Leukocyte-Associated Ig-Like Receptor 1 Inhibits Th1 Responses but Is Required for Natural and Induced Monocyte-Dependent Th17 Responses

Leukocyte-Associated Ig-like Receptor 1 Inhibits T h1 Responses but Is Required for Natural and Induced Monocyte-Dependent T h17 Responses This information is current as of September 26, 2021. Vrushali V. Agashe, Ewa Jankowska-Gan, Melissa Keller, Jeremy A. Sullivan, Lynn D. Haynes, John F. Kernien, Jose R. Torrealba, Drew Roenneburg, Melanie Dart, Marco Colonna, David S. Wilkes and William J. Burlingham

J Immunol published online 8 June 2018 Downloaded from http://www.jimmunol.org/content/early/2018/06/07/jimmun ol.1701753 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/06/07/jimmunol.170175 Material 3.DCSupplemental

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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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 8, 2018, doi:10.4049/jimmunol.1701753 The Journal of Immunology

Leukocyte-Associated Ig-like Receptor 1 Inhibits Th1 Responses but Is Required for Natural and Induced Monocyte-Dependent Th17 Responses

Vrushali V. Agashe,*,† Ewa Jankowska-Gan,* Melissa Keller,‡ Jeremy A. Sullivan,* Lynn D. Haynes,* John F. Kernien,x Jose R. Torrealba,{ Drew Roenneburg,* Melanie Dart,‖ Marco Colonna,# David S. Wilkes,** and William J. Burlingham*

Leukocyte-associated Ig-like receptor 1 (LAIR1) is an ITIM-bearing collagen receptor expressed by leukocytes and is implicated in immune suppression. However, using a divalent soluble LAIR1/Fc recombinant protein to block interaction of cell surface LAIR1

with matrix collagen, we found that whereas Th1 responses were enhanced as predicted, Th17 responses were strongly inhibited. Downloaded from Indeed, LAIR1 on both T cells and monocytes was required for optimal Th17 responses to collagen type (Col)V. For pre-existing “natural” Th17 response to ColV, the LAIR1 requirement was absolute, whereas adaptive Th17 and Th1/17 immune responses in both mice and humans were profoundly reduced in the absence of LAIR1. Furthermore, the addition of C1q, a natural LAIR1

ligand, decreased Th1 responses in a dose-dependent manner, but it had no effect on Th17 responses. In IL-17–dependent murine organ transplant models of chronic rejection, LAIR1+/+ but not LAIR12/2 littermates mounted strong ﬁbroproliferative re-

sponses. Surface LAIR1 expression was higher on human Th17 cells as compared with Th1 cells, ruling out a receptor deﬁciency http://www.jimmunol.org/ that could account for the differences. We conclude that LAIR1 ligation by its natural ligands favors Th17 cell development, allowing for preferential activity of these cells in collagen-rich environments. The emergence of cryptic self-antigens such as the LAIR1 ligand ColV during ischemia/reperfusion injury and early acute rejection, as well as the tendency of macrophages/

monocytes to accumulate in the allograft during chronic rejection, favors Th17 over Th1 development, posing a risk to long- term graft survival. The Journal of Immunology, 2018, 201: 000–000.

ung transplantation is complicated by chronic rejection, APCs (4), and was independent of purinergic P2X7R function (5). which manifests itself as obliterative bronchiolitis (OB) or In contrast, the Th17 response required IL-17 and TNF-a, but not

L its clinical correlate bronchiolitis obliterans syndrome. IFN-g. It had an absolute requirement for IL-1b, as well as by guest on September 26, 2021 Improvements in immunosuppression have decreased acute re- monocytes as APCs, and was strictly P2X7R-dependent (2, 5, 6). jection, but chronic rejection remains a major obstacle for long- Besides the critical role of the Th17 response to ColV in bron- term survival (1). One reason for this is the development of chiolitis obliterans syndrome, a “mixed” Th1/17 ColV response + CD4 Th17 autoimmunity directed against collagen type V (ColV) was found in coronary artery disease patients and in models of (2) and K-a1-tubulin (3). Th17-driven autoimmune responses are atherosclerosis (5, 7, 8). different as compared with the Th1-driven response in several Recently, we have described pre-existing or natural Th17 re- aspects. When the Th1 response to tetanus toxoid/diphtheria tox- sponses to certain self-antigens, including ColV, vimentin, and oid (TT/DT) and the Th17 response to ColV were compared in a K-a1-tubulin in normal healthy individuals and in naive CBA trans-vivo delayed type hypersensitivity (Tv-DTH) bioassay, the mice (9). These pre-existing responses could be detected after the + + Th1 response required IFN-g (2) and IL-12 (V.V. Agashe, removal of CD39 or CD25 T regulatory (Treg) cells or by the E. Jankowska-Gan, and W.J. Burlingham, unpublished observa- neutralization of TGF-b1 (9). Interestingly, these Th17 cells tions), could use either dendritic cells (DCs) or monocytes as responded in an HLA-DR–speciﬁc fashion to 160-aa fragments of

*Division of Transplantation, Department of Surgery, School of Medicine and Public V.V.A. conceived, designed, and performed the experiments; analyzed the data; and Health, University of Wisconsin–Madison, Madison, WI 53792; †Comparative Bio- wrote the paper. E.J.-G., M.K., J.A.S., L.D.H., J.F.K., J.R.T., and D.R. performed medical Sciences Graduate Program, University of Wisconsin–Madison, Madison, experiments and histology analysis. M.C. provided technical (reagents) support and WI 53706; ‡Michigan State University, East Lansing, MI 48824; xDepartment of reviewed the paper. M.D. initiated the project. D.S.W. reviewed the paper. W.J.B. Medicine, School of Medicine and Public Health, University of Wisconsin–Madison, conceived and designed the experiments and helped write the paper. Madison, WI 53706; {Division of Renal Pathology, Department of Pathology, Uni- ‖ Address correspondence and reprint requests to Dr. William J. Burlingham, Univer- versity of Texas Southwestern Medical Center, Dallas, TX 75390; Promega Corpo- sity of Wisconsin–Madison, 600 Highland Avenue, Room G4/702, Clinical Science ration, Madison, WI 53711; #Department of Pathology and Immunology, Washington Center, Madison, WI 53792. E-mail address: [email protected] University School of Medicine, St. Louis, MO 63110; and **University of Virginia School of Medicine, Charlottesville, VA 22908 The online version of this article contains supplemental material. ORCIDs: 0000-0001-8800-5946 (V.V.A.); 0000-0002-9539-9768 (M.K.); 0000- Abbreviations used in this article: ACR, American College of Rheumatology; ColI, 0002-3971-8429 (L.D.H.); 0000-0002-4424-7713 (J.F.K.); 0000-0001-5429- collagen type I; ColV, collagen type V; DC, dendritic cell; LAIR1, leukocyte- 3695 (M.D.). associated Ig-like receptor 1; OB, obliterative bronchiolitis; pDC, plasmacytoid DC; SHP, Src homology region 2 domain–containing phosphatase; sLAIR1/Fc, sol- Received for publication December 19, 2017. Accepted for publication May 15, uble LAIR1/Fc protein; T , T regulatory; TT/DT, tetanus toxoid/diphtheria toxoid; 2018. reg Tv-DTH, trans-vivo delayed type hypersensitivity; WT, wild-type. This work was supported by National Institutes of Health Grants 1P01AI084853 (to V.V.A., E.J.-G., M.K., J.A.S., L.D.H., J.F.K., J.R.T., D.R., D.S.W., and W.J.B.), Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 1R01AI119140 (to V.V.A., E.J.-G., J.A.S., L.D.H., and W.J.B.), and R01AI110617 (to E.J.-G. and J.A.S.).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701753 2 LAIR1 INHIBITS Th1 BUT NOT Th17 RESPONSES

