of May 25, 2018.This information is current as NK CellsdimCells and CD319 on CD56

CD229 (LY-9) on Plasmacytoid Dendritic Family Members CD319 (CRACC) andComplexes Increase the Expression of SLAM Systemic Lupus Erythematosus Immune

RönnblomMaija-Leena Eloranta, Yenan T. Bryceson and Lars Niklas Hagberg, Jakob Theorell, Heinrich Schlums,

ol.1301022http://www.jimmunol.org/content/early/2013/08/16/jimmun

published online 16 August 2013J Immunol 

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Systemic Lupus Erythematosus Immune Complexes Increasethe Expression of SLAM Family Members CD319 (CRACC)and CD229 (LY-9) on Plasmacytoid Dendritic Cells andCD319 on CD56dim NK Cells

Niklas Hagberg,* Jakob Theorell,† Heinrich Schlums,† Maija-Leena Eloranta,*

Yenan T. Bryceson,† and Lars Ronnblom*

Patients with systemic lupus erythematosus (SLE) display an activated type I IFN system due to unceasing IFN-a release from

plasmacytoid dendritic cells (pDCs) stimulated by nucleic acid–containing immune complexes (ICs). NK cells strongly promote

the IFN-a production by pDCs; therefore, we investigated surface molecules that could be involved in the pDC–NK cell cross-talk.

In human PBMCs stimulated with RNA-containing ICs (RNA-ICs), the expression of the signaling lymphocyte activation molecule

(SLAM) family receptors CD319 and CD229 on pDCs and CD319 on CD56dim NK cells was selectively increased. Upregulation of

CD319 and CD229 on RNA-IC–stimulated pDCs was induced by NK cells or cytokines (e.g., GM-CSF, IL-3). IFN-a–producing

pDCs displayed a higher expression of SLAM molecules compared with IFN-a2 pDCs. With regard to signaling downstream of

SLAM receptors, pDCs expressed SHIP-1, SHP-1, SHP-2, and CSK but lacked SLAM-associated protein (SAP) and Ewing’s

sarcoma-activated transcript 2 (EAT2), indicating that these receptors may act as inhibitory receptors on pDCs. Furthermore,

pDCs from patients with SLE had decreased expression of CD319 on pDCs and CD229 on CD56dim NK cells, but RNA-IC

stimulation increased CD319 and CD229 expression. In conclusion, this study reveals that the expression of the SLAM receptors

CD319 and CD229 is regulated on pDCs and NK cells by lupus ICs and that the expression of these receptors is specifically altered

in SLE. These results, together with the observed genetic association between the SLAM locus and SLE, suggest a role for CD319

and CD229 in the SLE disease process. The Journal of Immunology, 2013, 191: 000–000.

Systemic lupus erythematosus (SLE) is an autoimmunedisease characterized by the presence of immune complexes(ICs) that deposit in tissue and cause inflammation (1). Such

SLE ICs are typically formed by autoantibodies and nucleic acid–containing autoantigens, which are released by dying cells (2). SLEICs promote tissue inflammation and immune activation throughseveral mechanisms, most importantly by complement activationand FcgR triggering of immune cells (3). In response to SLE ICs,macrophages and neutrophils produce proinflammatory cytokinesand proteolytic enzymes (4), whereas plasmacytoid dendritic cells(pDCs) produce type I IFN (5). The DNA- or RNA-containing SLE

ICs (interferogenic ICs) are internalized by pDCs through FcgRIIAand trigger TLR7 or TLR9 activation, ultimately leading to IFN-asecretion (5). A continuous activation of pDCs by interferogenicICs is a prominent feature of SLE and causes an increased ex-pression of type I IFN–induced genes (an IFN signature) in bothcirculating cells (6, 7) and affected tissues (8). An IFN signaturealso has been observed in other systemic autoimmune diseases thatexhibit interferogenic ICs (9, 10), suggesting that an activation ofthe type I IFN system may be important in the development of anautoimmune disease process. This assumption is also supported bythe observation that treatment of infectious or malignant diseaseswith IFN-a can trigger the development of several autoimmunediseases (11, 12).Previous studies showed that NK cells interact with pDCs to

potently enhance IFN-a production upon stimulation with viruses,synthetic oligonucleotides, or RNA-containing ICs (RNA-ICs)(13–15). Mechanistically, NK cells promoted RNA-IC–inducedIFN-a secretion by pDCs via soluble factors, such as MIP-1b, andvia LFA-1–dependent cell–cell interactions. In addition to IFN-a,the secretion of several cytokines and chemokines implicated inthe pathogenesis of SLE (e.g., IFN-g, IL-6, IL-8, MIP-1b, andTNF-a) was also enhanced in RNA-IC–stimulated pDC–NK cellcocultures (15). Consequently, pDC–NK cell cross-talk could beimportant in promoting sustained type I IFN production and theinflammatory response in systemic autoimmune diseases.In the current study, we aimed to identify cell surface molecules

of possible importance in the interaction between pDCs and NKcells when activated by RNA-ICs. By screening pDCs and NK cellsfor molecules that were regulated by RNA-ICs, consisting of SLE-IgG and U1snRNP particles, we identified two members of thesignaling lymphocyte activation molecule (SLAM) family that

*Section of Rheumatology, Department of Medical Sciences, Uppsala University, S-75185 Uppsala, Sweden; and †Department of Medicine, Center for Infectious Medicine,Karolinska Institute, Karolinska University Hospital Huddinge, S-141 86 Stockholm,Sweden

Received for publication April 16, 2013. Accepted for publication July 22, 2013.

