of March 25, 2018.This information is current as

Autoimmune Encephalomyelitisand Progression of Experimental

Producing T Cells−Differentiation of IL-17 SAP Signaling Promotes−SLAM

Vallance, John J. Priatel and Rusung TanYu-Hsuan Huang, Kevin Tsai, Caixia Ma, Bruce A.

http://www.jimmunol.org/content/193/12/5841doi: 10.4049/jimmunol.1301435October 2014;

2014; 193:5841-5853; Prepublished online 31J Immunol 

MaterialSupplementary

5.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/10/31/jimmunol.130143

Referenceshttp://www.jimmunol.org/content/193/12/5841.full#ref-list-1

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The Journal of Immunology

SLAM–SAP Signaling Promotes Differentiation ofIL-17–Producing T Cells and Progression ofExperimental Autoimmune Encephalomyelitis

Yu-Hsuan Huang,*,† Kevin Tsai,*,† Caixia Ma,*,‡ Bruce A. Vallance,*,‡ John J. Priatel,*,†,1

and Rusung Tan*,†,x,1

IL-17 plays critical roles in host defenses, combating bacterial and fungal infections, as well as the pathogenesis of autoimmune

diseases such as experimental autoimmune encephalomyelitis (EAE). The signaling adaptor SAP is essential for normal immune

homeostasis and mutations within SH2D1A, the locus encoding this protein, result in serious and sometimes fatal syndromes,

including X-linked lymphoproliferative disease and severe cases of common variable immunodeficiency. However, the precise

cellular basis of how SAP deficiency contributes to immune dysfunction remains incompletely understood. In this study, we found

that CD4 and CD8 T cells lacking SAP had a diminished capacity to differentiate into IL-17–producing Th17 and T cytotoxic (Tc17)

cells relative to wild-type lymphocytes. The use of costimulating SLAM Abs was found to augment the differentiation of IL-17–

secreting effectors in wild-type but not Sh2d1a2/2 splenic T cells under IL-17–polarizing conditions. In addition, SAP’s regulation of

IL-17–secreting T cells was shown to be a T cell–intrinsic role, as purified naive Sh2d1a2/2 CD4 and CD8 T cells were inherently

defective at converting into Th17 and Tc17 cells in vitro and in vivo. Furthermore, Sh2d1a2/2 mice were protected from EAE and

exhibited greatly decreased numbers of CNS-infiltrating Th17 and Tc17 effector T cells and reduced disease severity. Collectively,

these results suggest that SLAM–SAP signaling drives the differentiation and function of Th17 and Tc17 cells in vitro and in vivo and

contributes to the pathogenesis of autoimmunity in EAE. The Journal of Immunology, 2014, 193: 5841–5853.

Pathogen clearance and immunity require generating theappropriate adaptive immune responses for a given type ofinfectious challenge. This immune specificity is mediated

in part through the actions of the innate immune system that rec-ognizes the invading pathogen and tailors specific immune responsesthrough instruction of naive CD4 and CD8 T cells to differentiateinto various types of effector Th or T cytotoxic (Tc) cell subsets,respectively. The effector Th lineages were initially defined as Th1or Th2 on the bases of cytokines produced, IFN-g or IL-4,

respectively, and the types of responses elicited, promoting

clearance of intracellular pathogens or augmenting Ab-mediated

humoral responses and parasite removal. However, it is now clear

that a much greater diversity of effector T cell lineages exists and

that their differentiation is regulated through a complex integra-

tion of signals received from the TCR, costimulatory molecules,

and cytokine receptors (1).Th17 and Tc17 effector lineages have recently been described

that possess unique developmental requirements and immuno-

logical functions (2). The differentiation of IL-17–secreting T cells

is dependent on specific cytokines (TGF-b and IL-6 in mice and

TGF-b, IL-1b, and IL-6, IL-21, or IL-23 in humans) and STAT3

activation to induce their master transcription factor retinoic acid–

related orphan receptor-gt (RORgt) (3–6). Besides their signature

cytokine IL-17, Th17 and Tc17 cells also produce IL-21, IL-22,

and TNF-a and have important immune roles in the regulation of

microbial defense and leukocyte recruitment (7–11). However,

Th17 and Tc17 effector cells have also been suggested to be

pathogenic given their associations with sites of tissue inflammation

and autoimmunity in psoriasis, rheumatoid arthritis, inflammatory

bowel disease, systemic lupus erythematosus (SLE), multiple

sclerosis (MS), and type 1 diabetes (9, 10, 12–21). Murine studies,

using neutralizing IL-17 Ab (22) or IL-17a2/2 mice (23), strongly

imply that IL-17 has a causative role in the T cell–dependent MS

model experimental autoimmune encephalomyelitis (EAE). The

cytokine IL-23 has been shown to be critical for promoting the

stabilization and pathogenicity of Th17 and Tc17 effector cells in

models of EAE and autoimmune diabetes although dispensable for

the generation of IL-17–secreting T cell effectors (9, 24).Cell-surface signaling lymphocytic activation molecule (SLAM)

