Characterization of the Expression and Function of the C-Type Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration This information is current as of September 29, 2021. Tsun-Ho Lo, Pablo A. Silveira, Phillip D. Fromm, Nirupama D. Verma, Phi A. Vu, Fiona Kupresanin, Rhonda Adam, Masato Kato, Victoria C. Cogger, Georgina J. Clark and Derek N. J. Hart

J Immunol 2016; 197:885-898; Prepublished online 17 June Downloaded from 2016; doi: 10.4049/jimmunol.1600259 http://www.jimmunol.org/content/197/3/885 http://www.jimmunol.org/

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Characterization of the Expression and Function of the C-Type Lectin Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration

Tsun-Ho Lo,*,†,1 Pablo A. Silveira,*,†,1,2 Phillip D. Fromm,*,† Nirupama D. Verma,*,† Phi A. Vu,* Fiona Kupresanin,* Rhonda Adam,* Masato Kato,‡ Victoria C. Cogger,†,x Georgina J. Clark,*,† and Derek N. J. Hart*,†,{,2

C-type lectin receptors play important roles in immune cell interactions with the environment. We described CD302 as the simplest, single domain, type I C-type lectin receptor and showed it was expressed mainly on the myeloid phagocytes in human blood. CD302 colocalized with podosomes and lamellopodia structures, so we hypothesized that it played a role in or migration. In this study, we used mouse models to obtain further insights into CD302 expression and its potential immunological Downloaded from function. Mouse CD302 transcripts were, as in humans, highest in the liver, followed by lungs, lymph nodes (LN), spleen, and bone marrow. In liver, CD302 was expressed by hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells. A detailed analysis of CD302 transcription in mouse immune cells revealed highest expression by myeloid cells, particularly macrophages, granulocytes, and myeloid dendritic cells (mDC). Interestingly, 2.5-fold more CD302 was found in migratory compared with resident mDC populations and higher CD302 expression in mouse M1 versus M2 macrophages was also noteworthy. CD302 knockout (CD302KO) mice were generated. Studies on the relevant immune cell populations revealed a decrease in the frequency and http://www.jimmunol.org/ numbers of migratory mDC within CD302KO LN compared with wild-type LN. In vitro studies showed CD302KO and wild-type DC had an equivalent capacity to undergo maturation, prime T cells, uptake Ags, and migrate toward the CCL19/CCL21 chemokines. Nevertheless, CD302KO migratory DC exhibited reduced in vivo migration into LN, confirming a functional role for CD302 in mDC migration. The Journal of Immunology, 2016, 197: 885–898.

ells of the immune system use various pattern recognition defined by the presence of at least one C-type lectin-like domain receptors to distinguish molecular patterns displayed on (CTLD) (1). Although most CLR bind specific types of carbohy- C pathogens or damaged cells. The C-type lectin receptors drates using calcium-dependent interactions, some bind carbohy- by guest on September 29, 2021 (CLR) are a large superfamily of pattern recognition receptors drates in a calcium-independent manner and others have evolved to bind lipid or ligands. In addition to pathogen recognition, *Dendritic Cell Research, ANZAC Research Institute, Sydney, New South Wales certain CLR can contribute to other immune cell functions, in- 2139, Australia; †Sydney Medical School, University of Sydney, Sydney, New South cluding cell adhesion and migration (2–4), lymph node (LN) ex- Wales 2006, Australia; ‡Mater Medical Research Institute, Brisbane, Queensland x pansion and contraction (5), and glycoprotein turnover (6). 4101, Australia; Biogerontology Laboratory, ANZAC Research Institute, Sydney, New South Wales 2139, Australia; and {Department of Haematology, Royal Prince CD302 (also known as DEC-205–associated C-type lectin-1 Alfred Hospital, Sydney, New South Wales 2050, Australia [DCL-1] or CLEC13A) is a recently identified CLR, which was 1T.-H.L. and P.A.S. contributed equally to performing the research in this paper. discovered originally as the 39 end of a transcript of an intergenic 2P.A.S. and D.N.J.H. share senior authorship. splice variant cloned from the human Hodgkin Reed–Sternberg ORCIDs: 0000-0003-0229-6004 (T.-H.L.); 0000-0001-5074-8824 (P.A.S.); 0000- lymphoma cell line, L428 (7). This fused transcript encoded a 0001-7047-1113 (P.D.F.); 0000-0002-6041-6804 (M.K.); 0000-0002-6346-9928 protein with the CD205 extracellular domain, containing 10 (V.C.C.); 0000-0001-7894-232X (G.J.C.); 0000-0003-2620-1612 (D.N.J.H.). CTLD, spliced to the single CTLD, transmembrane, and intra- Received for publication February 12, 2016. Accepted for publication May 23, 2016. cellular regions of CD302. The mRNA of CD205-CD302 fusion This work was supported by National Health and Medical Research Council Program protein was expressed in two forms with either exon 33 or 34 of Grant 543727 and by the Rebecca Cooper Foundation. D.N.J.H. is a National Health and Medical Research Council Senior Professorial Research Fellow. the CD205 transcript fused to exons 2–6 of CD302. A later study Address correspondence and reprint requests to Prof. Derek N.J. Hart, Dendritic Cell demonstrated that the CD205-CD302 intergenic fusion protein Research, ANZAC Research Institute, Gate 3 Hospital Road, Concord Hospital, was also expressed by activated primary human dendritic cells Sydney, NSW 2139, Australia. E-mail address: [email protected] (DC), although the conventional single CD205 transcript and The online version of this article contains supplemental material. protein were the dominant isoforms expressed by these cells (8). Abbreviations used in this article: AF, Alexa Fluor; B6, C57BL/6; BM, bone marrow; CD302 was subsequently characterized as a unique protein, and it BMDM, bone marrow–derived macrophage; CD302KO, CD302 knockout; CLR, C-type is the simplest type I transmembrane CLR described (9). It has been lectin receptor; CT, threshold cycle; CTLD, C-type lectin-like domain; CTV, CellTrace Violet; DBP, dibutyl phthalate; DC, dendritic cell; DN, double-negative; EST, expressed classified into the group XV C-type lectin family (10). The human sequence tag; FL-DC, Flt3L–derived DC; GM-DC, GM-CSF plus IL-4–derived DC; IFC, CD302 gene consists of 29 kbp, containing six exons, and is located interfollicular channel; KC, Kupffer cell; KO, knockout; Lin, lineage; LN, lymph node; ∼ LSEC, liver sinusoidal endothelial cell; Mf, macrophage; mDC, myeloid DC; MHC II, 5 kbp downstream of the CD205 gene on 2q24 (9). It MHC class II; Mo, monocyte; pDC, plasmacytoid DC; qPCR, quantitative real-time encodes a protein consisting of 232 aa, containing a signal peptide, a PCR; SCS, subcapsular sinus; viSNE, visualized t-distributed stochastic neighbor em- single CTLD, short spacer, transmembrane region, and a cytoplas- bedding; WT, wild-type. mic tail. In nonreduced conditions, the protein was found to exist in Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 24 and 30 kDa states, presumably reflecting different glycosylated www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600259 886 CD302 EXPRESSION AND FUNCTION forms. The extracellular region is predicted to contain eight b strands Female mice were used for experiments except where indicated. All mouse and two a helices using nuclear magnetic resonance (11). experiments were approved by the Sydney Local Health District Animal The highest expression of CD302 transcripts in humans was ob- Ethics Committee. served within liver followed by lungs, PBMC, and spleen (9). Among Human hepatic cell lines and tissue samples human leukocytes, CD302 transcription was restricted to the myeloid- HepG2 and SK-HEP-1 hepatic cells lines were purchased from American derived populations, including monocytes (Mo), macrophages (Mf), Type Culture Collection (Manassas, VA) and grown in DMEM with 1 or 4.5 myeloid DC (mDC), and granulocytes. Activation of Mo-derived Mf g/l D-glucose, respectively, supplemented with 10% FCS, 2 mM Gluta- or DC led to decreased expression of CD302. Several mouse mAbs MAX, 100 U/ml penicillin, and 100 mg/ml streptomycin (Thermo Fisher). (MMRI-18, 19, 20 and 21) specific for human CD302 were devel- Human PBMC were obtained by venesection from healthy donors at Concord Hospital following their informed consent. PMBC were isolated oped, and MMRI-20 was used to confirm CD302 protein surface by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare) expression on myeloid cells (9). Confocal microscopy colocalized according to the manufacturer’s protocols. For histology, liver sections CD302 with F-actin–rich filopodia, lamellopodia, and podosomes in were prepared from biopsies of normal donor tissue for liver transplanta- Mf and transfected cell lines. The association of CD302 with these tion obtained from the Liver Transplant Unit at the Royal Prince Alfred migratory structures (12) implicated a potential role for CD302 in cell Hospital. Spleens sections were prepared from cadaveric organ donors obtained from the Australian Red Cross Blood Services. All human ex- adhesion or trafficking. periments were approved by the jurisdictional Ethics Committees in A ligand for CD302 has yet to be identified, and no function Sydney as well as the Institutional Review Boards. beyond its potential role in cell migration has been described. Generating single-cell suspensions from mouse tissues Unlike other classical CLR such as CD206, CD209, or CD301, the