This work was supported by grants from the Swedish Research Council, the SwedishRheumatism Foundation, the Torsten and Ragnar Soderberg Foundation, King GustafV’s 80-Year Foundation, Controlling Chronic Inflammatory Diseases with CombinedEfforts, the Knut and Alice Wallenberg Foundation, Clas Groschinsky’s Memorial Fund,the Jeanssons Foundation, the Ake Olsson Foundation for Hematological Research, theAke Wiberg Foundation, and the Karolinska Institute Research Foundation.

Address correspondence and reprint requests to Niklas Hagberg, Systemic Autoimmu-nity Group, Department of Medical Sciences, Clinical Research Department 3, Entrance85, 3rd Floor, S-751 85 Uppsala, Sweden. E-mail address: niklas.hagberg@medsci.uu.se

The online version of this article contains supplemental material.

Abbreviations used in this article: EAT2, Ewing’s sarcoma-activated transcript 2;HAIG, heat-aggregated IgG; IC, immune complex; MFI, median fluorescence in-tensity; pDC, plasmacytoid dendritic cell; RNA-IC, RNA-containing immune com-plex; SAP, signaling lymphocyte activation molecule–associated protein; SLAM,signaling lymphocyte activation molecule; SLE, systemic lupus erythematosus.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301022

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were strongly upregulated on pDCs and NK cells by RNA-ICs:CD319 (SLAMF7, CRACC, CS1) and CD229 (SLAMF3, LY9).In humans, the SLAM family of receptors includes seven mem-bers (CD150 [SLAMF1], CD48 [SLAMF2], CD229, CD244[SLAMF4, 2B4], CD84 [SLAMF5], CD352 [SLAMF6, NTB-A],and CD319) encoded by a locus on chromosome 1 (16). With theexception of CD48, which interacts with CD244, all of the SLAMmembers interact via homophilic interactions. The SLAM recep-tors have important immunomodulatory effects, and the SLAMlocus has been genetically associated with human SLE and mousemodels of SLE (17–21). Therefore, we investigated the mecha-nism for the regulation of CD319 and CD229 and the expressionof signaling molecules downstream of these receptors in pDCs andNK cells. Finally, the expression of all SLAM family members inpDCs and NK cells was compared between healthy individualsand patients with SLE.

Materials and MethodsCells

PBMCs were isolated from healthy blood donors or patients with SLE(Rheumatology Unit, Uppsala University Hospital) by density-gradientcentrifugation. For the comparison of SLAM expression on PBMCs fromhealthy individuals and patients with SLE, PBMCs were resuspended in 90%FCS and 10% DMSO and stored at280˚C pending flow cytometry analysis.NK cells and pDCs were isolated by negative selection, according to themanufacturer’s instructions (NK or pDC cell isolation kit; Miltenyi Biotec,Bergisch Gladbach, Germany), and monocyte-depleted PBMCs were pre-pared using CD14 MicroBeads (Miltenyi Biotec). Isolated pDCs and NKcells were routinely .95% BDCA-2+ and CD32CD56+, respectively. If nototherwise stated, cells were cultured in 96-well V-bottom plates (Nunc,Roskilde, Denmark) using macrophage serum–free medium supplementedwith HEPES (20 mM), penicillin (60 mg/ml), and streptomycin (100 mg/ml)(all from Invitrogen, Carlsbad, CA) at a concentration of 1 3 106 PBMCs/well, 50 3 103 pDCs/well, or 100 3 103 NK cells/well. In some experi-ments, cells were preincubated for 30 min with chloroquine (1 mg/ml),cycloheximide (10 mg/ml), or actinomycin D (1 mg/ml) (Sigma-Aldrich,St. Louis, MO). All cell cultures were incubated at 37˚C with 5% CO2.

Patients

All patients fulfilled at least four of the American College of Rheumatologycriteria for SLE. Patients were 50.2 6 12.0 y old. The disease activity wasdetermined by the SLE disease activity index (22); the median score was 2(range, 0–13). The study was approved by the local ethics committee atUppsala University, and informed consent was obtained from all patientsand controls.

Flow cytometry and mAbs

For flow cytometry, fluorochrome-conjugated mAbs to CD3 (HIT3a or SK7),CD14 (MfP9), CD56 (NCAM16.2), CD19 (HIB19) (all from BD Bio-sciences, San Jose, CA), CD8 (3B5; Invitrogen), CD11c (Bu15; BioLegend,San Diego, CA), BDCA-2 (AC114), and BDCA-4 (AD5-17F6) (both fromMiltenyi Biotec) were used to identify different cell types, as specified. Afluorescent, fixable dead cell marker (LIVE/DEAD near IR; Invitrogen) wasused to exclude dead cells from the analysis. Fluorochrome-conjugatedmAbs to CD69 (TP1.55.3; Beckman Coulter, Indianapolis, IN), CD150(A12[7D4]; eBioscience, San Diego, CA), CD48 (MEM102; BioLegend),CD229 (HLy9.1.25; BioLegend), CD244 (C1.7; BioLegend), CD84(CD84.1.21 [BioLegend] and 2G7 [eBioscience]), NTB-A (#292811; R&DSystems, Minneapolis, MN), and CD319 (162.1; BioLegend) were used todetermine surface expression. If not otherwise stated, pDCs were definedas CD32CD142CD562BDCA-2+, and NK cells were defined as CD32

CD142CD56+. Flow cytometry data were acquired on a FACSCanto II oran LSR Fortessa (BD Biosciences) and analyzed using FlowJo software(version 7.6 and 9.4; TreeStar, Ashland, OR).

For functional studies of pDCs, unlabeled mAbs to CD319 (162.1;BioLegend), CD229 (HLy9.1.25; BioLegend), or BDCA-2 (AC114; Mil-tenyi Biotec) were used.