family receptors are drivers of immune cell activation and dif-

ferentiation, acting in concert with the intracellular adaptor SLAM-

associated protein (SAP) to mediate certain critical immune functions

*Child and Family Research Institute, BC Children’s Hospital, Vancouver, BritishColumbia V5Z 4H4, Canada; †Department of Pathology and Laboratory Medicine,University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada;‡Division of Gastroenterology, Department of Pediatrics, University of BritishColumbia, Vancouver, British Columbia V5Z 4H4, Canada; and xDepartment ofPathology, Sidra Medical and Research Center, Doha, Qatar

1J.J.P. and R.T. are cosenior authors.

Received for publication May 31, 2013. Accepted for publication October 6, 2014.

This work was supported by grants from the Canadian Institutes of Health Research.Y.-H.H. is a recipient of a Multiple Sclerosis Society of Canada studentship. B.A.V. isthe Children with Intestinal and Liver Disorders Foundation Research Chair inPediatric Gastroenterology. R.T. is a scholar of the Michael Smith Foundation forHealth Research.

Address correspondence and reprint requests to Dr. John J. Priatel or Dr. Rusung Tan,Child and Family Research Institute, BC Children’s Hospital, Room A4-148, Trans-lational Research Building, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada(J.J.P.) or Department of Pathology, Sidra Medical Center, PO Box 26999, Doha,Qatar (R.T.). E-mail addresses: jpriatel@cfri.ubc.ca (J.J.P.) or rtan@sidra.org (R.T.)

The online version of this article contains supplemental material.

Abbreviations used in this article: DAG, diacylglycerol; EAE, experimental autoim-mune encephalomyelitis; aGalCer, a-galactosylceramide; HCV, hepatitis C virus;iTreg, induced Treg; MFI, mean fluorescence intensity; MLN, mesenteric lymphnode; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; RORgt,retinoic acid–related orphan receptor gt; SAP, signaling lymphocyte activation mol-ecule–associated protein; SLAM, signaling lymphocyte activation molecule; SLE,systemic lupus erythematosus; Tc, T cytotoxic; Treg, regulatory T cell; WT, wild-type; XLP, X-linked lymphoproliferative disease.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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(25–27). Loss of SLAM–SAP signaling pathways due to mutationswithin the locus encoding SAP (called SH2D1A) result in X-linkedlymphoproliferative disease (XLP), a condition noted for its ex-quisite susceptibility to EBV but not other pathogens (25, 26).SAP contains a single SH2 domain that interacts with theimmunoreceptor tyrosine-based switch motifs (TxYxxI/V, “x”representing any amino acid) located in the cytoplasmic regions ofSLAM family receptors such as SLAM (CD150), CD48, CD84,Ly9 (CD229), NK–T–B Ag (in humans; Ly108 in mouse), and2B4 (CD244). SLAM family receptors, principally expressed byhematopoietic cells, have Ig-like extracellular domains involvedin homotypic (e.g., SLAM–SLAM) or heterotypic interactions(e.g., 2B4–CD48). Engagement of SLAM family receptors byself family ligands results in tyrosine phosphorylation of theirimmunoreceptor tyrosine-based switch motifs and the subsequentrecruitment of SAP is thought to regulate signaling by two distinctmechanisms: 1) SAP transduction of signaling by binding thetyrosine kinase Fyn and protein kinase Cu (25, 26, 28, 29); and 2)SAP inhibition of signaling by sterically hindering the docking ofSH2 domain-containing phosphatases 1 and 2 and SHIP-1 (25, 26,30–33). Thus, the absence of SAP may negate positive signalingand reinforce inhibitory signaling by SLAM family receptors.SAP is expressed by most lymphocytes, including NK cells,

NKT cells, CD4 T cells, and CD8 T cells, but absent from B cells(34, 35) and plays key roles in lymphocyte development, differ-entiation, and function (25). Studies of XLP patients and SAP-deficient (Sh2d1a2/2) mice reveal a matching range of immunecell deficits including severely impaired B cell differentiationresulting in diminished Ig levels, defective isotype switching, andthe lack of germinal centers and memory B cells (25). DefectiveB cell differentiation and humoral responses in SAP-deficientmice are attributed to a fault in CD4 T cell differentiation ratherthan a B cell–intrinsic defect (35, 36). More recent work indicatesthat SAP within CD4 T cells is important for sustained CD4T–B cell contact, necessary for the generation of T follicular helpercells and the formation of germinal centers (37, 38). Besides itseffects on CD4 T cells, SAP has also been found to be essential forthe development of NKT cells in both mice and humans (39–41)and for the effector functions of NK cells and CD8 T cells (32, 33).In this report, we have studied the effect of SAP on the dif-

