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Th17 DifferentiationErythematosus T Lymphocytes PromotesSLAMF3 and SLAMF6 in Systemic Lupus Increased Expression of SLAM Receptors

and George C. TsokosVasileios C. Kyttaris, Christian M. Hedrich, Cox Terhorst Madhumouli Chatterjee, Thomas Rauen, Katalin Kis-Toth,

http://www.jimmunol.org/content/188/3/1206doi: 10.4049/jimmunol.1102773December 2011;

2012; 188:1206-1212; Prepublished online 19J Immunol 

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3.DC1http://www.jimmunol.org/content/suppl/2011/12/19/jimmunol.110277

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The Journal of Immunology

Increased Expression of SLAM Receptors SLAMF3 andSLAMF6 in Systemic Lupus Erythematosus T LymphocytesPromotes Th17 Differentiation

Madhumouli Chatterjee,* Thomas Rauen,* Katalin Kis-Toth,* Vasileios C. Kyttaris,*

Christian M. Hedrich,* Cox Terhorst,† and George C. Tsokos*

Altered T cell function in systemic lupus erythematosus (SLE) is determined by various molecular and cellular abnormalities,

including increased IL-17 production. Recent evidence suggests a crucial role for signaling lymphocyte activation molecules

(SLAMs) in the expression of autoimmunity. In this study, we demonstrate that SLAMF3 and SLAMF6 expression is increased

on the surface of SLE T cells compared with normal cells. SLAM coengagement with CD3 under Th17 polarizing conditions results

in increased IL-17 production. SLAMF3 and SLAMF6 T cell surface expression and IL-17 levels significantly correlate with disease

activity in SLE patients. Both naive and memory CD4+ T cells produce more IL-17 in response to SLAM costimulation as

compared with CD28 costimulation. In naive CD4+ cells, IL-17 production after CD28 costimulation peaks on day 3, whereas

costimulation with anti-SLAMF3 and anti-SLAMF6 Abs results in a prolonged and yet increasing production during 6 d. Unlike

costimulation with anti-CD28, SLAM costimulation requires the presence of the adaptor molecule SLAM-associated protein.

Thus, engagement of SLAMF3 and SLAMF6 along with Ag-mediated CD3/TCR stimulation represents an important source of

IL-17 production, and disruption of this interaction with decoy receptors or blocking Abs should mitigate disease expression in

SLE and other autoimmune conditions. The Journal of Immunology, 2012, 188: 1206–1212.

For optimal T cell activation, recognition of the Ag/MHCcomplex by the TCR is accompanied by signals medi-ated through costimulatory pathways (1, 2). CD28 co-

stimulation is best characterized for T cell activation (3), butthere is evidence for other costimulatory molecules, includingsignaling lymphocyte activation molecule (SLAM) receptor fam-ily members (4, 5). Recently, the SLAM family of type I trans-membrane receptors has been reported to mediate importantregulatory signals between immune cells through their homophilicor heterophilic interactions. SLAM receptors are expressed onhematopoietic cells, including cells of the innate immune system,as well as T and B cells. By virtue of their ability to transducetyrosine phosphorylation signals through immunoreceptor tyrosine-based switch motif sequences, SLAM receptors play an importantrole in regulating both innate and adaptive immune responses.Upon activation, SLAM molecules associate with intracellularadaptor proteins, for example, those of the SLAM-associated

protein family (6–11). SLAM-associated proteins (SAPs) con-tribute to SLAM receptor activation as they mediate dimerizationof SLAM receptors and compete with SLAM-induced signals.SAP deficiency is associated with severe NK, T, and B cell ab-normalities and reduced Ab production (12).Recent evidence indicates that SLAM signaling is also involved