Stimulation of cells

SLE IgG was purified by protein G chromatography from a patient serumcontaining autoantibodies to SmB, SmD, U1-RNPA, U1-RNP C, ribosomalPAg, histone, and dsDNA (2). U1snRNP particles were purified from HeLa

cells, as previously described (23). U1snRNP particles were used togetherwith SLE IgG at a final concentration of 2.5 mg/ml and 1 mg/ml, re-spectively. Heat-aggregated normal IgG (HAIG) was prepared by heatingOctagam (Octapharma, Stockholm, Sweden) at 63˚C for 60 min at a con-centration of 50 mg/ml and used at a final concentration of 1 mg/ml. TheTLR9 agonist ODN2216 (Cybergene, Stockholm, Sweden) was used at afinal concentration of 3 mg/ml.

For stimulation of cells with cytokines, GM-CSF (2 ng/ml; Leukine;Berlex, Montville, NJ), IFN-a (500 U/ml; Intron A; Schering-Plough,Bloomfield, NJ), IL-3 (10 ng/ml; R&D Systems), IFN-g (100 U/ml),IL-12 (10 ng/ml), IL-18 (100 ng/ml), MIP-1b (50 ng/ml), or TNF-a (10ng/ml; all from PeproTech, Rocky Hill, NJ) was used.

For receptor cross-linking of pDCs, cells were stimulated with RNA-ICsin 96-well flat-bottom ELISA plates coated with F(ab9)2 goat anti-mouseIgG Ab (10 mg/ml; Southern Biotechnology Associates, Birmingham,AL) and 10 mg/ml anti-CD319, anti-CD229, or anti–BDCA-2 or relevantisotype-control mAb.

IFN-a detection

Concentration of IFN-a in culture supernatants was determined using adissociation-enhanced lanthanide fluoroimmunoassay, as previously de-scribed (24).

For detection of intracellular IFN-a, PBMCs depleted of monocytes(1 3 106/well) were stimulated with RNA-ICs for 9 h, with the addition ofGolgiPlug (BD Biosciences) after 6 h. Cells were spun down and stainedfor surface markers, fixed in 1% paraformaldehyde, and permeabilizedwith 0.5% saponin (Sigma-Aldrich) before being stained with allophyco-cyanin-conjugated anti-human IFN-a (LT27:295; Miltenyi Biotec) andanalyzed by flow cytometry.

Western blot analysis

Isolated pDCs or NK cells were resuspended in lysis buffer (50 mMHEPES[pH 7.5], 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, and 1% Triton X-100) supplemented with protease inhibitor mixture (Pierce, Rockford, IL).After lysis, cells were centrifuged at 4˚C for 10 min at 14,000 3 g. Pro-teins were separated by SDS-PAGE (NuPAGE; Invitrogen) and transferredto a polyvinylidene difluoride membrane (Millipore, Billerica, MA) byWestern blotting. A rat anti–SLAM-associated protein (SAP) mAb (XLP1D12; Cell Signaling Technology, Danvers, MA) and HRP-conjugatedgoat anti-rat IgG (H+L) F(ab9)2 fragments (Invitrogen) were used to de-tect SAP. Rabbit polyclonal Abs to Ewing’s sarcoma-activated transcript 2(EAT2), SHIP-1, SHP-2, CSK (Proteintech Group, Chicago, IL), anda rabbit mAb to SHP-1 (EPR5519; Epitomics, Burlingame, CA), followedby HRP-conjugated goat anti-rabbit IgG (H+L) (Invitrogen), were used todetect the indicated signaling molecules.

ResultsCD319 and CD229 expression is increased on both pDCs andNK cells after RNA-IC stimulation

In a screen of receptors and ligands potentially involved in theRNA-IC–induced pDC–NK cell cross-talk, we examined expres-sion of 42 surface molecules (summarized in Supplemental TableI) on pDCs and CD56dim and CD56bright NK cells in PBMCscultured with or without RNA-ICs for 6 h.As expected, strong upregulation on pDCs and NK cells was seen

for the early activation marker CD69 (median fluorescence in-tensity [MFI] was increased 37- and 7-fold on pDCs and CD56dim

NK cells, respectively), confirming that RNA-ICs are potentactivators of both pDCs and NK cells (Fig. 1A). Remarkably, inaddition to CD69, CD319 showed a strong increase in surfaceexpression on both pDCs and CD56dim NK cells (2.7- and 2.2-foldmean increase in MFI, respectively; Fig. 1A). In contrast, RNA-ICs did not induce an increase in expression of CD69 or CD319 onB cells or T cells (Supplemental Fig. 1A).The finding of dynamic regulation of CD319 expression, a re-

ceptor belonging to the immunomodulatory SLAM receptor familyassociated with SLE, prompted us to examine the expression of allseven SLAM family members on pDCs and NK cells before andafter RNA-IC stimulation. In freshly isolated PBMCs, nearly allpDCs expressed CD48, CD229, and CD319, whereas expression ofCD84 and NTB-A was lower. CD56dim and CD56bright NK cells

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expressed a similar profile of SLAM molecules as did pDCs, butthey displayed higher levels of NTB-A, lower levels of CD84, andexpressed CD244. Of note, in contrast to CD56dim NK cells,CD56bright NK cells expressed very low levels of CD319 (Fig. 1B,1C). Upon RNA-IC stimulation, we found that, in addition toCD319, the expression of CD229 was increased on pDCs (1.9-fold) and CD56bright NK cells (2.0-fold) (Fig. 1D). In contrast,none of the SLAM receptors was regulated by RNA-ICs on B cellsor T cells (Supplemental Fig. 1B).To assess the dynamics of the regulation of CD319 and CD229,