CD302 CTLD is devoid of the known amino acid residues essential Single-cell suspensions were made from mouse spleen, thymus, and LN by Downloaded from for calcium-dependent sugar binding, for example, the EPN motif mechanical disruption through 70-mm cell strainers in RPMI 1640 media supplemented with 2% FCS (Thermo Fisher; 2% FCS/RPMI 1640). RBC (for mannose binding), the QPD motif (for galactose binding), and 3 2+ were removed from spleens with 1 RBC lysis buffer (eBioscience). In the WND motif (for Ca binding), suggesting that CD302 does the indicated experiments, LN or spleens were digested with 1 mg/ml not have classic sugar binding capacity (9). CD302 was found to collagenase type III (Worthington Biochemical) and 20 mg/ml DNase I mediate the of microbeads coated with anti-CD302 (Roche) solution for 30 min at 37˚C before mechanical disruption. Bone marrow (BM) cells were collected by flushing femurs and tibiae with 2% Abs (9), indicating that, similar to many other CLR, it can act as http://www.jimmunol.org/ FCS/RPMI 1640 using a 25-gauge needle. Skin cells were extracted from an Ag uptake receptor. However, it was significantly less efficient mouse ears cut at the base. Dorsal and ventral sides were separated, cut than mAb loading via the CD205 or CD206 molecules. into small pieces, and digested with collagenase type III/DNase I solution There is a high degree of sequence identity (76%) and similarity for 1 h at 37˚C. Liver leukocyte suspensions for flow cytometry were (81%) between human and mouse CD302 orthologs, consistent obtained from the right lateral lobe, which was perfused (see below), diced, with a conserved function (9), and this encouraged us to investi- and digested in collagenase type III/DNase I solution for 30 min at 37˚C. Digested cells were passed through a 70-mm filter followed by a 40-mmfilter gate this CLR in mouse models to gain a better understanding of to obtain a single-cell suspension. RBC were removed as above. To enu- its function. We confirmed the gene structure for mouse Cd302 merate, cells were counted in a fixed volume (10–50 ml) using the Accuri C6 and examined the expression of Cd302 mRNA in isolated leuko- flow cytometer (BD Biosciences). by guest on September 29, 2021 cyte populations and other tissues. We developed and validated a Flow cytometry CD302 knockout (CD302KO) mouse to test how CD302 con- tributed to Mf and DC function. We found a very similar CD302 Single-cell suspensions were stained in FACS buffer (0.1% BSA and 2 mM EDTA in PBS) using optimized concentrations of Fc Block (2.4G2; BD distribution in mice to humans, with CD302 mainly expressed in Biosciences) and then combinations of the following fluorochrome-labeled the liver, lungs, and myeloid immune populations, including Mf, anti-mouse mAbs: CD4 (RM4-5), CD11b (M1/70), CD45 (30F-11), granulocytes, and mDC. Our functional studies showed that higher CD45R/B220 (RA3-6B2), CD80 (16-10A), CD103 (2E7), H-2Kb (AF6-88.5), CD302 expression was associated with the migratory DC subsets IA-IE (M5/114-15.2), and Ly6C/G (RB6-8C5) from BD Biosciences; CD8a and its deletion reduced their ability to migrate into draining LN. (53-6.7) and Ly6C (HK1.4) from eBioscience; and CD11c (N418), CD24 (M1/69), CD40 (3/23), CD83 (Michel-19), CD86 (GL-1), CD90.1 (OX-7), CD302 therefore has an important role to play in cell migration. and CD205 (NLDC145) from BioLegend. To distinguish rare myeloid However, other functions for this CLR seem likely, as, in the liver, populations, cells were also stained with a lineage (Lin) mixture containing CD302 is produced by hepatocytes and liver sinusoidal endothelial biotinylated mAbs: CD3e (145-2C11) and CD19 (1D3) from BD Biosci- cells (LSEC) and Kupffer cells (KC). ences, and Ly6G (1A8) and NK1.1 (PK136) from BioLegend. Lin Abs were detected using streptavidin–Brilliant Violet 421 (BD Biosciences). For hu- man studies, fluorochrome-conjugated anti-human Abs specific to the fol- lowing molecules were used: CD1c (L161), CD11c (B-ly6), CD14 (M5E2), Materials and Methods CD16 (3G8), CD20 (2H7), CD56 (B159), and HLA-DR (L243) from BD Mice Biosciences; CD56 (HCD56), CD86 (IT2.2), and CD127 (A01D5) from BioLegend; and CD141 (AD5-14H12) and CD304 (AD5-17F6) from All mice were housed at the ANZAC Research Institute under specific Miltenyi Biotec. Human CD302 was detected with the FITC-conjugated pathogen-free conditions. The gene construct for the CD302KO mice was mouse mAb, MMRI-20 (9). The gating strategy for human DC/Mo is generated and the microinjections were performed in partnership with described in Kassianos et al. (13). Intracellular CD302 in HepG-2 and Ozgene (Bentley, Australia). The construct with loxP sites flanking the SK-HEP-1 lines was determined by blocking surface CD302 with unla- Cd302 start codon and the downstream intronic region of exon 1 was in- beled MMRI-20 prior to fixation and permeabilization with the Foxp3 troduced into embryonic stem cells of C57BL/6 (B6) mice using PGKneo staining buffer set (eBioscience) according to the manufacturer’s in- as a reporter. Mice with the integrated construct were crossed with structions. MMRI-20–FITC was used to label intracellular CD302. DAPI ROSA-Cre-deletor mice to remove the loxP flanked region, thereby (3 mM) was added to samples to exclude dead cells. Data were collected deleting exon 1 of Cd302 in germline cells to creating the founder het- on the Accuri C6 flow cytometer or Influx cell sorter instruments (BD erozygous wild-type (WT)/knockout (KO) mice. Founder animals were Biosciences) and analyzed using FlowJo software (Tree Star). Visualized intercrossed to produce homozygous CD302 KO/KO animals without t-distributed stochastic neighbor embedding (viSNE) (14) was performed ROSA-Cre. The following PCR primers were used to distinguish CD302 on arcsinh-transformed fluorescent parameters using Cytobank (15). WT and KO genotypes: CD302 forward, 59-CCAGGAGGGAATTGT- GAAAA-39, reverse 1, 59-ACACCGCAGACTCCAACTTC-39, reverse 2, Mouse immune cell purification and stimulation 59-GCCTGGCATGGTAGAGAGAG-39. B6 and BALB/c mice were pur- chased from the Animal Resource Centre (Perth, Australia). OT-I and OT-II For transcriptional analyses, splenic B, T, and NK cells, granulocytes, and TCR transgenic CD90.1-congenic (B6) mice were obtained from Robert Mf were isolated from RBC-depleted splenocytes. Cells were labeled Brink and Elissa Deenick, respectively (Garvan Institute, Sydney, Australia). with fluorochrome-labeled Abs to separate B cells (B220, CD19, and The Journal of Immunology 887