in the pathogenesis of autoimmune diseases, including systemiclupus erythematosus (SLE) (13, 14). SLE is a chronic autoimmuneinflammatory disease that is characterized by improper regulationof B and T cell function (15). The SLAM gene cluster encodesseveral costimulatory receptors, including SLAMF3 and SLAMF6.It is located within a genomic region, which entails genes with keyimmunological functions, including the Fc receptor cluster, theSLAM cluster, CD58, IL23R, TLR5, and complement receptor CR1(16, 17). Polymorphisms in the SLAM cluster have been associatedwith autoimmune diseases in mice and humans for which this re-gion was designated Sle1b locus and is considered as a geneticsusceptibility region for the development of SLE (16, 18, 19).Polymorphisms in the Ly108 gene, one of the members of theSLAM family receptors (corresponding to SLAMF6 in humans),result in the generation of an Ly108 splice variant in lupus-pronemice that is involved in the pathogenesis of SLE (16, 20).Differentiation of CD4+ Th cells into distinct effector pop-

ulations is one of the hallmarks of adaptive immune responses.Previous reports suggest that costimulation of Th cells throughSLAMF6 promotes a Th1 phenotype under polarizing and non-polarizing conditions and, furthermore, SLAMF6 appears to havesuperior costimulatory capacities when compared with CD28,especially on CD8+ and CD4/CD8 double-negative T cells (14,21). Another member of the SLAM family, SLAMF3 (also knownas CD229/Ly9 in mice), which is expressed on T and B cells, hasbeen reported to promote Th2 differentiation (22). The most re-cently discovered Th cell subset, denoted Th17 cells, is charac-terized by abundant production of IL-17A (referred to in thisarticle as IL-17), IL-21, and IL-22 and plays a major role in host

*Division of Rheumatology, Department of Medicine, Beth Israel Deaconess Med-ical Center, Harvard Medical School, Boston, MA 02215; and †Division of Immu-nology, Department of Medicine, Beth Israel Deaconess Medical Center, HarvardMedical School, Boston, MA 02215

Received for publication September 28, 2011. Accepted for publication November28, 2011.

This work was supported by National Institutes of Health Grant PO1 AI065687 andby Deutsche Forschungsgemeinschaft Grant RA1927/1-1 (to T.R.).

Address correspondence and reprint requests to Dr. George C. Tsokos, Division ofRheumatology, Department of Medicine, Beth Israel Deaconess Medical Center,Harvard Medical School, 330 Brookline Avenue, CLS 937, Boston, MA 02115.E-mail address: gtsokos@bidmc.harvard.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: SAP, signaling lymphocyte activation molecule-associated protein; siRNA, small interfering RNA; SLAM, signaling lymphocyteactivation molecule; SLE, systemic lupus erythematosus; SLEDAI, systemic lupuserythematosus disease activity index.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102773

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responses against bacterial infections and the development ofautoimmune diseases, including SLE (23, 24). Indeed, higher se-rum concentrations of IL-17 have been reported in SLE patients(25, 26), and studies in lupus-prone mice provide evidence forIL-17 as a crucial mediator of disease pathology in SLE (27–29).

Because SLAMF6 and SLAMF3 have been shown to be en-gaged in Th cell differentiation, we asked whether SLAMreceptors play a role in the pathogenesis of SLE, and whether theycontribute to Th17 differentiation. We report that surface ex-pression of SLAMF6 and SLAMF3 is increased in SLE T cells andmirrors disease activity. Our results indicate that coactivation ofSLAMF6 and SLAMF3 receptors along with CD3/TCR stimu-lation potently induces IL-17 production and thus accounts forTh17 generation in T cells from both healthy controls and SLEpatients.

Materials and MethodsStudy subjects and T cell culture

All SLE patients included in our studies were recruited from the Di-vision of Rheumatology at Beth Israel Deaconess Medical Center(Boston, MA) and diagnosed according to the revised SLE classificationcriteria of the American College of Rheumatology (30). SLE diseaseactivity index (SLEDAI) scores ranged from 0 to 10. Written informedconsent was obtained from all patients. Control blood samples wereobtained from healthy platelet donors at the Kraft Family Blood DonorCenter (Dana-Farber Cancer Institute, Boston, MA). Primary totalT cells were isolated from peripheral venous blood by negative se-lection as described previously (14). All primary T cells were keptin RPMI 1640 medium supplemented with 10% FBS. Naive (CD4+

CD45RA+) and memory (CD4+CD45RO+) T cells were purified usingT cell isolation kits from Miltenyi Biotec according to the manu-facturer’s instructions.