PBMCs were stimulated with medium or RNA-ICs and analyzed atdifferent time points. In pDCs, the increased expression of CD319and CD229 started 3 h after RNA-IC stimulation and continued forthe duration of the study (Fig. 1E). In CD56dim NK cells, the ex-pression of CD319 decreased during the first 3 h in both unsti-mulated and RNA-IC–stimulated cultures. This decrease continuedin unstimulated cultures, whereas there was increased CD319 ex-pression in RNA-IC–containing cultures. The CD229 expression inCD56bright NK cells decreased during the time course studied; after6 h only a very low percentage was positive for CD229 (data notshown). Therefore, the increased MFI after RNA-IC stimulation

only resulted in a minor increase in CD229+ CD56bright NK cells;therefore, we chose not to study this regulation further.In summary, stimulation of PBMCs with RNA-IC induces a

rapid and prominent increase in the expression of the SLAMfamily members CD319 and CD229 on pDCs, as well as CD319 onCD56dim NK cells.

RNA-ICs induce CD319 and CD229 via both FcgR and TLRstimulation

Next, we asked whether the ability to stimulate the increasedexpression of CD319 and CD229 on pDCs and NK cells wasspecific for interferogenic ICs or whether it could be triggered byICs without RNA or DNA content. Stimulation of PBMCs withHAIG from healthy donors also induced CD319 and CD229 ex-pression on pDCs and CD319 on CD56dim NK cells, but the in-duction by RNA-ICs was significantly higher (Fig. 2A; p =0.0001, p = 0.03, and p = 0.005, respectively). Because the syn-thetic TLR9 agonist ODN2216 also induced expression of thesemolecules (Fig. 2A), it seemed likely that the effect from RNA-ICs was a combination of FcgR stimulation and endosomal TLRstimulation.

FIGURE 1. RNA-IC increases the expression of CD69, CD319, and CD229 on pDCs and NK cells. PBMCs were analyzed for surface expression of CD69

and SLAM family members on pDCs and CD56dim and CD56bright NK cells by flow cytometry. (A) Expression of CD69 and CD319 on pDCs and CD56dim

and CD56bright NK cells in PBMCs cultured for 6 h in medium only (dashed line) or stimulated with RNA-IC (solid line) relative to isotype stainings (filled

graph). (B and C) Frequencies of SLAM family receptor–positive pDCs and CD56dim and CD56bright NK cells in freshly isolated PBMCs. Graphs show

expression of the indicated SLAM family member (open graph) relative to isotype staining (filled graphs) from one representative healthy volunteer in (B) and

cumulative data from three healthy volunteers in (C). (D) Fold increase in surface expression (MFI) of SLAM family members in 6-h RNA-IC–stimulated

PBMC cultures relative to culturing in medium only. (E) Surface expression of CD319 and CD229 on pDCs and CD319 on CD56dim NK cells at different

time points after culturing in medium only or with RNA-IC stimulation. Live single-cell lymphocytes were gated according to viability stain and forward light

scatter/side scatter characteristics. pDCs were gated as CD11c2CD142CD192CD32CD562BDCA-4+HLA-DR+ cells and NK cells were gated as CD32

CD56+ cells in (A) and (D) or as described in Materials and Methods in (B), (C), and (E). Data (mean + SD) in (C–E) are from four healthy volunteers.

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To verify TLR involvement in the regulation of CD319 andCD229, cells were treated with chloroquine prior to RNA-ICstimulation. Interfering with endosomal TLR signaling by chlo-roquine partially blocked the RNA-IC–induced upregulation ofCD319 and CD229 on pDCs (Fig. 2B; p = 0.03 and p = 0.03,respectively), whereas the difference observed on NK cells was notsignificant (p = 0.06). As expected, chloroquine treatment did notaffect HAIG-mediated induction of CD319 and CD229, but itcompletely blocked ODN2216-mediated induction of CD319 andCD229 (Fig. 2B). Furthermore, preincubation with cycloheximideor actinomycin D completely blocked the upregulation of CD319and CD229 on pDCs and NK cells stimulated by RNA-ICs (Fig.2C), demonstrating the requirements for both transcription andtranslation for CD319 and CD229 expression.Together, these results show that both FcgR stimulation and

endosomal TLR ligation induce CD319 and CD229 expression onpDCs. Moreover, both de novo transcription and translation arerequired to induce these molecules.

CD319 expression is upregulated directly on CD56dim NK cellsand indirectly on pDCs by RNA-ICs

To investigate the requirements for the increased expression of CD319and CD229 on pDCs and NK cells, we stimulated purified cells withRNA-ICs. Surprisingly, when isolated pDCs were stimulated neitherCD319 nor CD229 was upregulated (Fig. 3A). Because the stimu-

lation of pDCs by these ICs is strongly promoted by NK cells (14,15), we proceeded to investigate the regulation of CD319 and CD229in cocultures of pDCs and NK cells. Notably, the addition of NK cellsrestored the upregulation of CD319 and CD229 on pDCs (Fig. 3A).In contrast, in isolated NK cells, CD319 was upregulated by RNA-ICs on CD56dim NK cells, and this increase was even larger than thatseen on CD56dim NK cells in RNA-IC–stimulated PBMCs (Fig. 3A).To further dissect the mechanisms for the increased expression