ColV a1, but not to 15mer ColV peptides (9). Thus, some tertiary marrow was flushed from the tibia and femur into monocyte media (PBS plus 2% FBS plus 1 mM EDTA). The marrow was filtered, RBCs were structure was required for the natural Th17 response. Although a number of receptors might be involved, we chose the leukocyte- lysed, and cells were resuspended in monocyte media. associated Ig-like receptor 1 (LAIR1) for in-depth study, because Murine transplantation it is constitutively expressed and has been implicated in both in- 2 2 All transplants were between B10 donors and B6 LAIR1 / recipients or hibitory and activating functions (10, 11). B6 LAIR+/+ recipients. Heterotopic cardiac and orthotopic lung transplants LAIR1 is a transmembrane receptor expressed by a variety of were performed as described previously (18–20). Heart grafts were mon- immune cells (10). It binds collagenous domains via its extra- itored by abdominal palpitation (23). The hearts and the lung were col- cellular domain and contains two ITIMs in its cytoplasmic tail. lected 42 and 21 d posttransplant, respectively, and used for H&E and LAIR1 ligands include fibrillar collagens and collagenous do- trichrome staining. A blinded pathologist scored the transplants for rejection and fibrosis. The cardiac grafts were scored according to the criteria mains containing proteins, for example, C1q and mannose binding in the Heart Rejection Study Group (24). Briefly, the scoring included no lectin (10, 12, 13). Traditionally, LAIR1 has been described as an rejection (grade 0), focal (grade 1A) or diffuse (grade 1B) interstitial inhibitory receptor, because LAIR1 ligation by anti-LAIR1 Ab mononuclear infiltrates without myocyte necrosis, focal aggressive cellular recruited Src homology region 2 domain–containing phosphatase rejection with myocyte damage (grade 2), multifocal aggressive infiltrates with myocyte damage (grade 3A), diffuse inflammatory process with ne- (SHP)1 and SHP2 and decreased NK cell cytotoxicity (14). crosis (grade 3B), and diffuse aggressive polymorphous/mononuclear in- 2/2 LAIR1 also inhibited T and B cells (15, 16). However, LAIR1 filtrate with edema, hemorrhage, vasculitis, and necrosis (grade 4). Fibrosis mice were not hyperresponsive and were no more susceptible to was scored on a graded scale as follows: 0, no fibrotic areas; 1, increased experimental autoimmune encephalomyelitis and colitis than were numbers of interstitial collagen fibers; 2, 0–10% of area is fibrotic, 3, 10–50% of area is fibrotic; and 4, 50–100% of area is fibrotic. Acute their LAIR1+/+ littermates (17). Thus, the role of LAIR1 in vivo Downloaded from vascular rejection score was calculated from the severity of involved epi- remains unresolved. Because most data about LAIR1 inhibition cardial and intramyocardial vessels (0, negative; +1, minimal intimal in- were developed from in vitro studies and with an anti-LAIR1 Ab, flammation; +2, moderate intimal and medial inflammation; and +3, severe rather than the natural ligands, we sought to determine the role of transmural inflammation). LAIR1 in the context of T 17 responses, including chronic allo- The lung transplant recipients were euthanized, and the native and donor h lungs were harvested, glutaraldehyde fixed, and paraffin embedded. A graft rejection model (18–20), as well as a trans-vivo bioassay portion of the lower lobe of each lung was sectioned and used for H&E and system (21, 22) using natural ligands for LAIR1. trichrome staining. Lung transplants were scored following the Interna- http://www.jimmunol.org/ tional Society of Heart and Lung Transplant 2007 classification (25). Scores include the alveolar perivascular inflammation (American College Materials and Methods of Rheumatology [ACR]-A score) as minimal (A1), mild (A2), moderate Human samples (A3), and severe (A4), and bronchial inflammation (ACR-B score) as low grade and high grade. Additionally, peribronchial fibrosis in this model Blood was drawn from lung, lung/heart, or kidney transplant patients and was graded as negative (0), mild (1), moderate (2), and severe (3). When coronary artery disease patients after written informed consent was ob- OB lesions were identified, they were labeled as present (yes) or absent tained, in accordance with Institutional Review Board guidelines. PBMCs (no). The hydroxyproline assay was used to determine the collagen content were isolated using Ficoll-Hypaque and frozen for later use. of the lung grafts as previously described (26, 27).