ferentiation of Th17 and Tc17 differentiation using wild-type (WT)and Sh2d1a2/2 mice. We show that CD4 and CD8 T cells fromSAP-deficient mice have an impaired ability to differentiate intoTh17 and Tc17 cells relative to other Th and Tc subsets. In ad-dition, costimulation of T cells with a SLAM (CD150)-specific Abenhances IL-17 secretion from WT but not SAP-deficient splenicT cells. Additionally, in vitro and in vivo studies using purifiedWT and Sh2d1a2/2 naive T cells demonstrated a T cell–intrinsicrole for SAP’s control of IL-17 T cell differentiation. Finally,Sh2d1a2/2 mice were protected from EAE relative to WT mice,and the protected mice had reduced numbers of Th17 and Tc17effectors in both the spleen and infiltrates of the CNS. Collectively,these findings suggest that the SLAM/SAP signaling pathwaypositively regulates the differentiation of Th17 and Tc17 effectorT cells in vitro and can promote autoimmune pathogenicity.

Materials and MethodsMice

Sh2d1a2/2 mice have been previously described (42) and backcrossedonto C57BL/6J background at least 10 generations. WT (C57BL/6J) andSh2d1a2/2 mice were bred, intercrossed, and housed in a specificpathogen-free and Helicobacter-free facility at the Child and FamilyResearch Institute. B6.PL-Thy1a/Cy (Thy 1.1+) and B6.SJL-Ptprca

Pep3b/BoyJ (CD45.1+) mice were purchased from The Jackson Laboratory.

All experimentation followed protocols approved by the Animal CareCommittee at University of British Columbia in conjunction with theCanadian Council on Animal Care.

Flow cytometry

Spleens were mashed through nylon screens and the subsequent cell sus-pensions treated with RBC lysis buffer containing ammonium chloride. Absrecognizing CD3ε (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD44 (IM7),B220 (RA3-6B2), CD49b (DX5), IFN-g (XMG1.2), IL-4 (11B11), FOXP3(FJK-16s), and IL-17A (eBio17B7) were purchased from eBioscience. Fordetection of NKT cells, a-galactosylceramide (aGalCer)–loaded mouseCD1d tetramers were acquired from the National Institutes of HealthTetramer Core Facility (Emory University). For intracellular cytokinestaining, samples were stimulated for 4 h with 50 ng/ml PMA and 1 mg/mlionomycin (Sigma-Aldrich) in the presence of 3 mg/ml brefeldin A(eBioscience) to block cytokine secretion. Subsequently, cells were stainedwith Abs specific for surface markers, fixed with 2% paraformaldehyde/PBS solution for 15 min, treated with eBioscience permeabilization buffer,and stained with Abs specific for intracellular Ags. SAP (clone 12C4) Aband detection of SAP by intracellular flow cytometry have been describedpreviously (35, 43). Sample acquisition was performed using a BD LSR IIbenchtop cytometer and FACSDiva software (BD Biosciences). Sampledata were analyzed with FlowJo software version 8.8.6 (Tree Star).

In vitro Th1, Th2, and induced regulatory T cell differentiationassays

RBC-depleted single-cell suspensions from spleens of 6–12-wk-old WTand Sh2d1a2/2 mice (0.5 3 106/well) were cultured in IMDM supple-mented with 10% FBS, 13 nonessential amino acids, 100 U/ml penicillin/streptomycin, and 55mM 2-ME (all reagents from Invitrogen). Splenocyteswere stimulated for a 96-h period with 1 mg/ml plate-bound CD3 (145-2C11) Ab and 10 mg/ml soluble CD28 (37.51) Ab in the presence or ab-sence of polarizing cytokines and anti-cytokine Abs. For Th1- and Tc1-polarizing conditions, cells were treated with IL-2 (100 U/ml), IL-12(10 ng/ml), and anti–IL-4 Ab (1 mg/ml). To facilitate Th2 differentiation,cells were treated with IL-2 (100 U/ml), IL-4 (10 ng/ml), and anti–IFN-gAb (1 mg/ml). For induction of regulatory T cells (Tregs), splenocytes weretreated with the cytokines IL-2 (100 U/ml) and TGF-b (10 ng/ml). All Absand cytokines were purchased from eBioscience except for TGF-b (BDBiosciences).