of CD319 and CD229, we stimulated purified pDCs or NK cellswith RNA-ICs, HAIG, or ODN2216. HAIG did not induce CD319or CD229 on pDCs, whereas it was as efficient as RNA-ICs onCD56dim NK cells (Fig. 3B). Stimulation of pDCs with ODN2216resulted in an increased expression of CD319 and CD229, whereasstimulation of NK cells had no effect. In cocultures of pDCs andNK cells, both HAIG and RNA-ICs induced the expression ofCD319 and CD229 on pDCs, and ODN2216 induced CD319 onCD56dim NK cells. Of note, in cocultures of pDCs and NK cellsRNA-ICs induced a higher expression of the SLAM molecules onpDCs and CD56dim NK cells compared with HAIG.Thus, together these data suggest that ligation of FcgRs on NK

cells and stimulation of endosomal TLRs in pDCs induces theexpression of CD319 on NK cells and CD319/CD229 on pDCs,respectively. Moreover, the increased expression of these mole-cules seems to be further enhanced by a bidirectional cross-talkbetween pDCs and NK cells.To investigate whether soluble factors secreted by NK cells were

important for the regulation of CD319 and CD229, isolated pDCswere stimulated with RNA-ICs in the presence of supernatants fromRNA-IC–stimulated NK cells or unstimulated NK cells. Super-natants from RNA-IC–stimulated NK cells significantly enhancedthe expression of CD319 and CD229 on RNA-IC–stimulatedpDCs (Fig. 3C, p = 0.03 for both), whereas supernatants fromunstimulated NK cells had no effect (p = 0.38 and p = 0.63, re-spectively), indicating that secreted soluble factors were neededfor the regulation of CD319 and CD229. Of note, in comparisonwith NK cell supernatants, NK cells induced a significantly higherexpression of CD319 and CD229 (p = 0.03 for both).RNA-IC stimulation of pDC–NK cell cocultures induces the

production of multiple cytokines that can affect both pDCs andNK cells (15). To investigate which cytokines could be responsiblefor the increased expression of CD319 and CD229, purified pDCswere cultured with or without RNA-IC stimulation and the additionof a selection of recombinant cytokines, which are either producedin pDC–NK cell cocultures (15) or known to have a stimulatoryeffect on pDCs (25). A weak increase in CD319 and CD229 ex-pression on pDCs was seen after the addition of rTNF-a, rGM-CSF, and rIL-3 (Fig. 3D). However, when pDCs were stimulatedwith RNA-ICs together with GM-CSF or IL-3, an equally strongincrease in CD319 and CD229 expression was seen as when NKcells were used (Fig. 3D). To investigate whether CD319 andCD229 could be induced in the absence of RNA-ICs, pDCs werestimulated with combinations of GM-CSF, IFN-a, and IFN-g. Acombination of GM-CSF and IFN-a or IFN-g was as potent aninducer of CD319 and CD229 as was RNA-ICs in combinationwith GM-CSF (Fig. 3E). On CD56dim NK cells, expression ofCD319 was not affected by GM-CSF or IL-3, but it was increasedstrongly following IFN-a or IL-12/IL-18 stimulation. This effectwas seen even in the absence of RNA-ICs (Fig. 3D).In conclusion, RNA-ICs, IFN-a, and IL-12/IL-18 induce the

expression of CD319 directly on CD56dim NK cells, whereaspDCs require a combination of stimuli, such as RNA-ICs togetherwith NK cells, RNA-ICs together with cytokines (e.g., GM-CSFor IL-3), or multiple cytokines (e.g., GM-CSF and IFN-a) to in-crease their expression of CD319 and CD229.

FIGURE 2. Both IgG and the nucleic acid component of RNA-IC are

important for the regulation of CD319 and CD229 expression. Fold increase

in CD319 and CD229 expression (MFI) in PBMCs stimulated with RNA-

ICs, HAIG, or ODN2216 for 6 h (A) and the effect of preincubation with

chloroquine (CQ) (B) or cycloheximide (CHX) or actinomycin D (ActD) (C),

as determined by flow cytometry. In (C), data are shown only for RNA-IC

stimulation. Data (mean + SD) are from at least six (A, B) or at least four (C)

healthy volunteers from two or more independent experiments. *p , 0.05,

**p , 0.01, ***p , 0.001, Wilcoxon signed-rank test. ns, Not significant.

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IFN-a–producing pDCs have higher CD319 and CD229expression

Because the expression of CD319 and CD229 was upregulated byinterferogenic ICs, we asked whether there was a difference insurface expression of SLAM molecules between the pDCs that

produced IFN-a and the pDCs that did not. Because monocytesinhibit IFN-a production by pDCs (14), PBMCs were depleted of

monocytes before stimulation with RNA-ICs. After stimulation,

pDCs were evaluated for intracellular IFN-a and SLAM family

member expression. The frequency of pDCs that produced IFN-a

FIGURE 3. The increased expression of CD319 and

CD229 on pDCs is dependent on NK cells. Expression

(MFI) of CD319 and CD229 was determined by flow

cytometry in 6-h cell cultures with or without RNA-IC

stimulation. (A) Expression on pDCs and NK cells in

cell cultures of PBMCs, isolated pDCs, isolated NK

cells, or cocultures of pDCs and NK cells. (B) Fold

increase in MFI in isolated pDCs, NK cells, or cocul-

tures of pDCs and NK cells stimulated with RNA-IC,

HAIG, or ODN2216. (C) Fold increase in MFI in RNA-

IC–stimulated pDCs with or without the addition of

supernatants from unstimulated NK cells [NK(0)],

supernatants from RNA-IC–stimulated NK cells [NK

(IC)], or NK cells. (D) Isolated pDCs or NK cells were

cultured with the indicated cytokines, with or without

the addition of RNA-IC, and compared with pDC–NK

cell cocultures. (E) Isolated pDCs cultured with the

indicated cytokines, with or without the addition of

RNA-IC. Data (mean + SD) are from six individuals

from three independent experiments in (A), four indi-

viduals from two independent experiments in (B) and

(E), and two healthy volunteers in (D). *p , 0.05,

Wilcoxon signed-rank test.