T cell stimulation, Th17 differentiation assays, and ELISAs

Cell culture plates were precoated overnight with 0.5 mg/ml monoclonalanti-CD3 (clone OKT3; BioXCell,), 0.5 mg/ml anti-CD28 (BioLegend),0.5 mg/ml anti-SLAMF6 (clone 24D8.1H5.1F5; Genentech), 0.5 mg/mlanti-SLAMF3 Abs (clone HLy-9.1.25; BioLegend), or 0.5 mg/ml controlIgG1 as indicated. Naive and memory CD4+ T cells (1 3 106/well) weredifferentiated into Th17 cells in serum-free X-VIVO 10 medium (Bio-Whittaker) by the addition of IL-6 (25 ng/ml), TGF-b1 (5 ng/ml), IL-1b(12.5 ng/ml), IL-21 (25 ng/ml), and IL-23 (25 ng/ml) for the indicatedtimes. IL-6, IL-1b, IL-23, and TGF-b1 were obtained from R&D Systems.IL-21 was purchased from Cell Sciences. Supernatants were collected atdifferent time points and tested for IFN-g (Endogen) and IL-17 (eBio-science) by ELISA.

FIGURE 2. SLAM receptor costimulation enhances

IL-17 production and reflects disease activity in SLE

patients. A, Total T cells obtained from 11 healthy

controls and 11 SLE patients were cultured under Th17

differentiation conditions along with anti-CD3 Abs

and costimulatory anti-CD28, anti-SLAMF3, or anti-

SLAMF6 Abs as indicated for a total period of 6 d.

Subsequently, supernatants were subjected to IL-17

measurement (ELISA). B, Total T cells were cultured

as outlined under A. Intracellular IL-17 staining was

performed gating on CD4+ T cells. C, Total T cells

were isolated from SLE patients and cultured under

Th17 conditions along with anti-CD3 and anti-

SLAMF3 Abs for 6 d. Percentages of IL-17–producing

CD4+ T cells were correlated to the individual SLE-

DAI. (D) Percentages of IL-17–producing CD4+

T cells isolated from SLE patients (under anti-CD3/

anti-SLAMF6 costimulation) were correlated to the

individual SLEDAI scores. CON, controls.

FIGURE 1. Cell surface expression of SLAMF3 and SLAMF6 receptors

is increased on SLE T cells. A, Total T cells from 11 healthy controls and

11 SLE patients were analyzed for surface expression of SLAMF3 by

flow cytometry gating on CD4+ T cells. B, Total T cells from the same

individuals were analyzed for surface expression of SLAMF6 by flow

cytometry gating on CD4+ T cells. C, Surface expression of SLAMF3 on

CD4+ T cells obtained from SLE patients was correlated to the individual

SLEDAI. D, Surface expression of SLAMF6 on CD4+ T cells obtained

from SLE patients was correlated to the individual SLEDAI scores. CON,

controls; MFI, mean fluorescence intensity.

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Flow cytometry

Intracellular T cell staining for IL-17 and IFN-g was performed at theindicated time points using the BD Cytofix/Cytoperm kit and Alexa 647-labeled anti–IL-17 (BD Biosciences) and PE-labeled anti–IFN-g (Bio-Legend) Abs. Cells were stimulated with PMA (50 ng/ml) and ionomycin(0.5 mg/ml) for a total period of 6 h. GolgiStop (monensin) was added 30min after T cell stimulation was initiated. Surface staining was performedusing a Pacific Blue-labeled anti-CD4 Ab (BioLegend) for 20 min on ice.Samples were analyzed on an LSRII flow cytometer (BD Biosciences) andanalysis was performed with FlowJo software version 8.3.3 (Tree Star).