In vitro Th17/Tc17 cell differentiation assays

For the generation of Th17 and Tc17 lineages, WT and Sh2d1a2 /2

splenocytes (0.5 3 106 per well in 96-well flat-bottom plates) werestimulated with 1 mg/ml plate-bound CD3 (145-2C11) Ab, unless spec-ified, in the presence of IL-6 (10 ng/ml), TGF-b (0.5 ng/ml), and IL-23(20 ng/ml). Costimulation was provided to samples by adding 10 mg/mlsoluble CD28 (37.51) or SLAM family receptor Abs. SLAM family recep-tor Abs were purchased from eBioscience (CD150, 9D1; 2B4, eBio244F4;and CD48, HM48-1) or BioLegend (CD84, mCD84.7; Ly9, Ly9ab3; andLy108, 330-AJ). Neutralizing anti-cytokine Abs were not added to IL-17–polarizing experimental conditions unless otherwise indicated. In experi-ments shown in Fig. 5A, IL-17–polarizing conditions were performed inthe presence of 10 mg/ml anti–IFN-g (XMG1.2), 10 mg/ml anti–IL-4(11B11), and 1 mg/ml IL-2 (JES6-1A12) Abs. For IL-17 differentiationof naive CD4 and CD8 T cells, splenocytes from WT and Sh2d1a2/2 micewere first labeled with Abs specific to CD44 (IM7), CD25 (PC61), CD4(GK1.5), and CD8 (53-6.7), and naive (CD252CD44lo) CD4 and CD8T cells were sorted using a BD FACSAria flow cytometer (BD Bio-sciences). Subsequently, sorted naive CD4 or CD8 T cells (1 3 105/well in96-well flat-bottom plates) were activated with the indicated amount ofplate-bound CD3 (145-2C11) Ab under IL-17–polarizing conditions (IL-6,TGF-b, and IL-23 as above but no neutralizing anti-cytokine Abs) for 4 dprior to a 4-h PMA/ionomycin stimulation for the assessment of cytokineproduction.

Naive CD4 T cell adoptive transfers, bacterial infections, anddonor CD4 T cell analyses

Naive CD4 T cells were enriched from spleens and lymph nodes (brachial,inguinal, and mesenteric) of WT (Thy1.1+) and Sh2d1a2/2 (Thy1.2+) mice.Briefly, single-cell suspensions were incubated with Abs specific for CD8(53-6.7), CD25 (3C7), CD44 (KM114), CD19 (eBio1D3), and NK1.1(PK136) and Ab-bound cells removed using anti-mouse Ig/anti-rat Igcoupled Dynabeads (Invitrogen). Residual samples were labeled with Absspecific for CD44 (IM7), CD25 (PC61), CD4 (GK1.5), and TCRb (H57-597) to assess naive CD4 T cell purity and cell mixtures, containing 63 106

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CD4 T cells of each genotype, subsequently generated for their infusioninto recipient (CD45.1+) animals. The next day, mice were administered100 ml WT Citrobacter rodentium biotype 4280 strain DBS100 culture(∼2.53 108 CFU; grown overnight in Luria broth at 37˚C) by oral gavage.Infected mice were monitored daily, and no overt signs of distress wereobserved. At day 10 postinfection, lymphocytes, recovered from spleens,mesenteric lymph nodes, and colons as described (44), were restimulatedovernight with PMA plus ionomycin and subsequently surface stained withAbs recognizing CD4, CD45.1 (A20), CD45.2 (104), Thy1.1 (HIS51),Thy1.2 (53-2.1), and CD3 (145-2C11) prior to intracellular detection ofIFN-g and IL-17A.

EAE induction and CNS-infiltrating T cell isolation

To induce EAE disease, 6–8-wk-old age- and sex-matched WT andSh2d1a2/2 mice, bred within the Child and Family Research Instituteanimal facility, were injected s.c. with 200 mg myelin oligodendrocyteglycoprotein peptide (MOG35–55) emulsified in a 100-ml volume of a50:50 mixture of CFA (Sigma-Aldrich) and PBS. Also, 200 ng pertussistoxin (Cedarlane Laboratories) was administered i.p. at days 0 and 2 post–MOG immunization. For assessment of cytokine secretion by infiltratingT cells, mononuclear cells were isolated from the CNS when WT micereached a mean clinical score of 2. In brief, brains and spinal cords weredissected from mice perfused with PBS and mashed through nylon screens.Subsequently, cell mixtures were suspended in 30% Percoll/PBS solution(GE Healthcare), layered on the top of 70% Percoll/PBS, and centrifugedat 2000 rpm for 20 min. After collection of mononuclear cell fraction fromthe Percoll gradient, cells were stimulated for 4 h with PMA plus ion-omycin in the presence of brefeldin A, labeled with surface marker Abs,fixed, permeabilized, and stained intracellularly with Abs specific forIFN-g, IL-17, and FOXP3 as described above.

Statistical analyses

Statistical significance was determined by performing unpaired, two-tailedt tests. For EAE experiments, nonparametric Mann–Whitney U and Fisherexact tests were used to calculate significant differences between mousecohorts in disease severity and incidence. All statistical analyses wereperformed using GraphPad Prism4 software (GraphPad).