Antigens Tv-DTH assay by guest on September 26, 2021 Collagen type I (ColI) and ColV were a gift from Dr. D.S. Wilkes or A Tv-DTH assay was performed by the cotransfer of 7–10 3 106 human Dr. D.D. Brand (University of Tennessee Health Science Center, Memphis, PBMCs or mouse splenocytes and the appropriate Ag into the footpads of TN), vimentin was from R&D Systems (Minneapolis, MN), K-a1-tubulin CB17-SCID or B6 mice (21, 28). Murine footpad thickness was measured was a gift from Dr. T. Mohanakumar (Dignity Health St. Joseph’s Hospital with a gauge before and 18–20 h after the cotransfer of cells. Background and Medical Center, Phoenix, AZ), TT/DT vaccine was from Sanofi- swelling seen with the buffer alone was subtracted to determine the Ag- Aventis (Bridgewater, NJ), C1q was from Complement Technology specific response. Ag concentrations per injection were: for human studies, (Tyler, TX), and ColV peptides were from GenScript (Piscataway, NJ) or ColV peptide (5 mg), ColV peptide (1 mg), TT/DT (15 mg), donor or the University of Wisconsin–Madison peptide synthesis core. donor-matched alloantigen lysate (8–12 mg), and EBV (8 mg); for murine The soluble LAIR1/Fc protein (sLAIR1/Fc) construct (human LAIR1 studies, ColV and ColI peptides (20 mg), ColV peptide (5 mg), and TT/DT fused to Fc of human IgG expressed in the J558 cell line) was obtained 1 (25 mg). In certain experiments, sLAIR1/Fc (0.05–1 mg) or C1q (0.5–10 mg) from Dr. M. Colona and purified in-house. An ELSIA was used to deter- was titrated in with the cells and the Ag of interest. To determine the cy- mine the binding capabilities of sLAIR1/Fc. Plates were coated overnight tokine dependency of certain immune responses, neutralizing Abs were with various proteins of interest (10 mg/ml) and blocked with 2% BSA. added: purified anti-mouse IFN-g (551216; BD Biosciences), anti-mouse IL- sLAIR1/Fc was serially diluted onto the plate and incubated overnight. 17A (555068; BD Biosciences), anti-mouse TNF-a (14-7423-85; eBio- The bound sLAIR1/Fc was detected with an HRP-labeled anti-human IgG 1 science), anti-mouse IL-1b (14-7012-85; eBioscience), or anti-mouse TGF-b1 Ab (Sigma-Aldrich, St. Louis, MO). For inhibition studies, sLAIR1/Fc (555052; BD Biosciences) were added at 10 mg per injection. (1 mg/ml) was preincubated overnight with the inhibitor of interest. This mixture was then transferred to the ColV-coated plates and incubated Cell isolations overnight. sLAIR1/Fc bound to the ColV-coated plate was detected as described. A section of mouse footpad was stained with sLAIR1/Fc and Untouched CD3+ T cells were isolated and depleted of CD25+ cells the bound protein was detected with an anti-human Fc HRP-conjugated according to the manufacturer’s instructions (130-095-130, 130-091-072; secondary Ab. Miltenyi Biotec). Untouched murine CD11b+Ly6G2 monocytes were isolated from the bone marrow (19761 or 19861; Stemcell Technologies) Mice according to the manufacturer’s instructions. B6 and B10 mice were purchased from Envigo (Indianapolis, IN) or Flow cytometry Taconic Biosciences (Cambridge City, IN), LAIR1 knockout (LAIR2/2) mice were a gift from Dr. John E. Coligan (17), and CB17 SCID mice were PBMCs (1 3 106) were Fc blocked (130-059-901; Miltenyi Biotec), from Biotron Laboratory. Male and female mice were used. Animals were stained, and acquired on a BD LSR II or a BD LSRFortessa (5). For in- housed in a specific pathogen-free facility and experiments conducted in tracellular cytokine staining, 1 3 106 PBMCs were cultured in the pres- accordance with the Institutional Animal Care and Use Committee ence of the appropriate antiegn (ColI or ColV) in DMEM containing 5% guidelines. Mice were immunized s.c. or intradermally with 50–100 mgof FBS at 37˚C. After overnight incubation, the cells were stimulated with bovine ColV or ColV a1 peptide p599 emulsified in CFA. In certain ex- PMA (10 ng/ml) and ionomycin (1 mg/ml) for 5 h, with the addition of periments animals received a boost in IFA and some mice also received brefeldin A (420601; BioLegend) for the last 3 h. The following Abs were 75 ml of TT/DT pediatric vaccine i.p. Animals were euthanized 2–3 wk used: CD3-FITC or CD3–Alexa Fluor 700 (317306/100216; BioLegend or after immunizations, and spleen and lymph nodes were collected and 557943; BD Biosciences), CD11b-BV421 (562632; BD Biosciences), homogenized to get a single-cell suspension for experimental use. Bone CD56-allophycocyanin (555518; BD Biosciences), CD14–Alexa Fluor 700 The Journal of Immunology 3

(325614; BioLegend), CD16-allophycocyanin-H7 (560195; BD Biosciences), condition, and results are expressed as an average number of spots per LAIR1-PE (550811; BD Biosciences), HLA-DR–PE-Cy7 (25-9956-41; million cells producing cytokine (18). eBioscience), CD123-BV421 (306017; BioLegend), lineage-BV510 (348807; BioLegend), CD11c-allophycocyanin (559877; BD Biosci- Statistical analysis ences), PE-IgG1 k isotype control (55749; BD Biosciences), CD196/ Statistical analysis was performed using GraphPad Prism (GraphPad CCR6-FITC (11-1969-42; eBioscience), CD194/CCR4-PECy7 (561034; Software, La Jolla, CA). BD Biosciences), CD4-BUV395 (563552/563790; BD Biosciences), CD183/CXCR3-allophycocyanin (550967; BD Biosciences), IFN-g– BV421 (563376; BD Biosciences), IL-17–PE (559502; BD Biosciences), Results gd TCR–FITC (118105; BioLegend). Data were analyzed using FlowJo Pre-existing Th17 responses to ColV are abolished by genetic (Treestar). deletion of LAIR1 ELISPOT We first sought to determine whether B6 mice (background of the 2/2 Plates with coated with the appropriate primary Ab, that is, purified anti- LAIR1 mice) had pre-existing responses to ColV, similar to mouse IFN-g (551216; BD Biosciences), IL-17A (16-7175-85; eBio- those reported for the CBA mice (9). Using the trans-vivo bio- science), or IL-4 (554434; BD Biosciences), and incubated overnight at assay system we demonstrated that whole spleen cells from naive 4˚C. Wells were blocked with 1% BSA at room temperature for 2 h wild-type (WT) B6 mice did not respond to ColV (Fig. 1A). The and the washed with HL1 serum-free media (Lonza, Walkersville, MD) addition of an isotype control did not reveal any significant re- supplemented with penicillin/streptomycin and 2 mmol/l L-glutamine. Murine lymphocytes were added at 2.5 3 106 to 4 3 106 cells per well and sponses to ColV (Fig. 1A). However, the neutralization of TGF-b1 stimulated with media, ColI (10 mg per well), ColV (10 mg per well), or in the same system revealed a statistically significant increase in TT. Plates were incubated at 37˚C at 5% CO2 for 24–48 h. Following response to ColV (Fig. 1A). No responses to ColI were detected Downloaded from incubation, cells were lysed with H2O and wells washed four times with (Fig. 1A). PBS plus Tween 20, followed by four washes in PBS. Secondary Abs anti- mouse IFN-g (554410; BD Biosciences), IL-17A (13-7177-85; eBio- To determine whether this response could be recapitulated using 2 + 2 science), or IL-4 (554390; BD Biosciences) diluted in PBS containing 1% isolated cells (9), we isolated CD25 T cells and CD11b Ly6G BSA were added to the wells. Plates were incubated at room temperature monocytes and used them in the same assay. Whereas the un- for 2 h. After washing, spots were developed with streptavidin–alkaline separated T cell population and monocytes gave a higher response phosphatase and 5-bromo-4-chloro-3-indolyl phosphate substrate (SEL002; 2 to ColV than to ColI (Fig. 1A), the CD25 T cells and monocytes R&D Systems) according to the manufacturer’s instructions. Spots http://www.jimmunol.org/ were counted using the AID ELSIPOT plate reader system (Autoimmun revealed a strong natural response to ColV, but not to ColI Diagnostika, Strasburg, Germany). Triplicate wells were run for each Ag (Fig. 1A). by guest on September 26, 2021

FIGURE 1. Natural responses against ColV in C57BL/6 and LAIR1+/+ mice, but not in the LAIR12/2 littermate controls. Spleen cells from naive WT B6, LAIR1+/+, or LAIR12/2 littermate control mice were tested for their pre-existing natural immune responses to ColV. (A) Whole spleen cells from WT B6 mice (n = 8–12, pooled) were tested for their reactivity to ColI and ColV. The neutralization of TGF-b1 lead to the uncovering of a response against ColV but not ColI. Neither the whole spleen cells alone nor the addition of an IgG control isotype Ab revealed any responses to any Ag. Unseparated T cells and monocytes did not demonstrate a robust response to ColV. However, the CD252 T cells plus monocytes demonstrated a robust response to ColV, but not to ColI. #p , 0.0005 by unpaired t test. (B) Spleen cells from LAIR1+/+ and LAIR12/2 littermates (n = 7–9, pooled) were tested for their natural ability to respond to ColI and ColV. After neutralizing TGF-b1, the LAIR+/+ mice revealed strong responses to ColV, comparable to those seen with the WT B6 mice. No responses were detected in the LAIR12/2 mice. #p , 0.0001 by unpaired t test. (C) CD25-depleted spleen cells from LAIR1+/+ mice (n = 8, pooled) were tested for their reactivity to ColV in the presence of neutralizing Abs to various cytokines. Neutralization of IL-17, TNF-a, and IL-1b decreased the ColV response. Importantly, the neutralization of IFN-g did not affect the natural ColV response. #p , 0.05, ##p , 0.005 by unpaired t test. 4 LAIR1 INHIBITS Th1 BUT NOT Th17 RESPONSES