Small interfering RNA experiments

Naive (CD4+CD45RA+) T cells were purified from healthy donors andtransfected with different concentrations of small interfering RNA (siRNA)

oligonucleotides specific for SLAMF3, SLAMF6, SAP, or a nonspecificcontrol siRNA (all from Applied Biosystems) using the Amaxa transfec-tion system (Lonza). SAP siRNA transfection efficiency was confirmed byimmunoblotting of cytosolic protein lysates using anti-SAP (Cell SignalTechnology), anti–b-actin (Sigma-Aldrich), and suitable HRP-linked sec-ondary Abs. SLAMF3 and SLAMF6 siRNA transfection efficiency wasconfirmed by analyzing their surface expression by flow cytometry usinganti-SLAMF3 and anti-SLAMF6 Abs (both from BioLegend). All subse-quent assays were performed 4 d after transfection at the point of maximalknockdown.

Statistical analyses

The paired two-tailed Student t test and the Pearson product momentcorrelation coefficient (r) were used for statistical analyses.

FIGURE 3. SLAM costimulation promotes Th17 differentiation in naive CD4+ T cells. A–C, Naive (CD45RA+CD62Lhi) CD4+ T cells were stimulated

with anti-CD3 and costimulatory anti-CD28, anti-SLAMF3, SLAMF6, and control IgG under Th17 conditions for 7 d. IL-17 and IFN-g production was

analyzed by intracellular staining and ELISA. A, Secretion of IL-17 by naive CD4+ T cells on day 7 was measured by ELISA. B, Percentages of IL-17–

producing cells. The relative number of IL-17–producing cells is given as a percentage of naive CD4+ T cells. C, One representative experiment is

displayed (from day 7). D, Kinetics of IL-17 secretion by naive CD4+ T cells in response to costimulation through CD28, SLAMF3, and SLAMF6. On

days 3, 5, and 7 the IL-17 levels were monitored by ELISA. E, Naive CD4+ T cells were labeled with CFSE, then stimulated and differentiated until day

7. Staining on day 7 was as above. No differences in CFSE staining were detected between the different groups, indicating comparable proliferation

rates.

1208 SLAM COSTIMULATION PROMOTES Th17 DIFFERENTIATION

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ResultsSLAMF3 and SLAMF6 are upregulated on SLE T cells andserve as major costimuli for Th17 differentiation

To assess the involvement of SLAM costimulation in SLE patho-genesis, we performed flow cytometric analysis of SLAM re-ceptor surface expression on CD4+ T cells obtained from 11 SLEpatients at different levels of disease activity as measured by theSLEDAI and from 11 healthy control subjects. We observed asignificantly increased surface expression of both SLAMF3 andSLAMF6 on CD4+ T cells from SLE patients in comparison withthe healthy individuals (Fig. 1A, 1B). Of note, there was a robustcorrelation between the individual cell surface expression ofSLAMF6 on SLE CD4+ T cells and the corresponding SLEDAIindices (r = 0.9178; Fig. 1D); however, we did not find a signifi-cant correlation in the case of SLAMF3 (Fig. 1C).Because it has been reported that SLAM signaling is involved in Th