ResultsSAP is strongly expressed by Th17 cells

SAP protein is expressed in NK, NKT, and T cells of both humansand mice (34, 35). To characterize SAP expression withinother lymphocyte lineages, in particular, its distribution withinnaive, effector, and memory T cell lineages, we used an anti-SAPmAb, previously shown to detect cells transfected with SAP butnot the closely related adaptor molecule EAT-2 (35), and per-formed intracellular flow cytometry employing Sh2d1a2/2 miceas a control to establish background and autofluorescence staining(Fig. 1). Virtually all naive and memory CD4 and CD8 T cells,defined as CD44lo and CD44hi, respectively, exhibit detectableSAP levels with memory-phenotype T cells showing an appre-ciable increase relative to naive T cells (Fig. 1A, 1B; CD44hi

CD4+, 1.5-fold, mean fluorescence intensity [MFI] 4210 6 127versus 28856 144, p, 0.01; CD44hiCD8+, 2.2-fold, MFI 58126202 versus 2699 6 167, p , 0.001). By contrast, the splenic Treg(CD4+FOXP3+) subset displayed a greater variation in SAP ex-pression, with almost one-quarter of the population having low orundetectable SAP levels. In agreement with a previous study (35),NK (DX5+CD32) cells and especially NKT (aGalCer/CD1-tet-ramer+CD3+) cells were found to strongly express SAP, whereaswe were unable to discern any SAP expression within splenic B(B220+CD32) cells (Fig. 1A, 1B).Previous results implicating a role for SAP in Th differentiation

(25) led us to investigate SAP expression in effector T cell line-ages. Splenic single-cell suspensions were cultured in vitro underpolarizing conditions (see Materials and Methods) to induce Th1,Th2, Th17, or induced Tregs (iTregs), and SAP protein levels werequantified in CD4 and CD8 T cells that expressed IFN-g, IL-4, IL-17A, or FOXP3 (Fig. 1C, 1D). Notably, Th17 (IL-17+FOXP32)

cells were found to contain significantly higher levels of SAP ascompared with both Th1 (IFN-g+IL-17A2) and Th2 (IL-4+IL-17A2) cells (Th17/Th1, 2.0-fold, MFI 5030 6 102 versus 2470 6222, p, 0.01; Th17/Th2, 3.0-fold, MFI 50306 102 versus 16726436, p , 0.05). By contrast, SAP levels were similar between Tc1(IFN-g+IL-17A2) and Tc17 (IL-17+IL-42) effector cells. Theseresults demonstrate that SAP is expressed in most T cells and atvarious stages of T cell differentiation and that the Th17 celllineage, in particular, maintains high levels of SAP.

SAP positively regulates the differentiation of IL-17–producingCD4 and CD8 T cells

Given the high levels of SAP expression in Th17 cells, we hy-pothesized that SAP is especially critical for the formation of Th17cells. To address whether SAP plays a selective role in the dif-ferentiation of Th17 cells, WT and Sh2d1a2/2 splenocytes werecultured with anti-CD3 plus anti-CD28 Abs under Th1-, Th2-,Th17-, or iTreg-polarizing conditions for 4 d in vitro and subse-quently cytokine production assessed by intracellular cytokinestaining (Fig. 2A, 2B). Strikingly, Sh2d1a2 /2 CD4 T cells ex-hibited reduced potential to differentiate into Th17 effectors rel-ative to WT (Th17: 1.7-fold decrease; 12.5 6 0.6 versus 21.7 61.2%, p , 0.01). By contrast, lack of SAP did not affect thegeneration of Th1 cells (IFN-g+IL-172), Th2 cells (IL-4+IL-172),or iTregs (FOXP3+IL-172) under respective Th1-, Th2-, andiTreg-polarizing conditions, respectively. These observations areconsistent with previous findings documenting that Sh2d1a2/2

CD4 T cells have a similar capacity to WT cells to differentiateinto Th1 and Th2 cells under Th1- and Th2-polarizing conditionsin vitro (42, 45). In addition, fewer IL-17–secreting CD8 T cells,either IFN-g2IL-17+ (Tc17) or IFN-g+IL-17+ (Tc1/Tc17) subsets,were observed among Sh2d1a2/2 relative to WT splenocytesculturing under IL-17–polarizing conditions (Tc17: 3.2-fold de-crease; 2.7 6 1.3 versus 8.7 6 1.1%, p , 0.05; Tc1/Tc17: 2-folddecrease; 4.06 0.2 versus 7.96 1.4%, p, 0.05; Fig. 2C, 2D). Bycontrast, the frequencies of Tc1 cells (IFN-g+IL-172) were re-markably equivalent when WT and Sh2d1a2/2 splenocytes wereincubated under Th1-polarizing conditions. Together, these find-ings suggest that SAP plays a selective role in positively regu-lating the differentiation of IL-17–secreting CD4 and CD8 T celleffectors.