IA/IE), CD4+ and CD8+ T cells (CD3e, CD4, and CD8), NK cells (DX5 CAAGTGTGTCAAG-39 and 59-GGCACAGCCATCACT-GTAAGG-39), or and NK1.1), granulocytes (CD11b and Ly6C/G), and Mf (F4/80) by Gapdh (59-AGGCCGGTGCTGAGTATGTC-39 and 59-ATGCCTGCTTCAC- FACS using the BD Influx cell sorter (BD Biosciences). Mouse DC were CACCTTC-39). RT-PCR was performed on 1 mg cDNA with Taq polymerase enriched from collagenase/DNase I–treated spleen or LN suspensions according to the manufacturer’s instructions (Thermo Fisher), and amplified using the OptiPrep density barrier (Sigma-Aldrich) according to the products were electrophoresed on a 2% agarose gel. For quantitative real-time manufacturer’s instructions. Light density cells were labeled with bio- PCR (qPCR), primers specific for Cd302 (mouse, 59-CGCCGACTGTCCTT- tinylated Lin Abs (as before) and anti-biotin microbeads (Miltenyi CATC-39 and 59-TTCCACATTGATGGTTACT-TGAAGA-39;human,59- Biotec)toenrichDCbyMACSnegativeselectiononanautoMACSPro CTCCTGCTGCCGTTGCTG-39 and 59-CAA-CTGTCTTGGAACTGAATC- instrument (Miltenyi Biotec). DC subsets were subsequently sorted to CA-39)orHprt (mouse, 59-CAATGCAAA-CTTTGCTTTCCC-39 and 59- high purity (.90%) by FACS using the gating strategies outlined in AAATCCAACAAAGTCTGGCC-39; human, 59-AATTATGGACAGGACTG- Supplemental Fig. 1A and 1B (spleen) and Fig. 5C (LN). Activated AACGTCTTGCT-39 and 59-TCCAGCA-GGTCAGCAAAGAATTTATAGC- B cells, DC, and Mf for transcription studies were cultured for 4 h in 39) were mixed with triplicate samples of 1 mg cDNA and 13 Fast SYBR complete RPMI (RPMI 1640 with 10% FCS, 2 mM GlutaMAX, 100 U/ml Green Master Mix (Thermo Fisher). Amplifications were performed on a penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, 7500 Fast real-time PCR system (Thermo Fisher). Threshold cycle (CT) and 50 mM 2-ME from Thermo Fisher) with 1 mg/ml LPS (Escherichia coli values for Cd302 amplification were normalized to the Hprt house- K12, InvivoGen), and T cells with a 1:1 ratio of mouse T-Activator CD3/28 keeping gene and are presented as fold changes to the mean of a refer- 2 Dynabeads (Thermo Fisher). To isolate hepatocytes, LSEC, and KC for ence sample using the formula: fold change = 2 DDCT. Primer efficiencies transcriptional analysis, livers of WT or KO mice were perfused with col- of all mouse and human primers were .98%. lagenase III and extracted as previously described (16, 17). The resulting liver cell suspension was twice centrifuged at 54 3 g to pellet hepato- Development of a rat anti-mouse CD302 mAb cytes, and the residual supernatant was layered on a two-step Percoll Hybridomas were developed in partnership with the Monash Antibody 3 gradient (25–50%) and centrifuged for 30 min at 1350 g. Cells collected at Technologies Facility (Clayton, Australia). Rats were immunized i.p. with the interface were stained with CD11b, CD45, and F4/80 Abs to separate KC Downloaded from + + 2 + 2 2 16 mg recombinant mouse extracellular CD302 (aa 1–156) protein fused to (CD11b F4/80 )andCD45 /CD45 (CD11b F4/80 ) LSEC by FACS on a polyhistidine tag (Sino Biological) in Sigma Adjuvant System plus the Influx cell sorter. To examine upregulation of DC maturation markers by methylated CpG (Sigma-Aldrich). Splenocytes were fused to the mouse flow cytometry, WT or CD302KO splenocytes or LN cells were cultured myeloma cell line SP2/0-Ag14 using a conventional polyethylene glycol 18 h in complete RPMI with or without the presence of stimuli (1 mg/ml fusion protocol. IgG-producing hybridomas specific for recombinant ex- either LPS, poly(I:C), or CpG2006; all from InvivoGen). tracellular CD302 (aa 1–156) fused to a human Fc tag (Sino Biological) Human myeloid subset isolation were selected by ELISA. The DCR1 hybridoma, showing the highest specific binding to mouse CD302, was adapted to a serum-free medium http://www.jimmunol.org/ For human Mo, PBMCs were labeled with CD14 microbeads (Miltenyi (Hybridoma-SFM; Invitrogen Thermo Fisher). Supernatants containing Biotec), and CD14+ cells were isolated using a MACS positive selection high yields of the DCR1 mAb were used for Western blot and ELISA. The strategy. For DC, cells were Lin depleted on an autoMACS following DCR1 isotype was determined with the rat mAb isotyping test (AbD staining with an Ab mixture containing purified mouse anti-human CD3 Serotec). (HIT3a; Sony iCyt), CD14 (RMO52), CD20 (B9E9), CD56 (N901; Beckman Coulter), CD16 (3G8), and CD235a (GA-R2; BD Biosciences) ELISA + + + and anti-mouse IgG microbeads (Miltenyi Biotec). CD1c ,CD16,CD141 , To test the DCR1 mAb specificity, Nunc microplates (Thermo Fisher) and plasmacytoid DC (pDC) populations were isolated from Lin-depleted were coated overnight with 1 mg/ml CD302-Ig or CD300f-Ig (Sino Bi- cells by staining with CD1c (M5E2; eBioscience), CD141 (AD5-14H12), ologicals) and blocked with 5% BSA/PBS. Plates were overlaid with CD304 (AD5-17F6; Miltenyi Biotec), and HLA-DR (L243; BD Biosciences) DCR1 supernatant (100 ml) or LMIR3 (1 mg/ml; R&D Systems) and then by guest on September 29, 2021 . and sorting them to high purity ( 90%) on the Influx cell sorter. an HRP-conjugated goat anti-rat IgG Fc Ab (Sigma-Aldrich). Sigmafast Generation of cytokine-expanded Mf and DC populations OPD solution (Sigma-Aldrich) was used to measure binding of Abs through a colorimetric change read at 450 nm on a Victor3 multilabel plate reader To generate mouse GM-CSF plus IL-4–derived DC (GM-DC), BM cells (PerkinElmer). (1 3 106 cells/ml) were cultured with 20 ng/ml mouse rGM-CSF and 0.3 ng/ml mouse rIL-4 (PeproTech) in complete RPMI. Cells were Western blots supplemented on day 3 with fresh media containing cytokines. Loosely For Western blots, cell suspensions (at 2.5 3 107 cells/ml) were solubilized adherent cells were collected and replated in fresh complete RPMI on in modified RIPA buffer (1% Triton X-100, 0.25% sodium deoxycholate, day 6 and harvested on day 7. For mouse Flt3L-derived DC (FL-DC), BM 0.15 M NaCl, 50 mM Tris-HCl, 5 mM EDTA [pH 7.4]) containing com- 3 6 cells (2 10 cells/ml) were resuspended in complete RPMI containing 10% plete protease inhibitor (Roche). Lysates were centrifuged at 10,000 3 g supernatant derived from the B16 cell line expressing mouse Flt3L (18) and and the protein content was determined by bicinchoninic acid assay cultured for 9 d without intervention. Maturation of both types of DC was (Pierce). Lysates (5 mg) were fractionated on a 4–12% Bis-Tris gel (Bolt; achieved by adding 0.1 mg/ml LPS on day 6. To generate mouse BM-derived Invitrogen) under reducing conditions. were transferred to nitro- 3 5 Mf (BMDM), BM cells (3 10 cells/ml) were cultured in complete RPMI cellulose (Novex mini blot) using an iBlot according to the manufacturer’s containing 10% L929 cell line supernatant containing murine M-CSF (19). recommendations. Membranes were stained with Ponceau before over- Cells were supplemented with 10% M-CSF supernatant on day 3. After 6 d, night incubation in 5% skim milk (Diploma) in TBST. Membranes were adherent cells were treated with ice-cold 0.6 mM EDTA solution to detach incubated with 5 ml tissue culture supernatant from the DCR1 mAb (or in- BMDM from plates. M-CSF–expanded human Mf were generated from house rat isotype control) and then an HRP-conjugated goat anti-rat IgG Fc purified Mo as in Kato et al. (9). Mf cultures were differentiated into M1 Ab (Sigma-Aldrich). Protein bands were detected by chemiluminescence cells by addition of 0.1 mg/ml LPS with or without 50 ng/ml mouse using Clarity Western ECL substrate (Bio-Rad) and visualized on a Bio- (PeproTech) or human (Thermo Fisher) rIFN-g and M2 cells with 30 ng/ml Rad GelDoc. The molecular masses of visualized bands were determined mouse (PeproTech) or human (Thermo Fisher) rIL-4 for the final 16 h cul- by comparison with colorimetric standards (Precision Plus [Bio-Rad] and ture. All cultures were at 37˚C with 5% CO2. SeeBlue [Thermo Fisher]). Transcriptional analyses by RT-PCR and quantitative real-time Immunohistology PCR Sections of snap-frozen tissue (5–6 mm) were fixed with ice-cold acetone. Total RNA from tissues or purified cells was extracted using TRIzol reagent as Human tissues were stained with anti-human CD68–Alexa Fluor (AF)647 per the manufacturer’s instructions (Thermo Fisher). Integrity and quantity of (BD Biosciences), CD302 (MMRI-20; FITC for spleen and unconjugated extracted mRNA were assessed using an RNA 6000 Nano bioanalyzer for liver) (9), and biotinlayted HLA-DR (Sony iCyt). Secondary Abs in- assay (Agilent Technologies), and 100 ng DNase I (Thermo Fisher)–treated cluded goat anti-mouse AF488 (liver only) and streptavidin-AF555 RNA was synthesized into cDNA using the SuperScript III kit (Thermo Fisher). (Thermo Fisher). Mouse LN sections were stained with rat anti-mouse For RT-PCR, primers included: Cd302 exons 1–6 (59-TCCTCGCTCGTGC- Lyve-1 (clone 223322; R&D Systems) or CD3 (17A2), rat anti-mouse TGCTG-39 and 59-GGCACAGCCATCACTGTAAGG-39), Cd302 exons 2–6 CD11c-biotin (N418), and hamster B220-AF647 (RA36B2) from Bio- (59-TCATCTACCTGGGTCCAGTTCC-39 and 59-GG-CACAGCCATCACT- Legend. Secondary Abs included a goat anti-rat IgG Texas Red conjugate GTAAGG-39), Ly75 (59-GAGGCCAAACAAGTGTG-TCAAG-39 and 59- (SouthernBiotech) and streptavidin-AF488 (Invitrogen). Slides were TCGTTGGTTCCATGTTCATACC-39), Ly75-Cd302 (59-GAGGCCAAA- washed with 0.5% BSA in PBS between stains and blocked with 10% goat 888 CD302 EXPRESSION AND FUNCTION

(Invitrogen), mouse, or rat (Sigma-Aldrich) serum. Tissue sections were and peritoneal lavages performed with 10 ml of a 10 mM EDTA/PBS solution imaged on an EVOS-FL cell imaging system (Thermo Fisher). Mouse containing 1 3 105 TrueCount beads (Thermo Fisher). The frequency and GM-DC were attached to glass coverslips coated with or without 20 mg/ml number of CD11b+F4/80+ Mf in each lavage was determined by flow fibronectin (Sigma-Aldrich) for 1 h, washed three times in 37˚C PBS, and cytometry. the remaining attached cells were fixed with 4% paraformaldehyde and blocked and permeabilized with 10% BSA, 10% goat serum, and 0.1% Triton X-100 in Bioinformatics PBS. Cells were stained with biotinylated anti-mouse CD11c (N418), We used the Ensembl public domain database to analyze the mouse CD302 phalloidin-AF594, and 18 mM DAPI (Thermo Fisher) followed by streptavidin- transcript and protein sequences (accession no. ENSMUST00000074606; AF488 (Thermo Fisher). Coverslips were imaged on a Zeiss LSM510 confocal http://ensembl.org). Homology of the mouse CD302 CTLD region with microscope (Carl Zeiss). Images were processed and analyzed with ImageJ other proteins in the Mus musculus nonredundant protein database was (National Institutes of Health) or Photoshop (Adobe) software. established using the basic local alignment search tool (blastp) from the Mixed leukocyte reactions National Center for Biotechnology Information. Isoelectric point and molecular mass of mouse CD302 was predicted by the Compute pI/Mw LPS-activated WT or CD302KO splenocytes, LN cells, or GM-DC stim- program on the ExPASy server (20). ulators were generated from three individual mice. Splenocytes and LN cells were irradiated with 2500 cGy. Responder T cells were purified Statistical analysis . ( 90%) from pooled BALB/c spleens utilizing a MACS negative deple- All statistical analyses were performed using Prism 6.0 (GraphPad Soft- tion strategy using biotinylated CD11b, CD11c, CD19, and Ly6G Abs and ware). Means with SEM error bars are shown in graphs. Nonparametric anti-biotin microbeads (Miltenyi Biotec). Purified T cells were labeled t tests (with multiple test corrections) were used to determine statistical 3 5 with 0.5 mM CFSE (Thermo Fisher) and 1 10 were mixed with equal differences between groups, with p , 0.05 deemed significant. numbers of activated stimulators. After 3 d culture, proliferation by b + +