Cells from LAIR1+/+ littermates responded similarly to ColV upon dependency of the induced ColV response (5). To test this, TGF-b1 neutralizations as did WT B6 mice (Fig. 1B), but cells from LAIR1+/+ and LAIR12/2 littermates were immunized with ColV. naive LAIR12/2 mice failed to respond to ColV (Fig. 1B). The use ELISPOT analysis of spleen cells from LAIR1+/+ mice revealed of isolated CD252 T cells and monocytes from the LAIR12/2 mice the production of IL-17 and IFN-g in response to ColV stimula- also did not reveal any significant responses to ColV, ruling out tion. Conversely, the production of both cytokines was decreased 2/2 changes in cytokine production by Treg cells (Supplemental Fig. 1A). in the LAIR1 mice (Fig. 2A). The IL-17 and IFN-g spots were Additionally, to determine the cytokines responsible for the natural similar in magnitude in the LAIR1+/+ mice, indicating that the + ColV response, we depleted CD25 cells from the spleen of naive induced ColV response is a Th1/17 response, not a Th17 response. LAIR1+/+ mice. The response of the CD25-depleted fraction to ColV Additionally, the neutralization of IL-17, TNF-a, and IFN-g was analyzed in the presence of neutralizing Abs against IL-17, IL-1b, significantly decreased the response to ColV (Fig. 2B) in WT B6 TNF-a,andIFN-g. The neutralization of the former three, but not mice. Neutralization of IL-1b was trending toward significance. IFN-g, decreased the natural response to ColV (Fig. 1C), indicating Interestingly, note that IFN-g was found to be significant, but the that the natural ColV response is a Th17 response. Further in vitro results varied between individual mice (Fig. 2B). This supported +/+ 2/2 analysis of cells from LAIR1 and LAIR1 mice revealed the the idea that the induced ColV response was more Th1/17. + + 2/2 source of IL-17 to be ⍺b TCR CD4 T cells (Supplemental Fig. 1B). To confirm the lack of Th17 responses in LAIR1 mice, we + These results together suggest that the Th17 ColV response is LAIR1- isolated CD3 T cells and monocytes from TT/DT- and ColV- dependent, and that the lack of LAIR1 abolishes this response. immunized LAIR1+/+ and LAIR12/2 mice and tested them in various combinations. The absence of LAIR1 on either the

LAIR1 is also required for the induced ColV and ColV peptide monocyte or the T cell alone did not abrogate the ColV response Downloaded from response in mice (Fig. 2C). Only when LAIR1 was absent on both cell types did we Having established that the natural Th17 response to ColV is absent ﬁnd a statistically signiﬁcant drop in ColV responses (Fig. 2C). in LAIR12/2 mice, we next aimed at determining the LAIR1 No responses to ColI were detected, and the TT/DT response http://www.jimmunol.org/ by guest on September 26, 2021

+/+ FIGURE 2. LAIR1 is required for optimal induced Th1/17 responses to ColV and ColV peptide in WT B6 and LAIR1 mice. Cells from immunized B6 WT, LAIR1+/+,andLAIR12/2 mice were tested in the ELISPOT and the Tv-DTH assay. (A) Lymphocytes from ColV-immunized WT B6 LAIR1+/+ (n =7)andB6 LAIR12/2 (n = 11) mice were cultured in vitro with ColV and analyzed for the expression of three cytokines, IL-17, IFN-g,andIL-4.Astatisticallysignificant decrease in IL-17 spots. #p , 0.05, B6 LAIR12/2 mice as compared with the B6 LAIR1+/+ mice (by unpaired t test; values .1000 were excluded in the analysis). (B) Lymphocytes from ColV-immunized WT B6 mice (n = 38) were tested for their ability to respond to ColV in the presence of neutralizing Abs to various cytokines. Neutralization of IL-17, TNF-a,andIFN-g and gave statistically significant decreases in ColV reactivity. #p , 0.05, ##p , 0.005, ###p , 0.0005 by +/+ 2/2 paired t test. (C) Using isolated T cells and monocytes, robust Th1 responses to TT/DT were seen in the LAIR1 and the LAIR1 mice. However, a statistically significant decrease in ColV response was seen in the LAIR12/2 group as compared with the LAIR1+/+ group. No significant differences in TT/DT and ColV responses were noted when only one of the cell type, either the T cell or the APC, was LAIR12/2 (n = 3 separate experiments, with cells pooled from four mice in each group). No responses to ColI were detected in either group. #p , 0.005, by unpaired t test. (D) Lymphocytes from p599-immunized WT B6 mice were tested for their ability to respond to ColI and ColV Ags and to ColV peptide p599 in the presence of neutralizing Abs to various cytokines. Left, Lymphocytes from p599-immunized WT B6 mice (n = 25) responded equally well to p599 and whole ColV, but not to ColI and p909 (28). #p , 0.05, ##p , 0.0001, unpaired t test. Right, Neutralizing IL-17, IL-1b,andTNF-a, but not IFN-g, decreased the response to ColV peptide p599 (n =4).#p , 0.05, ##p # 0.005 by paired t test. The Journal of Immunology 5

FIGURE 3. sLAIR1/Fc binds to collagenous domain–containing proteins, the 160-aa fragments, but not the 15mer peptides of ColV a1. The sLAIR1/Fc construct was tested for its ability to bind various proteins and peptides of interest in an ELISA assay. (A) sLAIR1/Fc bound all the proteins of interest (collagens and C1q) but failed to bind ﬁbrinogen and ﬁbronectin. sLAIR1/Fc also bound the larger 160-aa fragment of ColV a1, F5, but not the 15mer ColV peptides p629 and p1049 (n = 2–3 individual experiments; a representative plot is shown). (B) Inhibition of sLAIR1/Fc binding to a plate coated with ColV after preincubation of sLAIR1/Fc with various proteins and peptides of interest is shown. Preincubation with certain proteins (CollV, ColXI, and C1q) but not others (tetanus Ag, ColV peptides) inhibited sLAIR1/Fc from binding to the plate-bound ColV (n = 2–3 experiments; representative plots from one

experiment are shown). Downloaded from remained consistent in all groups (Fig. 2C). These results sug- protein was purified and characterized in-house (Supplemental gested that the absence of LAIR1 on both the T cell and the Fig. 2A). Consistent with the published data on LAIR1, sLAIR1/Fc monocyte is required for the decrease in Th17 ColV responses. bound all collagens tested, including the larger 160-aa-long fragment We also determined the response to the I-Ab–specific ColV a1 of ColV a1, along with C1q, but did not bind fibrinogen, fibronectin,