SLAM–SAP signaling positively modulates the differentiationof Th17 and Tc17 cells

SAP regulates lymphocyte differentiation and function by trans-mitting signals from surface SLAM family receptors. Conse-quently, we hypothesized that the addition of costimulating SLAMfamily receptor Abs could drive the differentiation of Th17 andTc17 lineages. Previous reports have indicated that the SLAM familyreceptors SLAM, Ly108, Ly9, CD48, and CD84 are expressed on theT cell surface (25) and that SLAM ligation can regulate T cellactivation (46). To investigate the potential of SLAM familyreceptors to promote the differentiation of IL-17–secreting Teffectors, WT and Sh2d1a2/2 splenocytes were incubated underIL-17–polarizing conditions with anti-CD3 Ab alone (TCR), anti-CD3 plus SLAM family receptor Abs, or anti-CD3 plus CD28(TCR/CD28) Abs for comparison’s sake (Fig. 3). After 4 d, cultureswere restimulated with PMA/ionomycin for 4 h prior to labelingwith surface markers, fixation, and intracellular cytokine staining. Asimilar fraction of WT and Sh2d1a2/2 CD4 T cells were found toexpress IL-17 when polarized with anti-CD3 Abs alone (Fig. 3A,3B). By contrast, treatment with SLAM Ab (CD150; clone 9D1),an activating Ab shown to costimulate TCR-induced proliferationin a SAP-independent fashion (27), plus anti-CD3 Abs increased

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the proportion of Th17 cells among WT but not Sh2d1a2/2 CD4T cells (WT, 1.6-fold; 20.3 6 1.2 versus 12.9 6 0.5%, p , 0.01).However, Abs directed against Ly108 (330-AJ), Ly9 (Ly9ab3),CD48 (HM48-1), CD84 (mCD84.7), or 2B4 (eBio244F4) wereunable to synergize with anti-CD3 Ab to boost the frequency ofIL-17–secreting T cells regardless of T cell genotype (data notshown). Interestingly, anti-CD28 Ab costimulation also was foundto increase the proportion of WT but not Sh2d1a2/2 CD4 T cellssecreting IL-17 (WT, 1.7-fold; 21.7 6 1.2 versus 12.9 6 0.5%,

p , 0.001). Regardless of genotype or the presence or absence ofCD28 or SLAM costimulation, very few FOXP3-positive CD4T cells were detected under tested culture conditions (data notshown). The failure of other SLAM family receptor Abs to supportthe generation of IL-17 effector T cells could be a consequence ofthe tested Abs lacking sufficient stimulatory activity under testconditions or nonredundant functioning of SLAM family recep-tors. Thus, these findings suggest that SLAM (CD150)–SAP sig-naling can augment the differentiation of Th17 cells.

FIGURE 1. SAP is strongly expressed by Th17 cells. (A) Splenocytes fromWT (C57BL/6J) mice were labeled with Abs specific for the indicated surface

markers directly ex vivo, fixed, made permeable, and stained intracellularly with SAP (clone 12C4) Ab. To account for autofluorescence and nonspecific

SAPAb labeling, splenocytes from Sh2d1a2/2 mice were used as a negative control, being surface stained, fixed, permeabilized, and treated with SAPAb

in an identical fashion (gray-shaded histograms). (B) SAP expression is presented in bar graph format as background-subtracted MFI (WT SAP MFI 2Sh2d1a2/2 SAP MFI), and regulatory (CD4+ FOXP3+, thymus-derived Treg [tTreg]), naive (CD44lo, nCD4, or nCD8), and memory (CD44hi, mCD4 or

mCD8) T cells, NK (CD49b+CD32), NKT (aGalCer/CD1d Tet+CD3+), and B cells (B220+CD32) were electronically gated as shown. (C) Splenocytes,

treated with the indicated polarizing conditions to generated Th and Tc lineages (seeMaterials and Methods), were stimulated with PMA/ionomycin for 4 h

to facilitate detection of intracellular cytokine. SAP levels in various Th and Tc cells, distinguished through cytokine expression (shown in contour plots),

are presented by open histograms. Gray-shaded histograms (Sh2d1a2/2 Th and Tc cells) represent background fluorescence. (D) Cumulative data of SAP

expression in Th and Tc are shown as bar graphs with MFI calculated as in (B). Data reflect results from three independent experiments. Statistical

significance was calculated using unpaired, two-tailed t tests. Error bars denote the SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

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By selective gating of CD8+ T cells, we observed that Sh2d1a2/2