H2-K -negative CD4 or CD8 BALB/c T cells were examined by CFSE Downloaded from dilution using flow cytometry. Results Mouse Cd302 gene and protein DC Ag uptake assays The mouse Cd302 gene consists of six exons spanning 32.5 kbp on 3 5 GM-DC (2 10 cells) were incubated in complete RPMI at 37˚C/5% CO2 (60,251,993–60,284,488 bp), producing a full- or on ice in the presence of 1 mg/ml FITC dextran for 30 min or 10 mg/ml DQ-BSA for the times indicated. Cells were washed three times with FACS length transcript of 1354 bp and a protein of 228-aa residues.

buffer and labeled with anti-mouse CD11c and IA/IE Abs to identify DC The isoelectric point and molecular mass of the mature protein are http://www.jimmunol.org/ populations by flow cytometry. predicted to be 4.35 and 23.46 kDa, respectively. The mouse protein Chemotaxis assay contains a signal peptide (aa 1–20, encoded by exon 1), a single CTLD (aa 21–153, encoded by exons 2–4), a spacer (aa 154–166, 3 5 Myeloid cells (5 10 ) purified from collagenase/DNase I–treated spleens encoded by exon 5/6), a transmembrane (aa 167–195, exon 6), and or LN by MACS negative selection with the biotinylated Lin mixture described above were added to the upper chamber, and CCL19 or CCL21 an intracellular region (aa 196–228, exon 6) (Fig. 1A). A compar- (0.1 mg/ml; R&D Systems) was added to the lower chamber of 5-mm ative BLAST of the mouse CD302 CTLD region to a nonredundant Transwell plates (Corning). After 4 h incubation at 37˚C and 5% CO2, mouse proteins database found it had the greatest similarity to migrated cells in the lower chamber were enumerated and stained with Abs CTLD of the CLR subfamily, with the highest recognizing B220, CD4, CD8, CD11b, CD11c, F4/80, IA/IE, and Ly6C/G identity/similarity to the eighth CTLD of the adjacent CD205 and by guest on September 29, 2021 to distinguish myeloid populations by flow cytometry using the gating in Supplemental Fig. 1A and 1B and Fig. 5C. Chemokine-specific migration PLA2R1 (29/49% and 26/47%, respectively; Fig. 1B). was calculated as cells migrating with chemokine minus migration with A search of murine expressed sequence tags (EST) found that in media only and is presented as a percentage of input. addition to a full-length transcript, a transcript lacking 27 bp encoded by exon 5 had been entered (9). We performed an RT-PCR Ear DC emigration on various B6 mouse tissues (including liver, lungs, LN, spleen, Mouse ears were excised and split into dorsal and ventral sides. The right and BM) to test for CD302 mRNA variants (Supplemental Fig. dorsal side was placed in complete RPMI with or without 1 mg/ml CCL19 1C). We used two sets of Cd302 primers that amplify regions (R&D Systems). After 24 h culture at 37˚C, emigrated cells were harvested, enumerated, and stained with mouse anti-CD11c and IA/IE Abs to establish between exons 1–6 or 2–6 and detected a single band with each of DC frequency by flow cytometry. the expected sizes (624 and 573 bp, respectively) in all organs. We did not detect a smaller band corresponding to the reported splice FITC painting DC migration assay variant. Additionally, we looked for a mouse CD205-CD302 fu- Mice were painted with 25 ml 0.5% FITC in 1:1 acetone/dibutyl phthalate sion transcript producing the fusion protein that we (7) and others (DBP) solution on the left ear and a 1:1 acetone/DBP control solution on (8) described in humans. To detect mouse Ly75 (gene producing the right ear. After 24 h, mice were euthanized to remove cervical LN, which were digested with collagenase/DNase I and the cell suspension was CD205) transcripts by RT-PCR, we designed primers spanning labeled with mAbs specific for CD11c and IA/IE to identify FITC+ mi- exons 34 to 35 (expected size 464 bp). The forward and reverse gratory DC by flow cytometry. For immunohistological studies, cervical primers for Ly75 and Cd302, respectively, were then combined to LN were removed from FITC/DBP-painted and nonpainted mice after test for intergenic mouse transcripts (expected size 900 bp). 24 h, snap frozen, and stained with anti-Lyve-1 and B220 Abs, as above. Whereas we could detect a Ly75 transcript in all mouse tissues In vivo T cell response tested, we could not detect a Ly75-Cd302 intergenic fusion tran- script (Supplemental Fig. 1C). MACS-purified T cells from pooled spleen and LN of OT-I and OT-II CD90.1 mice were respectively labeled with 5 mM CellTrace Violet Mice have similar CD302 tissue expression to humans (CTV) and CFSE (Thermo Fisher) and 1.25 3 106 cells of each were transferred i.v. into WT and CD302KO recipients. The following day, mice CD302 expression has not been examined in the mouse. We were immunized with a 1:1 vol of OVA (10 mg/mouse)/TiterMax Gold prepared additional primers amplifying a short region between adjuvant (Sigma-Aldrich) solution s.c. at the tail base. Inguinal LN were + + + exons 2 and 3 that enabled mouse CD302 transcripts to be quantified collected after 3 d to enumerate OVA-specific (CD90.1 ) CD4 and CD8 by qPCR. Transcripts in B6 mouse tissues were examined, con- T cells and assess CFSE/CTV dilution (proliferation) by flow cytometry. centrating on those that expressed CD302 in humans (9), that is, the Thioglycollate-induced Mf migration liver, lungs, spleen, LN, and BM (Fig. 2A). The highest expression Mice were injected i.p. with 1 ml of a 3% thioglycollate solution (BD of mouse Cd302 was found in liver, which was ∼16-fold greater Biosciences). At the indicated time points postinjection, mice were euthanized than the next highest, the lungs. Lymphoid tissues had marginally The Journal of Immunology 889

FIGURE 1. The mouse Cd302 gene. (A) Schematic diagram of the mouse Cd302 gene and transcript regions encoded by each exon, including the 59 untranslated region (59-UTR), signal peptide (SP), CTLD domain, spacer (S), transmembrane (TM) region, intracellular domain (IC), and 39 untranslated region (39-UTR). (B) Protein similarity tree diagram from a blastp of the CD302 CTLD region. less Cd302 transcripts: pooled LNs, spleen, and BM being 1.8-, 3.7-, stimulation. The levels of Cd302 declined both in stimulated CD8+ and 5-fold lower than lungs, respectively. This pattern mirrored the and CD82 DC. In contrast, mouse Mf increased their Cd302 ex- CD302 expression described in humans (9). pression 2.3-fold after LPS culture, a different result to that seen with human Mf (9). We also compared Cd302 expression in CD302 is primarily expressed by myeloid cell populations in unstimulated and LPS-stimulated FL-DC and GM-DC cultured the mouse immune system from mouse BM cells (Supplemental Fig. 1E). Flt3L cultures pro- Human CD302 was expressed by myeloid populations within duce FL-DC, containing two mDC subsets, CD11b+ and CD24+, Downloaded from healthy human PBMC, including Mf, Mo, granulocytes, and DC which share the characteristics of splenic resident CD82 and CD8+ + (9). We generated cDNA from highly purified B cells, CD4 and mDC, respectively (21). Unstimulated CD11b+ DC expressed more + CD8 T cells, DC subsets, granulocytes, Mf, and NK cell subsets Cd302 than did CD24+ DC, akin to CD82 and CD8+ DC (Fig. 2B, from B6 mouse spleens to compare their Cd302 transcription Supplemental Fig. 1D). Cd302 transcription was decreased by LPS levels by qPCR (Fig. 2B). Cd302 transcripts were most abundant stimulation of CD11b+ cells, but was not altered on CD24+ DC. + 2 2 in Mf, followed by granulocytes, CD4 /CD4 CD8 (double- GM-CSF plus IL-4 mouse BM cultures produce cell populations