cell differentiation and that SLE patients display increased serum

levels of IL-17, we hypothesized that the observed upregulation of

SLAMF3/SLAMF6 expression in SLE CD4+ T cells contributes to

an augmented production of IL-17. Thus, we stimulated T cells from

SLE patients and healthy controls with anti-CD3 and costimulatory

anti-CD28, anti-SLAMF3, or anti-SLAMF6 Abs in the presence of

Th17 polarizing stimuli (i.e., IL-6, IL-1b, IL-21, IL-23, and TGF-

b1). On day 6 after stimulation and differentiation had been initiated,

we measured IL-17 levels in cell culture supernatants (Fig. 2A) and

the percentage of IL-17–producing CD4+ T cells (Fig. 2B, Supple-

mental Fig. 1). Overall, SLE CD4+ T cells were found to produce

more IL-17 under these conditions. Intriguingly, costimulation

through both SLAM receptors was more potent in the induction of

IL-17 production when compared with CD28 costimulation. We

observed a strong correlation between the clinical disease activity

and the corresponding percentage of IL-17–producing CD4+ T cells

as induced through costimulation with either anti-SLAMF3 or anti-

SLAMF6 Abs (r = 0.9361 and 0.9701, respectively; Fig. 2C, 2D). Todetermine whether Th17 polarizing cytokines were necessary forthe production of IL-17 following engagement of SLAMF3 andSLAMF6 molecules on the surface of SLE T cells, T cells from SLEpatients and healthy controls were stimulated with anti-CD3 andanti-CD28, anti-SLAMF3, or anti-SLAMF6 Abs in the absence ofTh17 polarizing cytokines. We observed an increase in IL-17Aproduction (Supplemental Fig. 2A, 2B) following SLAM costim-ulation compared with stimulation with CD28, suggesting thatSLAMF3/6 costimulation can cause IL-17 production in SLET cells in the absence of Th17 polarizing cytokines.Taken together, our data suggest that T cells from active SLE

patients are characterized by an increased SLAM receptor ex-pression, which is linked to increased IL-17 production.

SLAM-mediated costimulation promotes Th17 differentiation inhuman T cells

Next, we studied the involvement of SLAM receptors in humannaive (CD45RA+) and memory (CD45RO+) CD4+ T cells. Thus,we stimulated naive CD4+ T cells with plate-bound anti-CD3 andcostimulatory anti-CD28, anti-SLAMF3, or anti-SLAMF6 Absunder Th17 differentiation conditions for 7 d and subsequentlyanalyzed IL-17 production by flow cytometry and ELISA. In linewith our observations in total CD4+ T cells, Th17-differentiatednaive CD4+ T cells produced significantly more IL-17 in responseto SLAM costimulation when compared with CD28 costimulation(Fig. 3A–C). Interestingly, time kinetics of CD28 and SLAMcostimulation were profoundly different. Whereas IL-17 proteinexpression peaked on day 3 following T cell activation throughCD3/CD28 costimulation and gradually decreased over time untilday 7, it further increased in response to either SLAMF3 orSLAMF6 costimulation throughout the observation period (Fig.3D). To exclude the possibility that IL-17 production is an effectof increased proliferation versus Th17 differentiation in response

FIGURE 4. SLAM costimulation

promotes Th17 differentiation in mem-

ory CD4+ T cells. A–C, Memory

(CD45RO+) CD4+ T cells were stim-

ulated with anti-CD3 and costimu-

latory anti-CD28, anti-SLAMF3,

SLAMF6, and control IgG under

Th17 conditions for 7 d. IL-17 pro-

duction was analyzed by intracellu-

lar staining. A, Secretion of IL-17 by

memory CD4+ T cells on day 7 was

measured by ELISA. B, Percentages

of IL-17–producing cells. The rela-

tive number of IL-17–producing

cells is given as a percentage of

memory CD4+ T cells. C, One re-

presentative experiment is displayed

(from day 7). D, Kinetics of IL-17

secretion by memory CD4+ T cells

in response to costimulation through

SLAMF3, SLAMF6, and CD28.

IL-17 levels were monitored on days

3 and 6 by ELISA.

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to SLAM costimulation, we performed comparative proliferationanalysis of SLAM and CD28 costimulated naive CD4+ T cells.Naive CD4+ T lymphocytes exhibited comparable rates of celldivision in response to CD28 and SLAM costimulation as assessedby CSFE staining, whereas IL-17 production was increased uponSLAM costimulation (Fig. 3E). This suggests that activatedSLAM signaling transfers enhanced differentiation capacities tonaive CD4+ T cells toward a Th17 phenotype under polarizingconditions, rather than solely exerting proliferative effects onIL-17–producing cells.Next, we performed similar studies in memory CD4+ T cells.