CD8 T cells stimulated under IL-17–polarizing conditions withanti-CD3 Ab alone exhibited a reduced propensity to differentiateinto IL-17–secreting effectors, Tc17 or Tc1/Tc17 phenotype, ascompared with WT CD8 T cells (Fig. 3C, 3D; Tc17, 2.6-fold; 1.860.2 versus 4.6 6 0.2%, p , 0.01; Tc1/Tc17, 1.9-fold; 3.5 6 0.3%versus 6.56 0.9%, p, 0.05). In addition, activation with anti-CD3plus anti-SLAM Abs was found to increase the proportion of WTbut not Sh2d1a2/2 CD8 T cells that express IL-17, both Tc17 andTc1/Tc17 subsets (WT; Tc17, 1.7-fold; 7.8 6 0.8 versus 4.6 60.2%, p , 0.05; Tc1/Tc17, 2-fold; 12.9 6 2.4 versus 6.5 6 0.9%).Again, stimulation with anti-CD3 plus anti-CD28 Abs was found toboost IL-17 secretion by WT but not Sh2d1a2/2 CD8 T cells (WT;Tc17, 1.9-fold; 8.7 6 1.1 versus 4.6 6 0.2%, p , 0.05; Tc1/Tc17,1.2-fold; 7.9 6 1.4 versus 6.5 6 0.9%). These results indicate thatSAP–SLAM (CD150) signaling can also support the differentiationof IL-17–secreting CD8 T cell effectors.

SAP–SLAM controls the differentiation of naive CD4 and CD8T cells into Th17 and Tc17 effectors

Next, we sought to determine whether SLAM–SAP signaling di-rectly influences the differentiation of naive CD4 and CD8 T cells

into Th17 and Tc17 cells. Sorted naive (CD252CD44lo) WT andSh2d1a2/2 CD4 and CD8 T cells were cultured with various con-centrations of anti-CD3 Ab alone or anti-CD3 plus costimulatingSLAM Abs under IL-17–polarizing conditions for 4 d (Fig. 4A,4B). SLAM Abs were found to drive the conversion of WT but notSh2d1a2/2 naive CD4 and CD8 T cells into Th17 and Tc17effectors at a low TCR Ab (1 mg/ml) concentration (Th17: 3.8-fold,15.46 1.8 versus 4.16 0.1%, p, 0.01; Tc17: 1.8-fold, 13.96 0.2versus 7.86 0.3%, p, 0.001). In addition, colligation of SLAM atintermediate (3 mg/ml) TCR Ab concentrations also fostered WTnaive CD4 and CD8 T cells to become IL-17–secreting effectors(Th17: 2.7-fold, 16.6 6 1.1 versus 6.2 6 0.2%, p , 0.001; Tc17:1.3-fold, 15.8 6 0.4 versus 11.9 6 0.6%, p , 0.001), whereas thiseffect was not apparent at higher (10 mg/ml) TCR Ab concen-trations. Collectively, these findings suggest that SLAM–SAPinteractions are effective at promoting naive CD4 and CD8 T cellsinto Th17 and Tc17 cells under weaker TCR signaling conditions.

SAP–SLAM signaling promotes Th17 cell differentiationthrough an IFN-g– and IL-4–independent mechanism

Given the critical role that cytokines play in the programming of Thsubsets (1), we next investigated whether SAP’s regulation of Th17

FIGURE 2. SAP positively regulates the differentiation of IL-17–producing CD4 and CD8 T cells. Splenocytes from WT and Sh2d1a2/2 mice were treated

with plate-bound anti-CD3 and soluble anti-CD28 Ab under various polarizing Th/Tc conditions for 4 d (seeMaterials and Methods). Subsequently, cells were

stimulated for 4 h with PMA plus ionomycin prior to being labeled for surface markers and stained for intracellular Abs. (A) Contour plots, generated by

electronically gating on CD4+ T cells, present relative amounts of cytokine and FOXP3 expression. (B) Proportions of the indicated Th lineage among total

CD4+ T cells are shown after 4 d of in vitro polarization. (C) Contour plots indicate relative amounts of cytokine production by CD8+ T cells after 4 d of in vitro

polarization. (D) Proportions of the indicated Tc lineage among total CD8+ T cells are shown after 4 d of in vitro polarization. Data represent findings from three

independent experiments. Statistical significance was calculated using unpaired, two-tailed t tests. Error bars denote the SEM. *p , 0.05, **p , 0.01.