+ http://www.jimmunol.org/ negative [DN]) DC, and CD8 DC (24.7-, 4.3-, and 2.7/2.9-fold resembling Mf (CD11c+MHC class II [MHC II]lo)orinflammatory + respective to the CD8 DC reference sample). B cells had low but DC (CD11c+MHC IIhi) (21, 22). Consistent with this, CD11c+ + detectable Cd302 expression, 1.8-fold lower than CD8 DC. No MHC IIlo cells expressed high levels of Cd302, which were further Cd302 transcripts were detected in pDC, T cell, or NK cell upregulated by LPS activation. In contrast, CD11c+MHChi cells samples. Therefore, CD302 transcription in mice, as in humans, is expressed lower levels of Cd302 that were not changed substantially mainly restricted to myeloid cell populations. by LPS activation. Migratory DC express higher levels of CD302 than resident Differential expression of CD302 by mouse M1 and M2 Mf DC Mf exposed to certain cytokines or stimulants can differentiate by guest on September 29, 2021 Our former study found that CD302 colocalized with podosomes into alternate states that either promote inflammation (classical, in human Mf (9), suggesting a potential role in cell adhesion or M1) or repair (alternate, M2) (23). Given the high Cd302 ex- migration. Resident mouse spleenDCexpressedsignificantlyless pression exhibited by mouse Mf in our ex vivo studies (Fig. 2B), Cd302 than did Mf (Fig. 2B), but we reasoned that migratory DC we investigated its expression by M1 and M2 Mf. WT and trafficking from peripheral sites to draining LN might have higher CD302KO BM cells cultured with M-CSF produced a highly int hi levels of CD302. To test this hypothesis, migratory (CD11c IA/IE ) purified CD11b+F4/80+ Mf population. These were cultured for hi int and resident (CD11c IA/IE ) DC populations were purified from an additional 24 h with no additional cytokines/stimulants (M0), pooled B6 LN. RNA was extracted from these and compared with or with LPS with and without IFN-g (M1) or with IL-4 (M2). Our splenic resident DC samples using the Cd302 qPCR. Cd302 ex- qPCR analysis showed that LPS-treated M1 BMDM cells upregu- pression was indeed 4-fold higher in migratory DC compared with lated Cd302 transcripts 5-fold (Fig. 2E) to levels that were similar to resident LN DC (Fig. 2C) and was similar (1.4-fold lower) to the LPS-activated primary splenic Mf (Supplemental Fig. 1D). Inter- levels found in splenic Mf (Fig.2B).ResidentLNDCCd302 levels estingly, addition of IFN-g to LPS abrogated Cd302 upregulation by were similar to those of their splenic counterparts. To determine M1 BMDM. M2 BMDM reduced their Cd302 expression 1.5-fold whether Cd302 expression varied in different migratory DC sub- relative to M0 cells (Fig. 2E). Cd302 expression can therefore change 2 hi populations, we purified CD103 CD205 DC (Langerhans cells), in alternative Mf activation states. the CD103+, CD1032CD205int, and CD205lo dermal DC subsets, as well as the resident DC from pooled B6 LN and extracted their RNA. CD302 is expressed by various cell types in mouse liver The qPCR analysis showed that all four LN migratory DC subpop- Liver contained the highest level of Cd302 transcripts in mice ulations examined expressed equivalent high levels of CD302 (∼2.5- (Fig. 2A) and humans (9), so we took advantage of the mouse fold greater than resident DC; Fig. 2D). The data suggest that CD302 model to investigate which liver components expressed Cd302. contributes to the function of all migratory DC subpopulations. Three predominant mouse liver cell populations were isolated from B6 mice: hepatocytes, CD452/+ LSEC, and the resident Mf CD302 expression by activated immune cells population or KC. KC expressed almost twice as much Cd302 Human Mf and DC downregulate CD302 transcription and trans- transcripts than did splenic Mf (Fig. 2F). Even higher expression lation upon LPS activation (9). To investigate the effect of activation of Cd302 transcripts were present in purified hepatocytes (4.8- on mouse immune cell CD302, we sorted B6 splenic B cells, Mf, fold) and LSEC (3.4 to 4.7-fold) relative to splenic Mf. CD8+ DC, CD82 DC (combined DN and CD4+ DC), and T cells and compared their Cd302 transcription before (unstimulated) and Mouse CD302 protein detection with a novel rat anti-mouse after 4 h stimulation (LPS for DC, Mf, and B cells and anti-CD3/ CD302 mAb 28–coated microbeads for T cells; Supplemental Fig. 1D). The low We generated hybridomas producing mAbs to mouse CD302 by to absent Cd302 expression in B and T cells persisted, despite immunizing rats with the extracellular portion of mouse CD302. 890 CD302 EXPRESSION AND FUNCTION

Fig. 1F). Western blots from liver, lungs, spleen, LN, and BM were probed with DCR1. This detected a single band of ∼29 kDa in liver extracts but not in other tissues (Fig. 2G). The low fre- quency of myeloid cells in immune organs made it difficult to detect CD302 protein in whole tissues. The DCR1 mAb did not bind to known CD302 mRNA expressing cells in flow cytometry experi- ments (data not shown), so we performed Western blots on protein extracts from BM-expanded DC or Mf populations in M0, M1, or M2 differentiation states (Fig. 2G). An ∼31- to 36-kDa band was most abundant in LPS-stimulated BMDM and reduced in LPS plus IFN-g M1 BMDM. The higher molecular mass of the band in Mf compared with the liver may represent distinct glycosylation states. The low sensitivity of the DCR1 Western blots probably accounted for the fact that we could not detect discernable bands in the GM- DC or other BMDM populations. CD302 expression in human tissues and myeloid cell populations

We added to our previous analysis of CD302 expression on human Downloaded from peripheral blood DC, Mo, and Mf (9) by examining their subsets. Human CD302 transcriptional analysis using qPCR demonstrated high expression in classical Mo and 2-fold less in nonclassical Mo. The CD1c+ DC and CD16+ populations had similar CD302 expression to nonclassical Mo, whereas the CD141+ mDC and pDC

had the lowest expression (Fig. 3A). We then compared CD302 http://www.jimmunol.org/ transcription to its cell surface protein expression by performing flow cytometry with the mouse mAb to human CD302, MMRI-20 (9, 24). Human blood DC and Mo analysis of healthy donors found the highest CD302 surface expression on classical Mo (Fig. 3B). Nonclassical Mo had 2-fold less CD302, compared with their classical counterparts, which was similar to that on myeloid DC, including the CD1c+, CD16+,andCD141+ DC subsets. CD302 staining was not detected on human pDC (Fig. 3B).

Given our mouse M1 and M2 Mf data (Fig. 2E), we compared by guest on September 29, 2021 CD302 expression by their human counterparts. Purified CD14+ healthy human blood Mo were cultured with human M-CSF to generate Mf, which were left inactivated (M0) or activated with LPS with and without IFN-g (M1 phenotype) or IL-4 (M2 phe- notype) for 24 h. CD302 expression was examined by qPCR (Fig. 3C) and flow cytometry (Fig. 3D). Human M1 Mf activated with LPS reduced their CD302 expression by 4.3- and 2.1-fold compared with M0 cells at the transcript (Fig. 3C) and protein level (Fig. 3D), respectively. Addition of IFN-g to the LPS made no difference to CD302 expression by M1 Mf. Moreover, human M2 Mf differentiated with IL-4 increased expression of CD302 at FIGURE 2. CD302 expression in cellular populations of B6 mice. Quan- the transcript level by 1.7-fold compared with M0 cells. Cell surface titation of CD302 transcripts by qPCR in (A) the indicated B6 mouse tissues; expression was greater in human M2 versus M1 Mf, broadly (B) purified immune cell populations from pooled B6 spleens; (C)purified reflecting the mRNA levels. These results indicate an apparent migratory and resident mDC populations from pooled peripheral (inguinal, species difference in CD302 expression during Mf M1/M2 dif- axillary, brachial, cervical, and mesenteric) LN compared with CD8+ and 2 ferentiation, although it must be acknowledged that the starting cell CD8 (CD4+ and DN) resident mDC populations from pooled B6 spleens; (D) migratory mDC (Lin2CD11cintIA/IEhi) subpopulations sorted from pooled B6 populations are necessarily different. peripheralLNbasedonexpressionofCD103andCD205andcomparedwith We performed immunohistological staining of human liver and sorted resident DC (Lin2CD11chiIA/IEint); (E) BMDM cultured in media alone spleen sections using the MMRI-20 mAb. MMRI-20 staining (M0), LPS with and without IFN-g (M1) or IL-4 (M2) for 16 h; and (F) showed that CD302 was present on hepatocytes and other cell types isolated hepatic populations from perfused livers of B6 mice. Hprt was used as in human liver sections (Fig. 3E), as predicted by the high levels of the reference gene in all qPCR experiments. (G) Western blots loaded with CD302 transcripts in human hepatocyte cell lines, mouse hepato- protein extracts from the indicated B6 mouse tissues; BMDM in M0/M1/M2 cytes, and LSEC (Fig. 2F) as well as our Western blot data differentiation states or FL-DC were probed with the rat anti-mouse CD302 (Fig. 2G). Staining of CD68+ KC within the liver sinusoids (see mAb, DCR1. The first lane contains molecular mass bands with the indicated arrows in Fig. 3E) was evident but less intense and required double sizes. Arrowheads mark the location of the bands. labeling to visualize them. No staining was observed in control liver sections stained with a mouse isotype control (Fig. 3F). A similar After screening .3000 hybridomas by ELISA, we established one CD302 staining pattern was observed in mouse liver sections IgG2a,k-producing clone (DCR1), which bound to mouse CD302-Ig stained with DCR1 (data not shown). To establish whether CD302 protein, but not an irrelevant Ig fusion protein (Supplemental was on the surface or in the cytoplasm of human hepatocytes and The Journal of Immunology 891 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Tissue and cellular distribution of the CD302 transcript and protein in humans. (A) Comparison of CD302 transcription in purified DC or Mo subsets from human PBMC by qPCR. (B) PBMC from three healthy donors were labeled with Abs to differentiate Mo and DC subsets, which were each examined for surface expression of CD302 protein with the MMRI-20 mAb via flow cytometry. Mo-derived Mf in M0, M1, and M2 differentiation states were analyzed for CD302 expression of (C) transcript by qPCR or (D) surface protein by flow cytometry. For (A) and (C), CT values from three replicates were normalized to the transcripts of the HPRT housekeeping gene. For (B) and (D), D mean fluorescence intensity (DMFI) = MFI of MMRI-20 2 MFI of isotype control. (E) Immunofluorescence staining of human liver sections with the CD302-specific MMRI-20 mAb and anti-CD68 mAb marking KC. Arrows mark the position of CD68+ KC within the CD302-only stain. (F) Composite staining of an adjacent human liver section with CD68 and a mouse IgG1 isotype control. (G) Immunofluorescence staining of human spleen sections for CD302 with MMRI-20, Mf with anti-CD68, and APC with an anti– HLA-DR mAb. Images were captured with a 320 objective. Scale bars, 50 mm. liver endothelial cells, we usedMMRI-20tolabelsurfaceand from WT and CD302KO mice amplifying the Cd302 region en- intracellular CD302 in the hepatocarcinoma (HepG2) and coded by exons 2–6 (Fig. 4C). Cd302 transcripts were detected in hepatoma-derived endothelial cell (SK-HEP-1) lines. Both cells WT but not CD302KO cDNA from spleen and liver. Western blots lines had readily detectable surface and intracellular CD302 of liver digests from WT and CD302KO mice with DCR1 detected (Supplemental Fig. 1G). a CD302 protein band in the former but not in the latter (Fig. 4D). MMRI-20 staining of human spleen sections was localized to CD302KO homozygous breeder mice produced equivalent lit- HLA-DRhi cells within the periarteriolar lymphoid sheath and ter numbers and sizes compared with WT mice. CD302KO mice CD68+ cells in extrafollicular regions and periarteriolar lym- exhibited normal development, with no outward signs of disease or phoid sheath (Fig. 3G), consistent with CD302 expression on aberrant tissue histology (data not shown). Serum levels of tri- Mf and DC in these locations. glycerides, protein, and glucose at 12 wk of age were not signif- icantly different between CD302KO and WT mice (n = 12 per group, Generation of CD302KO mice NS, data not shown). Additionally, no significant differences were The targeting vector used to generate our CD302KO mouse strain observed in liver function tests (including AST, ALT, ALP, bilirubin, was designed to remove part of the Cd302 exon 1 containing the and albumin) in the serum of CD302KO and WT mice (n =12per start codon (Fig. 4A) and prevent transcription of the gene. Ho- group, NS, data not shown), indicative of normal liver function. mozygous CD302ko/ko mice (hereafter referred to as CD302KO) were generated (see Materials and Methods) and deletion of the Analysis of CD302KO mice immune populations reveals CD302 genomic region in CD302KO mice was distinguished from reduced migratory LN DC populations that of WT mice by PCR (Fig. 4B). To confirm the lack of CD302 We examined the contribution of CD302 to myeloid cell differ- transcription, we performed RT-PCR on liver and spleen cDNA entiation using the CD302KO mouse model. Cell counts performed 892 CD302 EXPRESSION AND FUNCTION Downloaded from