peptide p599 in WT B6 mice (28). Lymphocytes from these p599 or the 15mer peptides of ColV (Fig. 3A). http://www.jimmunol.org/ immunized mice responded to p599 as well as ColV, but not to ColI The inability of sLAIR1/Fc to bind the peptides was further and p909 (Fig. 2D). Unlike the induced ColV response, which was confirmed with inhibition studies. Preincubation of a fixed con- Th1/17, the induced ColV peptide p599 response was dependent centration of sLAIR1/Fc with increasing concentrations of ColVor only on IL-17, IL-1b,andTNF-a,butnotonIFN-g (Fig.2D).These ColXI completely inhibited its binding to the plate-bound ColV results indicated that the LAIR1, although absolutely required for the (Fig. 3B). The preincubation with C1q only partially inhibited its pre-existing Th17 response to ColV, was also partially required for binding to the plate-bound ColV (Fig. 3B). However, the pre- the induced Th17 and induced Th1/17 responses to ColV. incubation with peptides did not inhibit its binding to the plate coated with ColV (Fig. 3B). Similar results were obtained when sLAIR1/Fc binds ColV, the larger 160-aa fragments, but not the increasing concentrations of sLAIR1/Fc were titrated into a fixed by guest on September 26, 2021 a 15mer peptides of ColV 1 inhibitor concentration (Supplemental Fig. 2B). To further understand the role of LAIR1 on Th1 and Th17 re- Staining of a mouse footpad with sLAIR1/Fc also demonstrated sponses, we employed an antagonist of LAIR1, sLAIR1/Fc. The its binding to collagens present in the tissue as evidenced by the

FIGURE 4. Effect of sLAIR1/Fc, an LAIR1 antagonist, and C1q, an LAIR1 agonist, on the Th1 tetanus and the Th17 ColV peptide response in immunizedWTB6mice.sLAIR1/FcandC1qwereusedtodeterminetheeffectofLAIR1onTh1andTh17 responses in cells obtained from TT/DT- and ColV-immunized B6 mice. (A) Addition of sLAIR1/Fc increased the Th1 TT/DT responses (black line) with increasing sLAIR1/Fc concentrations. Conversely, the Th17 ColV p599 peptide response (blue line) showed a decreasing trend with increasing sLAIR1/Fc concentrations (n =3 experiments with pooled splenocytes from two to four mice). Using a paired t test between Ag and each subsequent concentration of sLAIR1/Fc, for # ## p599, p = 0.0942 at 0.5 mg of sLAIR1/Fc. For TT/DT, p , 0.05, p , 0.005. (B) The introduction of C1q, an LAIR1 ligand, decreased the Th1TT/ DTresponse(blackline)butfailedtocauseanysigniﬁcantdifferenceintheTh17 ColV p599 peptide response (blue line) (n = 4–6 experiments, with cells pooled from two to seven immunized mice). Using a paired t test between Ag and each subsequent concentration of C1q, for p599, *p , 0.005. For TT/DT, #p , 0.05, ##p , 0.01, ###p , 0.005. 6 LAIR1 INHIBITS Th1 BUT NOT Th17 RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Effect of sLAIR1/Fc, an LAIR1 antagonist, and C1q, an LAIR1 agonist, on Th1 and Th17 responses in ColV-reactive heart or lung transplant patient PBMCs. PBMCs from ColV-reactive heart or lung transplant patients were tested for their ability to respond to various ColV peptides or donor responses and TT/DT or EBV in the presence of increasing concentrations of sLAIR1/Fc or C1q. (A) Addition of sLAIR1/Fc increased the Th1 TT/DT responses (black line) with increasing sLAIR1/Fc concentrations. Conversely, the Th17 ColV peptide response (blue line) decreased to baseline with increasing sLAIR1/Fc concentrations (n = 4 experiments with three patients). Using a paired t test between Ag and each subsequent concentration of # ## # sLAIR1/Fc, for ColV peptides, p , 0.05, p , 0.005. For TT/DT, p , 0.05. (B) Increasing concentrations of C1q decreased the Th1 tetanus response in a dose-dependent manner (black line) but did not affect the Th17 ColV peptide response (blue line) (n = 2 experiments with one patient). Using a paired t test between Ag and each subsequent concentration of C1q, for TT/DT, p = 0.09 at 5 mg, and p = 0.07 at 10 mg. (C). The antagonism of the surface LAIR1 receptor increased the Th1 EBV response but decreased the Th17 alloantigen response (n = 5 experiments with three patients. Using a paired t test between Ag and each subsequent concentration of sLAIR1/Fc, #p , 0.05, ##p , 0.005. trichrome stain (Supplemental Fig. 2C). Collectively, these results (Fig. 4A) in cells from immunized WT B6 mice. Conversely, in- indicate that the tertiary structure of its ligands is important creasing amounts of sLAIR1/Fc decreased the Th17 ColV peptide for LAIR1 binding and that LAIR1 binds collagens and C1q response (Fig. 4A). Utilizing C1q, an LAIR1 agonist due to its differently. collagen “tail” domain (29), decreased the Th1 TT/DT response but failed to affect the Th17 ColV peptide response (Fig. 4B) in Antagonism of collagen–LAIR1 interaction increases Th1 cells from immunized WT B6 mice. responses but decreases Th17 responses Similar results were seen with PBMCs from ColV-reactive To determine the in vivo role of LAIR1 in the effector end of the patients; that is, increasing sLAIR1/Fc concentrations signifi- immune response in a collagen-rich environment, we employed the cantly increased the Th1 TT/DT swelling response but decreased Tv-DTH assay. Lymphocytes from TT/DT- and ColV peptide– the Th17 ColV peptide response (Fig. 5A). The addition of C1q immunized mice or PBMCs from ColV-reactive patients were showed a trend toward a decrease in the Th1 TT/DT responses injected into the footpads of B6 or SCID mice along with the Ag (Fig. 5B), but it did not affect the Th17 ColV peptide response of interest, with increasing concentrations of sLAIR1/Fc. This (Fig. 5B). Owing to the lack of ColV peptide–reactive patient blocked the access of the injected cells to matrix collagen through samples, PBMCs from a single patient were used in Fig. 5B. their surface LAIR1 receptors, and the use of ColV a1 peptides in These results support the idea that LAIR1 differentially regulates the assay enabled us to rule out any effect ColV might have had as Th1 and Th17 responses, inhibiting the former but not the latter. an LAIR1 ligand. To determine whether this pattern holds true for other induced Because in the case of Th1 cells we were inhibiting an inhibitory Th17 versus Th1 responses, we used the Th1 EBV and the Th1/17 response, increasing sLAIR1/Fc concentrations gave a statistically anti-donor Ag response in chronic kidney graft rejection (5). In- significant increase in the Th1 TT/DT-induced swelling response creasing sLAIR1/Fc concentrations significantly increased the The Journal of Immunology 7