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after RNA-IC stimulation was 31.56 7.3% (mean6 SD). Nearly allIFN-a+ pDCs expressed CD319, CD229, and CD84, whereas onlya subpopulation of the IFN-a–producing pDCs expressed NTB-A(Fig. 4A). Interestingly, the IFN-a–producing pDCs had a signifi-cantly higher expression of CD319, CD229, and NTB-A comparedwith the IFN-a2 pDCs, as determined by the MFI (Fig. 4B).Thus, following RNA-IC stimulation, the IFN-a–producing

pDCs are characterized by a higher expression of CD319, CD229,and NTB-A compared with IFN-a2 pDCs.

pDCs do not express the activating, but do express theinhibitory, adaptor molecules of SLAM receptors

Given the strong regulation of CD319 and CD229 by interferogenicICs and their increased expression on IFN-a–producing pDCs, wenext investigated whether CD319 and/or CD229 affected IFN-aproduction. To this end, CD319 or CD229 was cross-linked onRNA-IC–stimulated pDCs through plate-bound mAbs. Anti–BDCA-2 mAb, which inhibits IFN-a production by pDCs (26),was used as a control. Although BDCA-2 cross-linking stronglyinhibited IFN-a production, cross-linking of CD319 or CD229had no effect (Fig. 5A).To better understand the function of CD319 and CD229 on

pDCs, we investigated the downstream-signaling molecules ofthese receptors. The signaling through SLAM receptors is mediatedvia the activating SH2 domain–containing adaptor molecules SAPand EAT2 (27). Although NK cells are known to express both, theexpression of SAP and EAT2 has not been studied in pDCs. Todetermine whether pDCs expressed SAP and EAT2, Western blotsof whole-cell lysates from isolated pDCs and NK cells wereperformed. No expression of SAP was observed in pDCs, whereasa strong expression was seen in NK cells (Fig. 5B). EAT2 washighly expressed in NK cells but barely detectable in pDCs. Asimilar weak signal from EAT2 was seen from all cell types tested(data not shown) and probably reflects background staining.In the absence of activating adaptor molecules, SLAM receptors

were suggested to signal via inhibitory SH2 domain–containingadaptor molecules (e.g., SHIP-1, SHP-1, SHP-2, and CSK) (27).Western blots of whole-cell lysates from purified pDCs and NK cellsrevealed that both cell types expressed these molecules (Fig. 5C).

In summary, cross-linking of CD319 or CD229 alone on pDCs doesnot influence IFN-a production. Furthermore, pDCs lack expressionof SAP and EAT2 but express SHIP-1, SHP-1, SHP-2, and CSK.

FIGURE 4. IFN-a1 and IFN-a2 pDCs differ in SLAM family member

expression. (A and B) Expression of indicated SLAM family members on

IFN-a+ and IFN-a2 pDCs in RNA-IC–stimulated CD14-depleted PBMCs

was determined by flow cytometry. Data from four healthy volunteers are

presented as frequency (%) in (A) and MFI in (B). pDCs were defined as

CD32CD11c2CD142CD192CD562HLA-DR+BDCA-4+ cells. Statistical

analysis was performed using the paired t test.

FIGURE 5. pDCs lack the activating adaptor molecules but express

inhibitory adaptor molecules of SLAM receptors. (A) IFN-a production by

RNA-IC–stimulated pDCs after cross-linking of CD319, CD229, or

BDCA-2 by plate-bound mAbs. IFN-a levels were determined in 20-h cell

culture supernatants by an immunoassay and normalized to the IFN-a

production in cultures without plate-bound mAb. The dashed line indicates

100%. Data (mean + SD) are from three donors from two independent

experiments. Expression of EAT2 and SAP (B) and SHIP-1, SHP-1, SHP-

2, and CSK (C) in cell lysates from isolated pDCs or NK cells from two

healthy donors was determined by Western blot. Expression of b-actin was

used as control.

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Altered expression of CD319 and CD229 on pDCs and NKcells from patients with SLE

Given the potent regulation of SLAM receptors on pDCs andNK cells by RNA-ICs in vitro, and the fact that patients with SLEhave similar interferogenic ICs in circulation, we compared theexpression of all SLAM receptors on pDCs and NK cells frompatients with SLE and healthy individuals.Patients with SLE have a reduced frequency of circulating pDCs

(24), and two of the patients in this cohort were excluded fromthe analysis of pDCs because they had too few cells (,100 events) toevaluate. Like pDCs from healthy individuals, almost all SLE pDCsexpressed CD48, CD229, and CD319 (data not shown). However,pDCs from patients with SLE had a significantly lower expressionof CD84 and CD319 (p = 0.04 and p = 0.001, respectively) (Fig.6A). A slightly lower expression of CD229 was also seen on SLEpDCs, but the differencewas not statistically significant (p = 0.102).

We observed a decreased expression of CD229 and an increasedexpression of CD150 on CD56dim NK cells from SLE patients

(p = 0.002 and p = 0.005, respectively), whereas CD56bright NK

cells had an increased expression of CD319 and CD48 (p = 0.01

and p = 0.009, respectively).Finally, PBMCs from patients with SLE were stimulated with

RNA-ICs, and the regulation of CD319 and CD229 on pDCs

and CD56dim NK cells was compared with that of healthy do-

nors.Similar to cells from healthy donors, pDCs and CD56dim NK

cells from patients with SLE responded to RNA-IC stimulation

with an increased expression of CD319 and CD229 on pDCs and

CD319 on CD56dim NK cells (Fig. 6B).Thus, patients with SLE have an altered expression of several

SLAM receptors, including both CD319 and CD229, on pDCs and

NK cells.