FIGURE 4. Production of CD302KO mice. (A) Schematic diagram of the genetic strategy used to generate the CD302KO mouse on the B6 background as described in the Materials and Methods. Arrows show the approximate positions of the PCR primers used to confirm the genotypes of mice. (B) PCR of genomic DNA from representative homozygous WT and CD302KO mice using WT-specific CD302-F/R1 and KO-specific CD302-F/R2 combinations of genotyping primers. (C)RT-PCRanalysisofCd302 transcripts (exons 2–6) in cDNA derived from spleen and liver of WT and CD302KO mice. Amplifications with reverse http://www.jimmunol.org/ transcriptase positive (RT+) and negative (RT2) cDNA preparations shown. Gapdh housekeeping gene is shown as a loading control. (D) Duplicate protein gels containing liver and lung protein extracts from WT and CD302KO mice were probed with an anti-mouse CD302 (DCR1) or an isotype control mAb. on WT and CD302KO female mice at 11–12 wk of age revealed a no differences were noted between the frequency or total numbers of significantly reduced cellularity of CD302KO pooled inguinal, B or T cells in CD302KO and WT mice (Supplemental Fig. 2C, 2D). brachial, axillary, and cervical LN relative to WT mice (Fig. 5A). The frequency and numbers of CD302KO and WT mice splenic However, no such differences between the KO and WT strains resident mDC and pDC (Supplemental Fig. 2A, 2B) were also

were observed in BM, spleen, or thymic cell numbers. Flow similar (spleen does not contain migratory mDC). Despite the high by guest on September 29, 2021 cytometry was performed to identify which immune populations expression of CD302 by splenic Mf and granulocytes, the frequency accounted for the reduction in overall CD302KO cellularity. Flow and numbers of these cells were similar in the spleen (Supplemental data were analyzed using viSNE, which uses a stochastic neighbor- Fig. 2A, 2B) and LN (data not shown) of CD302KO and WT mice. embedding algorithm to allow visualization of our nine dimensional To determine whether CD302 altered myeloid cells in the liver, data (including staining for CD4, CD8, CD11b, CD11c, CD45R, we undertook a flow cytometric analysis of the DC, KC, and Mo CD103, CD205, IA/IE, and Ly6C) in two-dimensional space (14). populations (Supplemental Fig. 2E) within the right hepatic lobe For this analysis, datasets from five CD302KO and WT mice were of eight 12-wk-old CD302KO and WT mice. We did not detect a segregated into Lin (CD3, CD19, Ly6G, NK1.1) positive and neg- significant difference in the absolute numbers of any of these ative events, permitting a detailed investigation of rare myeloid hepatic cell populations (Supplemental Fig. 2F). populations (including DC, Mf, and Mo) separately from the larger lymphocyte and granulocyte populations in these samples. CD302-deficient DC can upregulate activation markers and No differences were noted in the viSNE plots of Lin+ populations prime T cells in an MLR between the strains (Fig. 5B). In contrast, comparison of Lin2 plots The reduction of mDC in CD302KO mice led us to further in- revealed a reduction of a cell population expressing high levels of vestigate DC function in these animals. CD302KO and WT mDC CD11c, CD205, and MHC II, corresponding to DC, in CD302KO showed an equivalent capacity to upregulate the maturation mice (Fig. 5B). Conventional flow cytometry gating was used to markers CD80, CD83, and CD86 after overnight culture in the detail the frequency and total numbers of LN DC subsets in presence of various TLR stimulants, including LPS, poly(I:C), and CD302KO versus WT mice. A significant decrease in the frequency CpG (Supplemental Fig. 3A). Furthermore, LPS-stimulated WT or of migratory DC (Fig. 5C, 5D), but not the resident mDC or pDC CD302KO GM-DC showed similar abilities to prime allogeneic subsets, was observed within CD302KO LN (Fig. 5D). A significant CFSE-labeled BALB/c T cells in an MLR (Supplemental Fig. 3B, decrease in both frequency and total numbers was particularly ev- 3C). Comparable levels of T cell priming were also achieved using 2 int ident for the CD103 CD205 subpopulation of migratory DC, al- irradiated LN or spleen cell stimulators from WT or CD302KO 2 though a trend toward decrease (p =0.06)wasalsoseenforCD103 mice (Supplemental Fig. 3C). DC from CD302KO mice are lo CD205 migratory DC numbers in CD302KO LN (Fig. 5D, 5E). therefore able to mature and prime CD4+ and CD8+ T cell re- Conventional gating confirmed that there was no significant differ- sponses equivalently to WT DC. ence in the frequency of B cells, CD4+ T cells, or CD8+ T cells in LN of CD302KO and WT mice (Fig. 5F). However, owing to the CD302 does not contribute to the formation of F-actin–rich decreased cellularity of the CD302KO LN, there was a significant migratory structures in DC reduction in their total numbers (Fig. 5G). This was in contrast to the In our human studies, CD302 was shown to colocalize with F-actin–rich analysis of spleens (which lack an afferent lymphatic input), where migratory structures including podosomes and lamellopodia (9). To The Journal of Immunology 893 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Reduced lymphocyte cellularity and frequency of CD205int migratory DC in CD302KO LN. Lymphoid organs from WT and CD302KO female mice at 11–12 wk of age were examined by flow cytometry. (A) Scatter plot displaying the total cellularity in spleen, pooled (inguinal, brachial, axillary, and cervical) LN, thymus, and BM (femur and tibia). (B) Representative viSNE plots (n = 5 per strain) of LN flow (Figure legend continues) 894 CD302 EXPRESSION AND FUNCTION determine whether CD302 contributed to their formation, GM-DC CD302-deficient DC show decreased entry into the from CD302KO and WT mice were attached to glass coverslips and interfollicular channels of LN labeled with phalloidin to reveal F-actin–rich migratory structures at To determine whether CD302KO DC exhibit difficulties in entering the basal surface (Supplemental Fig. 3D). Comparison of CD302KO LN from the draining lymphatics, we performed an immuno- and WT DC on glass coverslips revealed a similar percentage of histological analysis on the draining cervical LN of WT and KO podosomes forming cells, numbers of attached cells, and attachment mice, painted with the FITC/DBP solution. DC migrating into the cell surface area (Supplemental Fig. 3E–G). F-actin–rich lamellopodia LN were labeled with FITC and the LN sections were costained for at the periphery of GM-DC could also be observed on both WT and Lyve-1 to mark the lymphatic vessels and the subcapsular sinus CD302KO GM-DC. These characteristics were also comparable when (SCS) as well as B220 to mark the cortical B cell follicular areas CD302KO and WT GM-DC were attached to fibronectin-coated that surround the interfollicular channels (IFC) used by migratory coverslips (Supplemental Fig. 3E–G). CD302 does not appear to be DC to enter LN (27). Interestingly, although FITC+ WT DC were critical for the formation of podosome or lamellopodia structures in readily observed within the IFC of the draining LN 24 h after DC despite its localization to these sites. painting, CD302KO DC had reduced capacity to enter the IFC and CD302KO and WT DC exhibit equivalent chemotaxis toward appeared to accumulate within the SCS (Fig. 6D). To determine CCL19/21 in vitro whether CD302 contributed to entry of DC into the IFC in the steady-state, we performed further immunohistological staining on We assessed the ability of CD302KO and WT DC to migrate untreated WT and KO mice LN. Inguinal LN sections were labeled toward the CCL19 and CCL21 chemokines, which are essential for for CD11c to distinguish DC, B220 to mark B cell follicles, and CCR7-mediated DC migration into the spleen and LN (25, 26). either CD3 (Fig. 6E) or Lyve-1 (Fig. 6F) to highlight the paracortical Downloaded from However, splenic and LN DC and Mf populations derived from T cell zones or lymphatic endothelium lining the SCS, respectively. CD302KO and WT showed comparable levels of chemotaxis to There was a reduction in DC located within the IFC of the both chemokines (Supplemental Fig. 3H). CD302KO LN sections compared with WT LN sections, concom- + Reduced in vivo migration of CD302KO DC from peripheral itant with an increase in CD11c cells within the SCS. These studies sites into draining LN suggest that CD302 plays an important role in the entry of DC into the LN by facilitating their migration from the SCS into the IFC. http://www.jimmunol.org/ The reduced proportion of migratory DC within CD302KO LN + raised the possibility that CD302 contributed to other processes, CD302 deficiency on APC reduces Ag-specific CD8 T cell apart from chemotaxis, that influence DC migration from the response in draining LN periphery to draining LN. Therefore, we examined the ability of The reduced trafficking of migratory CD302KO DC into LN DC within ear skin to migrate to the draining cervical LN. The suggested that the CD302KO mice might have a reduced capacity frequency and absolute numbers of DC within the ears of WT and to prime LN T cells to peripheral Ags. To investigate migratory DC CD302KO mice were the same (Supplemental Fig. 4A, 4B). We induction of T cell responses, we performed experiments in which then examined DC migration from CD302KO and WT dorsal ear purified OVA-specific OT-I (CD8+) and OT-II (CD4+) T cells sheet explants placed into medium containing CCL19. After 24 h, (CD302 sufficient) were labeled with different proliferation dyes by guest on September 29, 2021 similar numbers of DC migrated out of the CD302KO and WT and transferred into five WT or CD302KO mice. The following ears (Supplemental Fig. 4C), indicating that CD302 was not es- day, mice were immunized in the tail base s.c. with OVA in sential for DC to exit the skin via the lymphatics. To assess the TiterMax adjuvant and the draining inguinal LNs were harvested trafficking of migratory DC from the skin to draining LN, we used 3 d later to monitor OT-I and OT-II T cell proliferation by flow an in vivo migration model in which CD302KO and WT mice cytometry dye dilution. The OT-II T cells proliferated to a similar were painted with a FITC solution containing the irritant DBP on extent in the immunized CD302KO and WT mice (Fig. 7). In their ears. After 24 h, draining cervical LN were harvested to de- contrast, the OT-I T cells from immunized CD302KO mice had a + termine the frequency of FITC DC that migrated from the skin. modest but significant reduction in proliferation index compared + In both strains most FITC DC in LN displayed the migratory with the WT mice (Fig. 7A, 7B). The reduction in proliferation hi int (IA/IE )and not the resident (IA/IE ) phenotype (Fig. 6A). The resulted in a trend toward fewer OT-I T cell numbers in the levels of FITC contained by migratory DC within the draining LN draining LN of immunized CD302KO compared with WT mice were similar between WT and KO mice (Fig. 6B). Moreover, we (Fig. 7C). The reduced trafficking of migratory DC into draining did not observe significant disparities in the ability of WT LN in CD302KO mice is reflected in a decreased capacity to and CD302KO DC to take up or process Ags, as determined cross-present OVA Ags and expand the frequency of specific by FITC-dextran (Supplemental Fig. 4D) or DQ-BSA uptake CD8+ T cells within the draining LN. (Supplemental Fig. 4E), respectively. However, the frequency of f FITC+ migratory DC detected within the CD302KO draining LN CD302 deficiency does not affect in vivo M migration into the after skin painting was significantly reduced compared with WT peritoneal cavity (Fig. 6A, 6C). Taken together, these experiments indicate that To test whether CD302 contributed to the in vivo migration of Mf although CD302 does not contribute to Ag uptake and DC emi- into inflammatory sites, we examined the peritoneal cavity cell gration into lymphatics, it plays a role in the ensuing processes infiltrate in WT and CD302KO mice after i.p. injection of a 3% that enable migrating DC to move efficiently through lymphatics thioglycollate solution. The influx of Mf within the peritoneal or enter the draining LN. cavity over 24 h after the thioglycollate injection was similar