FIGURE 6. LAIR1 expression on chemokine receptor gated Th1 cells, Th17 cells, DCs, and monocytes. PBMCs from ColV-reactive patients (n = 6) were Downloaded from stained to distinguish between Th1 cells, Th17 cells, DCs, and monocytes. T cells were gated on CCR4 and then on CCR6 and CXCR3 for differentiating + + 2 + + +/2 2 Th1(CCR4 CXCR3 CCR6 ) and Th17 (CCR4 CCR6 CXCR3 ) cells. DCs were defined as lineage (CD3, CD14, CD16, CD19, CD20, CD56), HLA- DR+ and then differentiated as mDCs (CD11c+CD1232) and pDCs (CD11c2CD123+). Monocytes were defined as CD32CD14+CD11b+CD562 and then + 2 + + gated as CD16 or CD16 monocytes. (A) T cells with a Th17 phenotype (CCR4 , CCR6 ) had a higher percentage of cells that stained positive for LAIR1, + # as compared with the Th1 cells (CXCR3 ). The Th17 cells also had a higher MFI of LAIR1 as compared with the Th1 cells. p , 0.05 by paired t test. (B) Of all the APCs analyzed, mDCs, pDCs, and CD16+ and CD162 monocytes expressed LAIR1. The MFI, however, was higher on pDCs and CD16+ and 2 CD16 monocytes as compared with mDCs. #p , 0.05 by paired t test. mDC, myeloid DC; MFI, median fluorescence intensity. http://www.jimmunol.org/

EBV response (Fig. 5C) but significantly decreased the anti-donor develop chronic rejection in a Th17-dependent murine transplant response (Fig. 5C) in chronically rejecting kidney transplant pa- models (18) (Supplemental Fig. 4). B10 hearts or lungs were tients. These results strongly suggest that LAIR1 is differentially transplanted into LAIR1+/+ or LAIR12/2 recipients. regulating Th1 and Th1/17 responses. A significant decrease was seen in cellular infiltration in the heart 2/2 +/+ However, artificial LAIR1 ligands inhibited both Th1 and Th17 grafts from LAIR1 mice as compared with the LAIR1 mice, responses. For example, the anti-LAIR1 Ab completely inhibited along with a trend toward a lower amount of collagen deposition and both the T 17 ColV peptide response (Supplemental Fig. 3) as vascular rejection (Fig. 7A, 7B, Table I). Only one of the B6 LAIR12/2

h by guest on September 26, 2021 well as the Th1 response to tetanus toxoid (data not shown). No- mice demonstrated collagen deposition and vascular rejection and thus tably, the addition of natural ligands of LAIR1, for example, C1q a statistical significance could not be achieved for vascular rejection +/+ 2/2 or ColI, did not inhibit the Th17 responses (Supplemental Fig. 3). between the LAIR1 and LAIR1 groups (Fig. 7B, Table I). Similar results were seen in the lung transplant model (18). The The differential effects of LAIR1 on T 1 and T 17 cells cannot h h LAIR1+/+ mice with chronic rejection but no OB showed a low be attributed to the lower expression of LAIR1 on human T 17 h level of cellular infiltration and collagen deposition around the cells or their APCs airways (Fig. 7C). The LAIR1+/+ mice that developed OB dem- We hypothesized that the differential regulation of Th1 and Th17 onstrated high levels of cellular infiltration and collagen deposi- responses by LAIR1 could be explained by a lack of, or by a tion (Fig. 7C). Additionally, one can observe the OB lesions differential expression of, LAIR1 expression on these cells. within the airways (arrows). However, the LAIR12/2 mice did Therefore, we analyzed the LAIR1 expression on PBMCs from show cellular infiltration into the graft, but little collagen depo- + + ColV-reactive patients. CXCR3 CD4 Th1 cells were found to sition (Fig. 7C) and no OB lesions. Perivascular inflammation + have lower LAIR1 expression as compared with the CCR4 (ACR-A) scores were decreased in the LAIR12/2 mice as com- + +/+ CCR6 CD4 Th17 cells (Fig. 6A), both in terms of the percentage pared with the LAIR1 mice, but due to a single outlier the + of LAIR1 cells and the median fluorescence intensity of LAIR1 values were trending toward statistical significance (Fig. 7D, (Fig. 6A). Similar to the published data, both the DCs and Table II). However, a significant difference was visible in fibrosis monocytes expressed LAIR1 (Fig. 6B), and plasmacytoid DCs between the LAIR1+/+ and LAIR12/2 groups (Fig. 7D, Table II). (pDCs) also expressed the highest levels of LAIR1 (30). Sur- The transplanted lungs were analyzed for their collagen content, + 2 prisingly, both the CD16 and CD16 monocytes expressed with the native lung used as a baseline control (PBS). Lung al- LAIR1 at levels comparable to the pDCs (Fig. 6B). Because most lografts from non-OB LAIR1+/+ mice but not the LAIR12/2 mice of the ColV-specific T cells expressed the ⍺b TCR (Supplemental showed a significant increase in the amount of collagen (Fig. 7E). Fig. 1B), the expression of LAIR1 on gd T cells was excluded from this publication. These results indicate that the differential regulation of Th1 and Th17 cells seen in vivo is not due to a lack of Discussion LAIR1 expression on Th17 cells. In this study we analyzed the role of LAIR1 in Th1 versus Th17 2/2 responses. We set out to test the hypothesis that the ITIM-bearing LAIR1 mice mount deficient chronic rejection responses to LAIR1 receptor will inhibit both responses (10). Recently, LAIR1 heart and lung allografts has been found to have diverse roles in the immune system; for Given that the LAIR12/2 mice were completely unable to make example, LAIR1 signaling induced stem cell–like proliferative a natural Th17 response to ColV, and they were defective in capabilities to acute myeloid leukemia cells, resulting in worse their induced Th17 responses, we determined whether they would disease outcome (11). Additionally, LAIR1 was found to aid in the 8 LAIR1 INHIBITS Th1 BUT NOT Th17 RESPONSES Downloaded from http://www.jimmunol.org/ FIGURE 7. Murine organ transplantation from a B10 donor into B6 LAIR1+/+ or B6 LAIR12/2 recipients. Murine hearts were transplanted from a B10 donor to an LAIR1+/+ (n = 2) or LAIR12/2 (n = 4) recipient as described. (A) Representative H&E (top) and trichrome (bottom) staining. Original magnification 320. LAIR12/2 recipients consistently demonstrated a lower infiltration of cells into the graft, as well as lower collagen deposition around the blood vessels, as compared with the LAIR1+/+ littermates. (B) Murine left lungs were transplanted from a B10 donor to an LAIR1+/+ (n =2) or LAIR12/2 (n = 5) recipient as described. Left, A decrease in acute cellular and vascular rejection was seen between the LAIR1+/+ and LAIR12/2 mice. #p , 0.01 by unpaired t test. Right, Statistical significance for vascular rejection could not be achieved due to a single outlier. *p = 0.4466, by unpaired t test. (C) Native and transplanted lung at day 21 posttransplant, along with the corresponding H&E and trichrome staining (original magnification 340). OB lesions are indicated by arrows (18). The first and second columns shows representative histology from LAIR1+/+ mice, one that developed OB and one that did not develop OB. The LAIR12/2 (third column) grafts did show cellular infiltration, but they had markedly reduced collagen deposition around the airways as compared with the LAIR1+/+ recipients. (D) A decrease in both perivascular inflammation and peribronchial fibrosis was noted by guest on September 26, 2021 between the LAIR12/2 mice. A statistically significant difference in peribronchial fibrosis was noted between the LAIR1+/+ and LAIR12/2 mice, ##p , 0.05 by unpaired t test. Statistical significance was trending toward a decrease for perivascular inflammation, *p = 0.0522 by unpaired t test. (E) Quantity of collagen deposition in the grafts, using the hydroxyproline assay (26, 27). Native lung (n = 4) was used as a baseline control (PBS). The LAIR12/2 (n =4) mice had lower collagen content than the LAIR1+/+ mice that did not develop OB (n = 4) and was comparable to the baseline control. pathogenesis of the myeloid-based chronic myeloid leukemia similar to the WT B6 mice, the B6 LAIR12/2 littermates com- (31). These results clearly indicate that LAIR1 can have both pletely lacked these responses. (Fig. 1A, 1B). Neither the neu- + activating and inhibitory functions. Because we were analyzing tralization of TGF-b1 (Fig. 1B) nor the depletion of CD25 Treg the role of LAIR1 in the context of in vivo studies, we used the cells (Supplemental Fig. 1A) revealed any pre-existing ColV Tv-DTH assay, which enabled us to analyze the role of LAIR1 in responses in the LAIR12/2 mice, which were dependent on ⍺b the presence of its ligands (21). Additionally, the bioassay was TCR+ CD4+ T cells (Supplemental Fig. 1B), suggesting that more sensitive to the influence of IL-17/Th17 cells, as compared LAIR1 is indispensable for the natural Th17 response to ColV in with an in vitro assay (Figs. 1, 2). naive mice. Whether the lack of ColV response was due to a Similar to the previously published data on CBA mice (9), we functional unresponsiveness of the cells or the complete absence found that WT B6 mice also had pre-existing responses to ColV of a responder population remains to be determined. Tetramer regulated via a TGF-b1 mechanism (Fig. 1A). Although the B6 analysis of the T cell population in LAIR12/2 mice will answer LAIR1+/+ littermates had robust pre-existing responses to ColV these questions.