As expected, CD28 costimulation yielded higher IL-17 concen-trations when compared with naive CD4+ T cells. CD3/SLAM-mediated costimulation of memory T cells induced levels of IL-17comparable to CD3/CD28 costimulation (Fig. 4A–C); however,time kinetics revealed an increase in IL-17 production by memoryCD4+ T cells from day 3 to 6 in response to SLAM costimulation,whereas CD28 costimulation led to a mild decrease during theobserved time period (Fig. 4D).

SLAM depletion reduces IL-17 production in response to T cellstimulation

To investigate the specificity of SLAM-promoted Th17 differen-tiation, we silenced SLAMF3 and SLAMF6 expression in naiveCD4+ T cells with siRNA. Efficiency of SLAM knockdown wasassessed by surface staining (Supplemental Fig. 3A, 3C). Fourdays after siRNA transfection, cells were stimulated with anti-CD3 and anti-SLAM Abs under Th17 polarizing conditions. Na-ive CD4+ T cells that had been transfected with SLAM siRNAwere costimulated with anti-CD3/anti-SLAM Abs, which resultedin a severely impaired IL-17 production as determined by ELISAanalyses (Supplemental Fig. 3B, 3D).

SLAM-promoted Th17 differentiation in human T cells involvesSAP-dependent pathways

Next, we sought to analyze whether the adaptor protein SAP isinvolved in SLAM-mediated Th17 differentiation. Therefore, weknocked down endogenous SAP protein using siRNA transfectionsin naive CD4+ T cells (Fig. 5A). After SAP depletion, we observeda severely impaired IL-17 production following anti-CD3/anti-SLAM but not following anti-CD3/anti-CD28 stimulation underTh17 polarizing conditions (Fig. 5B, 5C). This suggests that SAPis engaged in SLAM-mediated Th17 differentiation of humannaive CD4+ T cells.

DiscussionIn the present study, we investigated classical/canonical andnoncanonical TCR costimulatory molecules and their contributionto the differentiation of total, naıve, and memory CD4+ T cellstoward a Th17 phenotype. We document that the noncanonicalcostimulatory molecules SLAMF3 and SLAMF6 exhibit morepotent costimulatory capacities in Th17 generation in both controland SLE T cells. This is of special interest, as SLE T cells displayincreased surface expression of SLAM receptor proteins. Thedegree of SLAMF3 and SLAMF6 expression correlated withdisease severity as assessed by individual SLEDAI scores.In the presence of Th17 polarizing cytokines, costimulation

through SLAMF3 or SLAM6 results in increased production ofIL-17 in naive CD4+ T cells and follows different time kineticsthan those elicited by CD28. IL-17 production following CD28costimulation peaks at day 3 and may relate to a normal immuneresponse elicited by the Ag involving the CD3/TCR complex. Incontrast, engagement of SLAMF3 and SLAMF6 after exposure toAg may relate to a prolonged inflammatory response, which may

cause organ damage. In SLE patients the CD3/TCR complex maybe engaged by autoantigens or circulating anti-CD3/TCR auto-antibodies (31).It is well documented that effector memory CD4+ T cells are

principal producers of IL-17 in vivo (32, 33). Our observationsprovide evidence that under Th17 polarizing conditions IL-17 pro-duction by memory CD4+ T cells is significantly increased uponTCR costimulation through SLAMF3 when compared with canon-ical TCR costimulation by CD28. This is of special interest becausepathogenic memory CD4+ T cells are expanded and naive CD4+