cytometry data displaying levels of B220, CD4, and CD8 expression in Lin+ (CD3+, CD19+, Ly6G+, and NK1.1+) populations and CD11c, CD205, IA/IE in Lin2 populations. Arrows point to the affected population. (C) Representative flow cytometry plots showing the gating of migratory DC populations from Lin2IA/IE+ cells in WT and CD302KO LN. Scatter plots comparing the absolute frequency (D and F) and number (E and G) of the Lin2 (D and E) and Lin+ (F and G) immune cell populations in LN of individual WT and CD302KO mice. *p , 0.05, **p , 0.01 between groups using nonparametric t tests with multiple test correction. The Journal of Immunology 895 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. CD302 deletion reduces DC migration into draining LN. (A–D) DBP-FITC solution was applied to the left ear of WT (n = 13) and CD302KO (n = 13) mice in four experiments. After 24 h, FITC+ migratory DC populations in the cervical LN were identified using flow cytometry or immuno- histology. (A) Representative dot plots of CD11c+ cells in LN, showing the gating of FITC+ migratory (IA/IEhi) DC in WT and CD302KO mice. Scatter plots comparing the (B) FITC mean fluorescence intensity (MFI) and (C) absolute frequency of FITC+ migratory DC in cervical LN of individual WT and CD302KO mice. *p , 0.05 between groups of WT and CD302KO mice using a nonparametric t test. (D) Representative images of cervical LN sections from FITC-painted WT or CD302KO mice or nonpainted WT mice stained with anti-Lyve-1 and B220 Abs imaged with a 310 objective. Scale bars, 100 mm. Arrows point to the bridging areas of LN. (E and F) Representative images of inguinal LN sections from three untreated 10-wk-old WT and CD302KO mice stained with Abs specific for CD11c, B220, and either (E)Lyve-1or(F) CD3. CD11c only and composite images are shown. Images were taken with a 320 objective. Scale bars, 100 mm. between WT and CD302KO mice (Supplemental Fig. 4F), suggesting understanding of CD302 expression and function from its original that CD302 did not play a role in the migration of Mf into this site. description and formal CD classification (24) as a potentially important leukocyte cell surface molecule. Our results indicate Discussion that not only are the CD302 gene structure and protein sequences CD302 is a group XV C-type lectin and is the simplest type I highly conserved between mice and humans, but so too is the transmembrane molecule of the CLR family (10). We defined tissue distribution. Within the immune system, CD302 is ex- CD302 originally in humans and showed that it was associated pressed on the surface of mouse and human myeloid cells, most with the F-actin cytoskeleton within podosome and lamellopodia notably Mf, granulocytes, and DC. Further analysis suggested of human Mf, structures that are known to be extremely impor- that CD302 was present in increased amounts on mouse migratory tant in cell adhesion and migration (9). This study builds on our DC, reinforcing the prospect that this CLR contributes to cell 896 CD302 EXPRESSION AND FUNCTION

product fuses the 10th CTLD from CD205 with the CTLD, trans- membrane, and intracellular region of CD302. We did not identify a similar intergenic Ly75-Cd302 EST in mouse tissues (Supplemental Fig. 1C) or purified mouse DC (data not shown). Thus, the effects we describe in this study for mouse CD302 on leukocytes appear to relate to the full-length six exon–encoded cell surface protein. Liver appears to express the same transcript, although our Western blot data (Fig. 2G) raise the possibility that liver CD302 has different posttranslational processing. Conserved CD302 function between humans and mice is also suggested by its similar tissue distribution in both species. The highest transcriptional expression was in liver, with intermediate expression in the lungs and the immune system in each (Fig. 2A) (9). CD302 was most abundant within the myeloid compartment of both species’ immune systems, with highest expression in Mo, Mf, granulocytes, and myeloid DC (Fig. 2B). Similarly, CD302 was not found on pDC, T cells, and NK cells in both species. The differential expression of CD302 by DC subsets is most interesting