FIGURE 6. Altered expression of SLAM family receptors on immune cells from patients with SLE. (A) PBMCs were stained for SLAM family receptors

and analyzed by flow cytometry. Expression (MFI) of indicated SLAM family receptor on pDCs, CD56dim NK cells, and CD56bright NK cells, as specified. Live

single-cell lymphocytes were gated according to viability stain and forward light scatter/side scatter characteristics. pDCs were gated as CD142CD32CD192

CD162CD562CD11c2BDCA-2+CD4+, and NK cells were gated as CD142CD32CD192CD56+. Data from 19 healthy donors (HD) and 19 patients with SLE

are shown for pDCs, and data from 19 healthy donors (HD) and 21 patients with SLE are shown for CD56dim and CD56bright NK cells. Horizontal lines denote

the median value. (B) PBMCs from six healthy donors (HD) or five patients with SLE were cultured for 6 h, with or without the addition of RNA-IC, and

expression of CD319 and CD229 was determined by flow cytometry on pDCs and CD56dim NK cells. Data from two independent experiments are presented as

fold increase in MFI in RNA-IC–stimulated cells relative to cells cultured in medium. *p , 0.05, **p , 0.01, ***p , 0.001, Mann–Whitney U test.

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DiscussionSLAM family receptors are important immunomodulatory re-ceptors involved in the cross-talk between a number of immune

cells (16). The SLAM locus is genetically associated with SLE

(17–21), and recent data suggest involvement of SLAM receptors

in the pathogenesis of lupus (28–30). In the current study, we

found that the expression of the SLAM family receptors CD319

and CD229 was dynamically regulated on pDCs and CD56dim NK

cells by RNA-ICs typically found in lupus. Importantly, there was

no general upregulation of all SLAM molecules on pDCs and NK

cells or an increase in SLAM family receptor expression on T and

B cells by RNA-ICs. Thus, lupus ICs seem to selectively regulate

the expression of certain SLAM molecules on two cell types,

which together have the capacity to produce large amounts of type

I IFN after activation.Interferogenic ICs, such as RNA-ICs, have the capacity to ac-

tivate both FcgRIIA and endosomal TLRs. We showed that sig-

naling induced by engagement of both the IgG component and the

nucleic acid–containing autoantigen in the RNA-ICs contributed

to the increased expression of CD319 and CD229 on pDCs and

CD319 on CD56dim NK cells. When the mechanisms behind the

regulation of CD319 and CD229 were investigated further, we

found that transcription and translation were required. Experi-

ments using isolated or mixed populations of purified pDCs and

NK cells stimulated with RNA-ICs revealed that the upregulation

of CD319 and CD229 on pDCs by RNA-ICs required NK cells.

This effect was partially mediated by soluble factors produced by

NK cells, because supernatant from RNA-IC–stimulated NK cells

increased CD319/CD229 expression. RNA-ICs in combination

with GM-CSF or IL-3, or GM-CSF together with IFN-a or IFN-g

was an equally strong inducer of CD319 and CD229 on pDCs as

NK cells and RNA-ICs. These results demonstrate that the regu-

lation of at least some SLAM molecules on pDCs is complex and

involve several pathways that need to act in concert to trigger an

increased expression. In support of this conclusion, a previous

study showed that the combination of HSV and IL-3 is necessary

to upregulate CD319 in pDCs (31). Whether the induction of

SLAM molecules is coupled to maturation of pDCs to efficient

Ag-presenting dendritic cells is unknown, but this clearly is a

possibility given the fact that both IL-3 and GM-CSF are stimuli

for pDC differentiation (25).In contrast to pDCs, purified NK cells activated by RNA-ICs

exhibited a strong increase in CD319 expression on CD56dim NK

cells. This increase was even more pronounced than the increase

in CD319 among CD56dim NK cells in PBMC cultures. Conse-

quently, inhibitory cells present in the PBMC population may

suppress the upregulation of SLAM molecules on NK cells, pos-

sibly via the release of reactive oxygen species, TNF-a, and PGE2,

which suppress NK cell function (14). We also noted that IFN-a

and IL-12/IL-18 could upregulate CD319 on CD56dim NK cells,

representing an interesting mechanism whereby inflammation may

augment NK cell recognition of, and activation by, hematopoietic

cells. Notably, RNA-ICs or proinflammatory cytokines increased

the CD319 expression on NK cells to comparable levels. Thus, it

appears that at least partially different signaling pathways lead to

CD319 expression in pDCs and NK cells. Furthermore, we noted

that IL-12/IL-18 increased the expression of CD319 on CD56bright

NK cells (data not shown). These cytokines are well-known acti-

vators of NK cells (32), and one mechanism by which both IL-12/

IL-18 and RNA-ICs might contribute to increased NK cell cyto-

toxicity is by upregulation of CD319.The pDC population is heterogeneous with respect to the ex-

pression of several molecules and the capacity to produce IFN-a.