T cells are decreased in SLE patients (34–37). This suggests that inaddition to increased Th17 differentiation from naive CD4+ T cells,SLAM signaling contributes to increased IL-17 expression frompathologically expanded memory CD4+ T cells in SLE patients.SLAM molecules associate with intracellular adaptor proteins,

such as SAP family members. SAP proteins contribute to SLAMreceptor activation as they mediate dimerization of SLAM receptors.SAP proteins have been reported to promote Th1 and Th2 differ-entiation (38–42). Our observation that SAP blockade with SAPsiRNA results in significantly reduced IL-17 expression from T cellssuggests a key role for SAP as a common proximal signal in SLAM-promoted IL-17 production. As for the aforementioned SLAM

FIGURE 5. SAP knockdown abrogates IL-17 production. Naive CD4+

T lymphocytes were transfected with SAP siRNA or control siRNA as in-

dicated. Efficacy of siRNA transfection was determined by SAP protein

levels using Western blot analysis (A). Three days after transfection, cells

were costimulated with anti-SLAMF3 (B) or anti-SLAMF6 Abs (C) in

addition to anti-CD3 under Th17 differentiation conditions for another 4 d.

Levels of secreted IL-17 were measured by ELISA.

1210 SLAM COSTIMULATION PROMOTES Th17 DIFFERENTIATION

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receptors, SAP proteins may be promising targets for pharmaco-logical blockade. Unlike further downstream molecules, such asMAPK, SAP proteins are limited to SLAM-associated pathways,which contribute to (pathological) Th17 generation and IL-17 ex-pression in SLE and presumably other autoimmune diseases (43).However, farther downstream molecules through which SLAMmolecules promote Th17 differentiation remain to be elucidated,and future studies are warranted to unravel mechanisms how SLAMreceptors promote and sustain an increased IL-17 production.Our observations that TCR costimulation with SLAMF3 and

SLAMF6 promotes Th17 differentiation, and that SLAMF3 andSLAMF6 surface expression on SLE T cells is significantly in-creased when compared with healthy controls and reflects diseaseactivity, may also link the genomic location of SLAM family geneswithin the lupus susceptibility locus Sle1b to reported SLAMpolymorphisms in lupus-prone mice and SLE patients (13, 16, 18,20). Previous studies indicated that SLAM polymorphisms con-tribute to SLE pathogenesis through increased gene expression(16). Polymorphisms in the Ly108 (SLAMF6) gene result inoverexpression of an Ly108 splice variant in lupus-prone mice.This is in agreement with our previous report that SLAMF6 co-stimulation of human SLE T cells results in reduced secretion ofIL-2 and Th1 cytokines resembling a SLE phenotype (14).To our knowledge, this is the first report to demonstrate that

the noncanonical costimulatory molecules SLAMF3 and SLAMF6promote Th17 differentiation. Both of these molecules are morepotent inducers of Th17 differentiation when compared with thecanonical costimulatory molecule CD28. Furthermore, SLAMF3and SLAMF6 surface expression is increased on SLE T cells ina disease activity-dependent manner. The superior capacities ofSLAM molecules in Th17 generation may be attributed to yetunidentified downstream signal transducers. We think that ourstudies hold the promise to create a new platform to study Th17cells from a different perspective, and the SLAM family of cos-timulators could be chosen as therapeutic targets to control SLEand autoimmune disorders. Future studies are needed to 1) in-vestigate SLAM receptor expression in different autoimmunediseases, 2) dissect SLAM propensities in Th cell differentiationunder polarizing and nonpolarizing conditions, and 3) analyzethe involvement of downstream factors that account for SLAM-promoted IL-17 gene expression.The significance of our findings is manifold. The strong cor-

relation between SLE disease activity and SLAMF3 and SLAM6expression on the surface of SLE T cells suggests that their ex-pression levels may gain biomarker value. The fact that SLAMengagement leads to increased and prolonged production of IL-17strongly suggests that blockade of their engagement either withblocking Abs or decoy receptors may suppress the inflammatoryresponse without affecting IL-17 production through the canonicalengagement of CD28.

DisclosuresThe authors have no financial conflicts of interest.

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