured alone with anti-CD3ε,anti-CD28,andTGF-b (2 ng/ml) in combination with RA (10 nM), IL-12 (1 ng/ml), or IFN-g (10 ng/ml) for 3 d. Representative plots and the percentages of Foxp3+,IFN-g+,and Foxp3+IFN-g+ cells are shown. 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The receptor tyrosine kinase enhanced production of IL-12 from DCs, but we did detect in- Flt3 is required for dendritic cell development in peripheral lymphoid tissues. creased production of IFN-g as a consequence of elevated expression Nat. Immunol. 9: 676–683. 4. Onai, N., A. Obata-Onai, M. A. Schmid, T. Ohteki, D. Jarrossay, and of IL-12Rb1 and autocrine production of IL-12. In the presence of M. G. Manz. 2007. Identification of clonogenic common Flt3+M-CSFR+ plas- TGF-b, IFN-g as well as IL-12 induced IFN-g production by macytoid and conventional dendritic cell progenitors in mouse bone marrow. Foxp3+CD4+ T cells that had been derived from naive CD4+ cells Nat. Immunol. 8: 1207–1216. 5. Naik, S. H., P. Sathe, H.-Y. Park, D. Metcalf, A. I. Proietto, A. Dakic, S. Carotta, (Fig. 6C). The addition of RA reduced IFN-g production by most M. O’Keeffe, M. Bahlo, A. Papenfuss, et al. 2007. 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Med. 206: 3115–3130. or decreased functionality of Lnk/Sh2b3 in DCs affected T cell 8. Liu, K., G. D. Victora, T. A. Schwickert, P. Guermonprez, M. M. Meredith, Downloaded from K. Yao, F.-F. Chu, G. J. Randolph, A. Y. Rudensky, and M. Nussenzweig. 2009. differentiation toward Th1 responses. The T cell priming skewed In vivo analysis of dendritic cell development and homeostasis. Science 324: toward Th1 cells from naive CD4+ T cells and developing Tregs 392–397. might facilitate autoimmune diseases such as CD and type I diabetes. 9. Varol, C., A. Vallon-Eberhard, E. Elinav, T. Aychek, Y. Shapira, H. Luche, H. J. Fehling, W.-D. Hardt, G. Shakhar, and S. Jung. 2009. Intestinal lamina Alternatively, the skewed priming toward Th1 cells might be bene- propria dendritic cell subsets have different origin and functions. Immunity 31: ficial for certain infections. The accumulation of LNK/SH2B3 single 502–512. 10. Onai, N., K. Kurabayashi, M. Hosoi-Amaike, N. Toyama-Sorimachi, nucleotide polymorphisms in European populations might be a result K. Matsushima, K. Inaba, and T. Ohteki. 2013. A clonogenic progenitor with http://www.jimmunol.org/ of selective pressure arising from infections. Further experiments will prominent plasmacytoid dendritic cell developmental potential. Immunity 38: be required to explore the relationship between Lnk/Sh2b3 function 943–957. 11. Huang, X., Y. Li, K. Tanaka, K. G. Moore, and J. I. Hayashi. 1995. Cloning and and autoimmune diseases or protection from infections. characterization of Lnk, a signal transduction protein that links T-cell receptor 2 2 CD103+ DC numbers were increased in Lnk / spleens com- activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol 3- pared with WT. Those DCs were able to activate CD8+ T cells by kinase. Proc. Natl. Acad. Sci. USA 92: 11618–11622. + 12. Takaki, S., J. D. Watts, K. A. Forbush, N. T. Nguyen, J. Hayashi, J. Alberola-Ila, cross-presentation (39). The induction of IFN-g–producing CD8 R. Aebersold, and R. M. Perlmutter. 1997. Characterization of Lnk. An adaptor T cells was more prominent in Lnk2/2 CD8a+ splenic DCs protein expressed in lymphocytes. J. Biol. Chem. 272: 14562–14570. 13. Takaki, S., K. Sauer, B. M. Iritani, S. Chien, Y. Ebihara, K. Tsuji, K. Takatsu, (Supplemental Fig. 4). We also examined cross-presentation of and R. M. Perlmutter. 