Table I. Histological analysis of murine cardiac allografts

Transplant No.a Donor Recipient POD Acute Cellular Rejection Myocardial Fibrosis Score Acute Vascular Rejection 1 B10 B6 LAIR12/2 42 2 1 2 2 B10 B6 LAIR12/2 41 1B 0 0 3 B10 B6 LAIR12/2 42 1A 0 0 4 B10 B6 LAIR12/2 41 1B 0 0

5 B10 B6 LAIR1+/+ 41 3A 0 3 6 B10 B6 LAIR1+/+ 40 3A 1 0 aHistological analysis of B10 cardiac allografts transplanted into a B6 LAIR1+/+ or B6 LAIR12/2 recipient. The cardiac grafts were scored according to the criteria in the Heart Rejection Study Group (24). POD, postoperative day. The Journal of Immunology 9

Table II. Histological analysis of murine lung allografts

Transplant No.a Donor Recipient POD ACR-A ACR-B Peribronchial Fibrosis OB 1 B10 B6 LAIR12/2 21 1 Low 0 No 2 B10 B6 LAIR12/2 21 2 Low 1 No 3 B10 B6 LAIR12/2 21 1 Negative 0 No 4 B10 B6 LAIR12/2 21 1 Low 1 No 5 B10 B6 LAIR12/2 21 0 Negative 0 No

6 B10 B6 LAIR1+/+ 21 3 Low 3 Yes 7 B10 B6 LAIR1+/+ 21 2 Low 2 No aHistological analysis of B10 lung allografts transplanted into a B6 LAIR1+/+ or B6 LAIR12/2 recipient. Lung transplants were scored following the International Society of Heart and Lung Transplant 2007 classiﬁcation (25). POD, postoperative day.

We have shown that the natural Th17 response to ColV can be inhibit Th17 cells. We are currently investigating whether the induced by the 160-aa-long ColV a1 fragments but not the 15mer pre-existing responses to ColV contribute to the development of peptides of ColV (9). Additionally, in this study, we have dem- fibrosis and chronic rejection following organ transplantation (2), onstrated that sLAIR1/Fc binds the ColV fragment but not the in atherosclerosis (8) or in chronic pulmonary hypertension caused peptide (Fig. 3, Supplemental Fig. 2). Therefore, our data strongly by hypoxia (36, 37) (L.V. Gonzalez-Bosc, E. Jankowska-Gan, and Downloaded from suggests that a secondary signal, generated by LAIR1 ligation, by W.J. Burlingham, unpublished observations). its natural GPO ligands, is required for the of Th17 responses to Despite an increased LAIR1 expression (Fig. 6) on Th17 cells, ColV. Interestingly, also note that the secondary signal, in case of they were not inhibited by LAIR1, supporting the concept that ColV responses, can be triggered by the Ag itself. Additionally, LAIR1 is actually required for Th17 responses. The differential C1q did not completely inhibit sLAIR1/Fc from binding the ColV regulation of Th1andTh17 by LAIR1-collagen engagement could coated on the plate, indicating differences in LAIR1 ligand be due to the differential recruitment of secondary messengers in http://www.jimmunol.org/ binding. Such differences have also been noted between the Th1andTh17 cells, similar to the recruitment of CAMK1 and LAIR1–collagen and the LAIR1–anti-LAIR1 Ab interaction (32). CREB in acute myeloid leukemia cells (11). Thus, the recruitment The induced Th17 response, which was completely dependent of secondary messengers, either inhibitory or activating, likely de- on IL-17, TNF-a,andIL-1b, was also diminished in the LAIR12/2 pends on the type of LAIR1-ligand engagement on the cell surface mice (Fig. 2A, 2C). The induced ColV response in patients fol- (receptor oligomerization due to GPO repeats in collagens versus lowing lung transplantation was shown to be completely depen- dimerization using an Ab). Recently, it was published that CREB dent on monocytes as APCs (2). Monocytes expressed high levels was essential for Th17 cells in vivo and that its absence in CD4 of LAIR1 (Fig. 6B), and the addition of ColV to purified mono- T cells lead impaired Th17 differentiation and the development of cytes induced the production of IL-1b and TNF-a (5). The need autoimmunity (38). We hypothesize that the CREB required for by guest on September 26, 2021 for monocytes as APCs, the indispensable requirement of IL-1b, Th17 development could be recruited by LAIR1 binding via SHP1. and the expression of PLZF T cells (9) indicate the ColV response Another possibility is that LAIR1 is in fact required for the to be a true innate Th17 response. It is highly possible that the inhibition of the corresponding ColV-specific Treg cells that inhibit deposition of ColV in the transplanted organ provides a damage- ColV-specific Th17 cells. It is possible that LAIR1 in fact inhibits associated molecular pattern signal to the monocytes inducing the the Treg cells, thus augmenting the Th17 responses. However, such production of IL-1b and TNF-a. These cytokines further support a phenomenon would not fully explain the lack of responses seen 2/2 the development of the Th17 response to ColV posttransplant. in LAIR1 mice. Another possibility is that LAIR1 might in- 2/2 Such a possibility is supported by the fact that LAIR1 mice hibit Treg cells but provides a positive signal to Th17 cells. If the had lower fibrous (i.e., collagen) deposition and chronic rejection, level of LAIR1 expression is inversely related to its inhibitory despite having cellular infiltration into the transplanted organ. function (Fig. 6), then a low level of LAIR1 on Treg cells as The addition of sLAIR1/Fc and C1q into the assay system further compared with the Th17 cells (V.V. Agashe and W.J. Burlingham, supported the notion that LAIR1 inhibits Th1 but is required for unpublished observations) predicts that exposure of LAIR1 +/+ Th17 responses (Figs. 4, 5). Similar results were seen with Th1/17 ligands might upset Treg/Th17 equilibrium in the LAIR1 mice, 2/2 anti-donor responses in kidney transplant patients with chronic leading to chronic rejection, but that in LAIR1 mice the Treg rejection (Fig. 5C). This suggests that if we could modulate the and Th17 cells would remain in equilibrium, thereby decreasing immune response with LAIR1 antagonists, it might be possible to chronic rejection. It is interesting to speculate that lower LAIR1 induce suppression of Th17 but maintain Th1 immunity, similar to expression on cells inhibits them, but increased LAIR1 induces a the P2X7R (5). One such approach is the use of LAIR2, a natu- positive signal. Lastly and importantly, note that LAIR1 ligands rally occurring soluble form of LAIR1. Use of LAIR2 inhibited bind differently to the receptor. Preincubation of sLAIR1/Fc with the complement-mediated cytotoxicity in an ex vivo model of Ab C1q did not completely inhibit sLAIR1/Fc binding to collagens mediated rejection (33). coated on the plate, where incubation with matrix collagen Vor XI In the B10-to-B6 model, the grafts downregulated complement did (Fig. 3B). These possibilities, however, do not answer all the regulatory proteins, resulting in complement activation and en- questions regarding LAIR1 but provide exciting new avenues for hanced C3a deposition. Increased C3a upregulated IL-17 pro- LAIR1 research. duction that, in a feedback loop, further decreased the complement Thus, the analysis of TT/DT and EBV responses confirmed that regulatory proteins (34). Thus, the intervention with a complement LAIR1 can be a powerful inhibitory receptor in the immune system inhibitor, for example, C1 inhibitor, will be helpful in acute re- through the phosphatase activity of SHP1, as described previously jection and prevent complement activation during chronic rejec- (10). However, it is essential for the development of Th17 re- tion (35). However, our data suggest that free C1q in the system sponses and our results argue for a unique nonredundant role of during chronic rejection, although not detrimental, might not LAIR1 in Th17 responses. 10 LAIR1 INHIBITS Th1 BUT NOT Th17 RESPONSES