To clarify whether there was any difference in the expression ofdifferent SLAM molecules between pDCs with and without theability to synthesize IFN-a, we stimulated the cells with RNA-ICsand investigated SLAM expression and IFN-a content among thepDCs. This experiment revealed that the IFN-a–producing pDCsdisplayed higher expression of CD319 and CD229, as well asNTB-A, compared with IFN-a2 cells. It is well known that onlyFcgRIIA-expressing pDCs have the capacity to produce IFN-a inresponse to interferogenic ICs (33, 34), and the differences inSLAM expression between IFN-a+ and IFN-a2 pDCs could bedue to the lack of FcgRIIA/TLR-7 activation of the latter pop-ulation. Thus, our observation may indicate that induction of IFN-aproduction and increased SLAM molecule expression by RNA-ICstimulation of pDCs are linked processes in the activation of pDCs,although this needs to be formally proven.On NK cells, CD319 and CD229 act as coactivating receptors,

which results in increased cytotoxicity against targets cells ex-pressing CD319 and CD229 (35, 36), whereas, on B cells, ligationof CD319 induces proliferation and cytokine production (37). Incontrast, no study of the function of SLAM receptors on pDCs hasbeen reported. When investigating the potential role of CD319 andCD229 on pDC function, we noted that cross-linking of thesereceptors by immobilized mAbs did not affect the RNA-IC–induced IFN-a secretion in isolated pDCs. This finding does notnecessarily exclude the involvement of CD319 and CD229 inregulating IFN-a production, because SLAM receptors functionas coreceptors. Thus, CD319 and CD229 may be dependent onconcomitant engagement of other activating or inhibitory recep-tors to exert their effect. Although such receptors are well char-acterized on NK cells, the identity of these receptors on pDCsremains to be determined. Blocking experiments might be a wayto circumvent the need for coligation of other receptors, but thelack of Abs that effectively block the homophilic binding ofCD319 or CD229 regrettably precludes these experiments. Giventhese circumstances, we cannot exclude a function for CD319 andCD229 in regulating IFN-a production.With the exception of CD48, the SLAM molecules contain in-

tracellular immuno-tyrosine switch motifs that bind and signal viathe SH2 domain–containing adaptor molecules SAP and EAT2(27). CD319 differs from the other SLAM family receptors in that itonly recruits EAT2 (38). SAP is expressed in T cells, NK cells,NKT cells, and perhaps some B cells, whereas EAT2 expression hasbeen described in NK cells, dendritic cells, and macrophages (27).The presence of SAP or EAT2 has not previously been investigatedin pDCs. When isolated pDCs and NK cells were examined for SAPand EAT2, no SAP and little to no EAT2 were detected in pDCs. Incontrast, and as expected, high levels of both SAP and EAT2 weredetected in NK cells. Ligation of CD319 in NK cells deficient inEAT2 was shown to abolish the activating effect of CD319 andresult in inhibition of NK cell cytotoxicity (39). Furthermore, li-gation of CD319 on CD4+ T cells or monocytes, which naturallylack EAT2 expression, inhibits Ag-induced proliferation and LPS-induced production of TNF-a, respectively (39, 40). The inhibitoryeffect of SLAM receptors in cells lacking SAP and EAT2 probablycan be explained by the fact that, in the absence of these molecules,SLAM receptor signaling couples to inhibitory adaptor proteins,such as SHIP-1, SHP-1, SHP-2, and CSK (27, 41). By Western blot,we showed that pDCs expressed these molecules, suggesting thatCD319 and CD229 act as inhibitory coreceptors on pDCs. Anotherpossibility is that these receptors promote the activation of othercells expressing the corresponding molecules. For instance, SLAMfamily receptors on NK cells enhance LFA-1–dependent conjugateformation (41); because IFN-a production is strongly promoted byLFA-1–mediated interaction between pDCs and NK cells (15),

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expression of CD229 and CD319 on pDCs may support the pDC–NK cell cross-talk.Finally, we investigated the expression of the SLAM molecules

on pDCs and NK cells from patients with SLE. Patients with SLEhad decreased expression of CD84 and CD319 on circulatingpDCs, which is at variance with the in vitro stimulation of PBMCsfrom healthy individuals with RNA-ICs. However, RNA-IC stimu-lation of SLE PBMCs increased the expression of CD319 andCD229 on pDCs, demonstrating that SLE pDCs retain their ca-pacity to upregulate these molecules. One explanation for thedecreased SLAM expression on circulating pDCs in SLE could bethat SLE IC-activated cells migrate from the circulation to inflamedtissues. Such redistribution from blood to tissues of pDCs thatproduce IFN-a was also described in SLE (42, 43) and mightexplain our observation. On SLE NK cells, decreased expressionof CD229 on CD56dim NK cells and increased expression ofCD319 and CD48 on CD56bright NK cells were observed. Theexact consequence of the altered expression of SLAM moleculesin SLE NK cells is unknown, but it might reflect several previousobservations. For instance, the lower expression of CD229 onCD56dim cells may contribute to the decreased cytotoxicity of SLENK cells (44, 45). In contrast, the increased expression of SLAMmolecules on CD56bright NK cells may play a role in the increasedproinflammatory cytokine production in SLE (46). Conceivablysupporting the latter interpretation is the recent observation thatpatients with SLE have increased expression of CD229 and NTB-A on T cells and that activation of CD229 or NTB-A, togetherwith CD3, promotes IL-17 production (30).In summary, our study shows that RNA-ICs regulate a subset of

SLAM molecules on pDCs and NK cells and that lupus patientshave an altered expression of CD319 and CD229 on these celltypes. Our observations are important in light of emerging datademonstrating that the SLAM family of receptors is implicated inautoimmune disease processes. Because SLAM molecules havebeen suggested as both possible biomarkers and therapeutic targetsin SLE (30), further studies of the precise function of SLAMreceptors on pDCs and their role in the pathogenesis of SLE seemhighly warranted.

AcknowledgmentsWe thank Gert Weber for kindly providing the U1snRNP complexes,

Gunnar Alm for reviewing the manuscript, and Anne Tronnberg, Charlotta

Jakobsson, and Rezvan Kiani Dehkordi for excellent technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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