2000. Control of B cell production by the adaptor protein GM-CSF–induced and OVA-loaded BMDCs. Proliferation of lnk. Definition of a conserved family of signal-modulating proteins. Immunity by guest on September 26, 2021 CD8+ T cells from OT-I transgenic mice was induced equally well 13: 599–609. 14. Takaki, S., H. Morita, Y. Tezuka, and K. Takatsu. 2002. Enhanced hematopoiesis by cocultivating with BMDCs from WT or Lnk-deficient BMDCs by hematopoietic progenitor cells lacking intracellular adaptor protein, Lnk. J. (data not shown). The expression levels of cell surface MHC class Exp. Med. 195: 151–160. I were comparable between Lnk2/2 BMDCs and normal BMDCs. 15. Velazquez, L., A. M. Cheng, H. E. Fleming, C. Furlonger, S. Vesely, A. Bernstein, C. J. Paige, and T. Pawson. 2002. Cytokine signaling and hematopoietic ho- We conclude that cross-presentation on DCs was not affected by meostasis are disrupted in Lnk-deficient mice. J. Exp. Med. 195: 1599–1611. Lnk deficiency. However, induction of IFN-g–producing CD8+ 16. Tong, W., and H. F. Lodish. 2004. Lnk inhibits Tpo–mpl signaling and Tpo- + mediated megakaryocytopoiesis. J. Exp. Med. 200: 569–580. T cells was enhanced when they were cocultured with CD8a 17. Tong, W., J. Zhang, and H. F. Lodish. 2005. Lnk inhibits erythropoiesis and Epo- 2/2 DCs from Lnk mice (Supplemental Fig. 4B). Several studies dependent JAK2 activation and downstream signaling pathways. Blood 105: proved that GM-CSF–producing T cells in so-called Th1 and 4604–4612. 18. Buza-Vidas, N., J. Antonchuk, H. Qian, R. Ma˚nsson, S. Luc, S. Zandi, Th17 cells are pathogenetic and responsible for autoimmune- K. Anderson, S. Takaki, J. M. Nygren, C. T. Jensen, and S. E. Jacobsen. 2006. mediated tissue inflammation (46, 47). Enhanced responses of Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles DCs to GM-CSF might contribute to an exacerbated inflammatory of thrombopoietin and LNK. Genes Dev. 20: 2018–2023. 19. Takizawa, H., K. Eto, A. Yoshikawa, H. Nakauchi, K. Takatsu, and S. Takaki. loop among DCs, naive T cells, and effector T cells. 2008. Growth and maturation of megakaryocytes is regulated by Lnk/Sh2b3 In conclusion, our results revealed a formerly unrecognized role adaptor protein through crosstalk between cytokine- and integrin-mediated signals. Exp. Hematol. 36: 897–906. of Lnk/Sh2b3. The data clearly showed that Lnk/Sh2b3 regulated 20. Bersenev, A., C. Wu, J. Balcerek, and W. Tong. 2008. Lnk controls mouse he- the production and function of DCs. 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Goltsev, K. D. Gibbs, Jr., Acknowledgments J. D. Merker, J. L. Zehnder, G. P. Nolan, and J. Gotlib. 2010. Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with We thank M. Ikutani and R. Hamamichi for helpful discussions and advice myeloproliferative neoplasms. Blood 116: 988–992. and A. Yoshikawa, J. Koyama, T. Norose, and M. Anraku for technical 24. Pardanani, A., T. Lasho, C. Finke, S. T. Oh, J. Gotlib, and A. Tefferi. 2010. LNK assistance. mutation studies in blast-phase myeloproliferative neoplasms, and in chronic- phase disease with TET2, IDH, JAK2 or MPL mutations. Leukemia 24: 1713– 1718. 25. Vainchenker, W., F. Delhommeau, S. N. Constantinescu, and O. A. Bernard. Disclosures 2011. New mutations and pathogenesis of myeloproliferative neoplasms. Blood The authors have no financial conflicts of interest. 118: 1723–1735. The Journal of Immunology 9

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