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2 Figure S1: Pre-existing responses to ColV required LAIR1 and the traditional ⍺β T cells.

-/- + 3 A. Pre-existing responses to ColV were not detected in the LAIR1 littermates after CD25 Treg

Whole spleen cells 40 4 cell depletion. ) s -

e CD25 T cells + Monocytes

h c

n * Value is 0

i 30

5 4

(

g 20

n i

6 l

l e

w 10

7 e

* N 0 I l lV 8 o o C C Cells + Antigen 9 B. Intracellular cytokine staining after overnight stimulation of mouse spleen cells.

16 (A) Naïve LAIR1-/- mice were used to determine the presence of pre-existing responses to ColV

17 in the CD25- T cells mixed with isolated monocytes. Similar to the results seen with TGFβ1

18 neutralizations (Fig 1B), whole spleen cells did not show a response to ColV, although there was

19 a higher background response. (B) Spleen cells from LAIR1+/+ and LAIR1-/- mice were

20 stimulated overnight in the presence of ColI or ColV and then stained to determine the

21 production of IL17 and INFγ. IFNγ and IL17 are produced by 2 distinct CD3+ CD4+ T cells. γδ T

22 cells did not contribute to the ColV response and were not a source of IL17. The traditional ⍺β T

23 cells produced IL17 in response to ColV.

1 24 Figure S2: Characterization of sLAIR1/Fc.

25 A. PAGE and Western blot of sLAIR1/Fc B. Inhibition of sLAIR1/Fc binding to ColV

26 Col V p599 (IAb/DQ peptide) 1.5 p909 (IAb/DQ peptide)

27 m p629 (DR peptide)

n 0

5 p799 (DR peptide)

4 1.0

28 a p1049 (DR peptide)

. D

. p1439 (DR peptide)

O 0.5 29 TT C1q 0.0 Col XI .0 .5 .0 .5 .0 30 0 0 1 1 2 Concentration of sLAIR1 in ug/ml 31

32 C. Histology of mouse footpad stained with sLAIR1/Fc

37 Trichrome sLAIR1/Fc staining

38 sLAIR1/Fc construct, purified in house in two different cell lines, was tested for its ability to

39 bind to various proteins and peptides of interest. (A) PAGE and Western blot demonstrate a band

40 at 60kDa, the expected molecular weight. Probing with commercially available LAIR1 Ab and

41 an -human IgG1 Ab, indicates the presence of sLAIR1/Fc. (B) Increasing the concentrations of

42 sLAIR1/Fc did not bind the peptides, but bound the ColV coated on the plate (n=2-3

43 experiments, representative plots from one experiment are shown). (C) Shows trichrome staining

44 (left) of a mouse foot pad, indicating the presence and location of collagens, in blue. On the

45 right, the footpad was stained with sLAIR1/Fc and detected with an α-human Fc HRP conjugated

46 secondary Ab, indicating the regions in the footpad where sLAIR1/Fc can bind.

2 47 Figure S3: Effect of αLAIR1 Ab and natural ligands of LAIR1 on ColV peptide responses.

48 )

s ColV peptide + Anti LAIR-1 e

h Ab

c n

49 i ColV peptide + ColI

4 - 20

0 ColV peptide + C1q

x (

i l

l 10 e

51 w

t e 52 N 0

g g g g g g A u u u u u 5 1 5 5 .1 . . 53 0 0 2 Cells + 54 Amount of Anti LAIR1 Ab or ColI or C1q 55

56 Lymphocytes from ColV peptide p599 immunized mice were used to determine the effect of

57 three different LAIR1 ligands (C1q, ColI and LAIR1 Ab) on the Th17 response. The addition of

58 ColI and C1q, two in-vivo ligands of LAIR1did not inhibit the Th17 ColV peptide response.

59 However, addition of an αLAIR1 Ab inhibited the Th17 ColV response down to baseline

60 (αLAIR1 Ab n= 2 separate experiments, performed with pooled splenocytes, ColI and C1q n=1,

61 performed with pooled splenocytes from 8 mice).

3 70 Figure S4: Murine cardiac transplants between B10 donors and B6 recipients.

s 400 l

l IL17A

72 6 IFNγ

0 300

73 e

s t 200

74 o

75 n 100

o t

76 y C 0 B10 Ag (Donor) B6 Ag (Self) 77 Antigen 78 Similar to the published B10 to B6 orthotopic lung transplant model (18), our lab explored and

79 established a B10 to B6 heterotopic cardiac model (Keller et al. Unpublished data). By day 40

80 post-transplant chronic rejection developed in the allografts (Keller et al. Unpublished data).

81 Spleen and lymph nodes were obtained from these animals (n=5) at day 40 post-transplant and

82 analyzed for the production of IL17 and IFNγ in response to the donor antigen. Unlike the lung

83 transplant model, the cardiac transplant model was found to be a Th1/17 donor-based model.