and relates at least in part to their function. In mouse spleens, the Downloaded from CD4+ and DN (CD11b+) resident subsets expressed twice the level of CD302 transcripts than did the specialized cross-presenting CD8+ DC resident subset (Fig. 2B). This aligned with our hu- man blood DC data, which noted lower CD302 transcript levels in the specialized cross-presenting CD141+ DC subset, compared + + with the CD1c and CD16 subsets (Fig. 3A). However, these http://www.jimmunol.org/ FIGURE 7. CD302 deficiency reduces the capacity to produce an op- differences did not necessarily translate to protein levels displayed + timal CD8 T cell response. T cells purified from spleens and LN of male on the cell surface (Fig. 3B). We proposed a potential role for OT-I and OT-II mice were labeled with CTV and CFSE, respectively. CD302 in migration, and this was reinforced by our data showing Labeled T cells were then mixed at 1:1 ratio and adoptively transferred that the mouse migratory DC trafficking into LN expressed ∼2.5- into male WT (n = 5) and CD302KO (n = 5) mice via i.v. injection. After to 4-fold higher levels of CD302 than did the resident DC 24 h, mice were immunized s.c. at the tail base with OVA in adjuvant. Draining inguinal LNs were collected 3 d after immunization to examine (Fig. 2C, 2D), and these elevated transcript levels were equivalent T cell proliferation by flow cytometry. (A) Representative histograms in all migratory DC subsets (Fig. 2D). showing proliferation index (PI) of Thy1.1+CD4+CFSE+ OT-II and Several CLR family members contribute to adhesion or migration Thy1.1+CD8+CTV+ OT-I T cells extracted from WT and KO mice. Graphs of Mf or DC through their ability to bind endogenous ligands (4). by guest on September 29, 2021 of (B) the proliferation index and (C) absolute numbers of OT-I and OT-II For example, in addition to binding to various exogenous Ags on T cells in inguinal LN of recipient WT and KO mice are also shown. *p , viruses such as HIV (29), CD209 (DC-SIGN) on DC has the ca- 0.05 between WT and CD302KO mice using a nonparametric t test. pacity to bind to ICAM-2 and regulates the transmigration of these cells across endothelium (30). Another more recently discovered adhesion and migration. This was confirmed by showing a de- example is CLEC2, whose ability to recognize podoplanin on crease in the frequency and numbers of migratory DC in LN of lymphatic vasculature allows DC to move along stromal surfaces CD302KO mice. Additional experiments indicated that CD302 into LN (3) as well as regulate LN expansion and contraction (3). contributed to the migration of DC from lymphatics through to the Our CD302KO data have provided key evidence to suggest that this IFC in draining LN. This had an apparent consequential effect on CLR is involved in regulating DC migration. First, the frequency the numbers of LN T and B cells. Finally, it was notable that and numbers of migratory mDC within CD302KO LN were re- CD302 was required to generate a full draining LN T cell response duced compared with WT mice (Fig. 5). Interestingly, other immune after s.c. vaccination. cells, notably T and B cells, were present at similar frequencies in The CD302 gene in humans and mice consists of six exons WT and KO mouse LN, but their overall numbers were diminished located on Chromosome 2 (Fig. 1A and Ref. 9). The proteins (Fig. 6F, 6G). Given the minimal levels of CD302 found on T and produced by both species share 76% identity and 81% similarity, B cells (Fig. 2B, Supplemental Fig. 1D), a possible explanation for suggesting a conserved function. Nonetheless, mice and humans the decrease in LN lymphocyte cellularity could involve the role exhibit greater dissimilarities within the CD302 intronic se- played by DC in controlling expansion/contraction of LN (3). The quences, which may give rise to the differences reported in second piece of evidence linking CD302 to DC migration was ob- splicing between the species. An EST for a shorter variant of tained in the FITC-painting studies, in which CD302KO migratory CD302, lacking the spacer region encoded by exon 5, has been DC had reduced in vivo capacity to reach draining LN (Fig. 6A–C). described in a cDNA library obtained from 18-d-old mouse em- We demonstrated that WT and CD302KO DC had equivalent ca- bryos (28), but this variant has not been detected in humans (9). pacity to enter lymphatics and emigrate from skin (Supplemental Removal of exon 5 does not change the reading frame, and the Fig. 4D), suggesting that CD302 controlled later processes in- variant is likely to encode a protein where the CTLD sits closer to volved in the migration of DC into LN. CLEC2-deficient DC are the membrane. We did not detect this smaller Cd302 variant unable to enter afferent lymphatics in the skin (3), indicating that this (Supplemental Fig. 1C) in our studies on adult mouse tissues, sug- molecule regulates DC migration into LN through a different gesting that it may be expressed only during embryonic develop- mechanisms than CD302. ment. A human intergenic splice product between CD205 and For DC to enter the parenchymal LN T cell compartments, they CD302 was described at the transcript and protein level in a Hodgkin must traverse the SCS wall that overlies the IFC (27). This sinus wall lymphoma line and subsequently primary human DC (7, 8). This consists of endothelial cells expressing Lyve-1 and interdigitating The Journal of Immunology 897

CD169+ Mf. Our immunohistology studies in FITC-painted or N-acetylglucosamine residues and, in doing so, asialoglycoprotein CD302KO mice suggest that CD302 deficiency affects the ability receptor contributes to the turnover of circulating glycoproteins as of DC to exit the SCS and enter the IFC (Fig. 6D-F), indicating a well as the degradation of lymphocytes and platelets expressing role for CD302 in this migratory process. A similar phenotype was these moieties as they pass through the liver. CD302 has the capacity described for DC lacking the CCR7 receptor (26), although we do to mediate the endocytosis of beads when bound by Abs, although not think that CD302 alters CCR7 receptor function, given the this was not as efficient as CD205 or CD206 (9). Nevertheless, this normal migration of CD302KO DC toward the CCL19/21 ligands raises the possibility that liver CD302 could fulfill a similar scav- in chemotaxis assays (Supplemental Fig. 3H). Unlike related CLR enger role to the CLR described above by binding to material or cells (Fig. 1B), CD302 has the capacity to colocalize with F-actin–rich expressing its specific ligand. migratory structures, including podosomes (9). CD302KO DC The ligands bound by CD302 remain to be discovered. The mouse retained the capacity to form podosomes and lamellopodia, indicat- CD302 CTLD was shown to have highest homology with CTLD ing that CD302 is not required for their formation (Supplemental from most members of the mannose receptor CLR subfamily, which Fig. 3D, 3E). Nevertheless, CD302 concentration within these pro- includes PLA2R, CD205, and CD206 and CD280 (Fig. 1B), sug- truding podosome structures could well be crucial in allowing CD302 gesting that it could bind similar ligands. However, the receptors in to bind to the yet to be determined lymphatic endothelial ligands this family have evolved to bind quite distinct ligands, and these involved in DC adhesion or migration (12). However, given the high CLR are much larger structures, containing 8–10 CTLD regions on levels of CD302 on migratory DC, Mf, and endothelial cells (Fig. 2), a single polypeptide backbone (39), compared with the simple one we cannot yet rule out that CD302 deficiency in any of these pop- CTLD structure of CD302. Our first attempts to define a ligand ulations contributes to the altered DC migration phenotype. The using agarose beads conjugated with mannan, mannose, N-acetyl Downloaded from decreased DC migratory capacity in CD302KO mice reduced their glucosamine, and N-acetyl galactosamine showed no binding to capacity to form an optimal CD8+ T cell response in the draining LN human CD302-Ig (data not shown). However, our data make more after immunization (Fig. 7). This is readily explained by the de- definitive studies to define the ligand using new carbohydrate or creased migration of the CD302KO dermal DC populations into the protein libraries very important. LN with the ability to cross-present Ags (31, 32). In conclusion, our study has advanced the knowledge of the

Despite the high levels of CD302 on mouse Mf, we did not find expression and function of the novel CLR, CD302, in mouse and http://www.jimmunol.org/ alterations of Mf numbers or distribution in different CD302KO humans. We have been able to describe an important CD302 function tissues (including spleen, liver, and peritoneal cavity; Supplemental associated with DC migration into LN via the IFC, which is a crucial Figs. 2A, 2B, 2F, 4F [time 0 h]), nor did we observe reduced entry process in the development of immunogenic and tolerogenic immune into the inflamed peritoneal cavity (Supplemental Fig. 4F). The responses. Given the potential redundancy of molecules involved in function of CD302 on Mf therefore remains a conundrum for now, controlling cell adhesion and migration through the lymphatic although we did note that mouse M0 Mf differentiated with LPS system, this result is very significant indeed. Additionally, we into M1 Mf upregulated CD302 transcription and protein expres- showed that CD302 is highly expressed on Mf, endothelial, and sion substantially (Fig. 2E, 2G). This contrasted with DC, which hepatocyte populations, but its function on these cell types will need decreased CD302 expression after LPS exposure (Supplemental to be elucidated in future studies. CLR are commonly used to target by guest on September 29, 2021 Fig. 1D, 1E). Curiously, this upregulation in M1 Mf was abro- Ags or drugs to different cellular populations, and ongoing inves- gated by exposure to IFN-g (Fig. 2E, 2G). CD302 expression was tigation of the unique properties of CD302 may well generate not changed by differentiating mouse M0 Mf into immunoregu- similar opportunities. latory M2 Mf by IL-4. Surprisingly, the opposite pattern of CD302 expression resulted when we generated human M1 and M2 Mf Acknowledgments equivalents (Fig. 3C, 3D). Higher CD302 expression by human M2 We acknowledge the staff of the ANZAC Research Institute Molecular versus M1 Mf was also reported in a microarray study (33). Note Physiology Unit for help with animal husbandry. We thank Robert Brink that the starting cell types differ (BM versus Mo), but these dif- and Elissa Deenick for the provision of OT-I/-II mice, Ben Roediger for ferences in CD302 expression between M1 and M2 Mf in mice and the B16-Flt3L cell line, and Maaike Kockx for the L929-M-CSF cell line. humans raises interesting questions as to potential species differ- We appreciate the assistance with histology experiments provided by Frederic ences in Mf function. Sierro,MainthanPalendira,andStuartTangye.Finally,wearegratefulto Mouse liver, as is the case in humans, produces the largest Barbara Fazekas for reviewing the manuscript and helpful suggestions. amount of the CD302 transcript and protein (Fig. 2A, 2G) (9), and we found that hepatocytes as well as LSEC and KC in mice all Disclosures expressed this CLR (Fig. 2F). Immunohistological studies on D.N.J.H. and G.J.C. are directors of a commercial company (DendroCyte human livers confirmed the widespread expression of CD302 in BioTech Ltd.) that manages and commercializes the Dendritic Cell Re- liver, including hepatocytes and KC (Fig. 3E). Our flow cytometry search group intellectual property. All of the authors acknowledge that our host institution (ANZAC Research Institute) has an agreement with studies showed that CD302 was on the surface of human hepatic DendroCyte BioTech Ltd. in relationship to the management and commer- cell lines, indicating that CD302 could be expressed on the surface cialization of our academic group’s intellectual property. of these cells (Supplemental Fig. 1G). Despite the abundant ex- pression of CD302 in liver, we did not detect abnormalities in its structure or function in the CD302KO animal. How liver CD302 References contributes to the host’s well-being therefore remains an open 1. Drickamer, K., and M. E. Taylor. 2015. Recent insights into structures and func- question. Some insight into this function might be gained from tions of C-type in the immune system. Curr. Opin. Struct. Biol. 34: 26–34. 2. Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, other CLR expressed by hepatocytes, LSEC, and KC. LSEC and G. J. 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