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differentiation is mediated through expression of the IFN-g and IL-4.WT and Sh2d1a2/2 splenocytes were cultured with two differentconcentrations of plate-bound anti-CD3 Ab (TCR, 0.3 or 1 mg/ml),with and without costimulating SLAM and CD28 Abs and in thepresence (Fig. 5A) or absence of neutralizing anti–IFN-g and anti–IL-4 Abs (Fig. 5B). As would be expected, the inclusion of neu-tralizing anti-cytokine Abs along with CD3/CD28 Abs resulted ina higher frequency of WT CD4 T cells differentiating into Th17cells (0.3 mg/ml: 3.4-fold change, 10.8 6 0.8 versus 3.2 6 0.3%,p , 0.001; 1.0 mg/ml: 1.8-fold change, 37.1 6 0.4 versus 20.9 60.9%, p , 0.001). Moreover, CD3/CD28-stimulated WT CD4T cells adopted Th17 fate at an increased frequency relative toSh2d1a2/2 CD4 T cells under these same conditions (0.3 mg/ml:1.6-fold change, 10.86 0.8 versus 6.96 0.9%, p, 0.05; 1.0 mg/ml:1.3-fold change, 37.1 6 0.4 versus 28.8 6 1.4%, p , 0.01). Theuse of SLAM Ab enhanced the generation of Th17 cells from WTCD4 T cells at a higher (1 mg/ml) concentration of plate-boundCD3 Ab but did not affect Sh2d1a2/2 CD4 T cells (2.1-fold change,38.5 6 3.0 versus 18.3 6 1.3%, p , 0.01). Furthermore, the ad-dition of SLAM Ab in tandem with CD28 Ab was found to actsynergistically to promote Th17 differentiation of WT CD4 T cells

at the lower concentration (0.3 mg/ml) of CD3 Ab (CD28/SLAMversus CD28 alone; 1.6-fold change, 17.56 1.6 versus 10.86 0.8%,p , 0.05). Finally, when higher concentrations (3.0 and 10 mg/ml)of plate-bound CD3 Ab was applied, the impact of costimulatoryCD28 and SLAM Abs was modest or even absent (SupplementalFig. 1). Collectively, these experiments suggest that SLAM–SAPboosts Th17 differentiation under weaker TCR signaling condi-tions and that this regulation is independent of the cytokines IFN-gand IL-4.

CD4 T cell–intrinsic SAP function is required for normal Th17cell differentiation in vivo

Previous studies have established that orogastric infection of micewith the natural rodent pathogen C. rodentium directs strong Th17responses that are dependent on both IL-23 and innate immunerecognition of apoptotic host cells (47–49). To establish whetherSAP plays a T cell–intrinsic role in the differentiation of Th17 cellsin vivo, naive (CD252CD44lo) CD4 T cells were purified from WT(Thy1.1+CD45.2+) and Sh2d1a2/2 (Thy1.2+CD45.2+) mice, mixedat a 1:1 ratio prior to i.v. infusing into lymphoreplete (nonirradiated;CD45.1+) recipient hosts and either left untreated or infected with

FIGURE 3. SLAM–SAP signaling positively modulates the differentiation of Th17 and Tc17 cells. Splenocytes from WT and Sh2d1a2/2 mice were

treated with CD3 Ab alone (TCR), CD3 plus SLAM Abs (TCR/SLAM), or CD3 plus CD28 Abs (TCR/CD28) under IL-17–polarizing conditions for 4 d

(see Materials and Methods). Subsequently, samples were stimulated for 4 h with PMA plus ionomycin and IL-17 and IFN-g cytokine profiles determined

for CD4 (A) and CD8 (C) T cells by electronic gating. Cumulative data are presented as bar graphs, representing the frequencies of IL-17A–producing CD4

(B) and CD8 (D) T cells. Data denote results three independent experiments. Statistical significance was calculated using unpaired, two-tailed t tests. Error

bars denote the SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

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C. rodentium by oral gavage (Fig. 6A–C). At day 10 postinfection,the numbers of donor WT and Sh2d1a2/2 CD4 T cells were notfound to be significantly different from one another in the spleenor mesenteric lymph node (MLN) (Fig. 6D, 6E). However, donorcolonic Sh2d1a2/2 CD4 T cell numbers were reduced relative todonor WT (2.6-fold, 27.4 6 8.1 versus 72.6 6 8.1%, p , 0.01;Fig. 6E). The proportions of donor Sh2d1a2/2 Th17 cells weremarkedly decreased in the spleens, MLNs, and colons relative todonor WT Th17 cells (spleen: 1.8-fold, 35.3 6 7.1 versus 64.7 67.1%, p , 0.05; MLN: 2.9-fold, 25.6 6 5.9 versus 74.4 6 5.9%,p , 0.001; colon: 3.6-fold, 21.6 6 7.8 versus 78.4 6 7.8%, p ,0.001; Fig. 6D–H). In addition, donor Sh2d1a2/2 Th1 cells weresignificantly decreased in the colon but not the spleen or mesen-teric lymph node relative to WT (Fig. 6H). Reduced colonicSh2d1a2/2 Th1 cells could be a consequence of decreased Agpriming or homing of effectors to the gut. Given that Th17 cellsmay transdifferentiate into Th1 cells (50), fewer Sh2d1a2/2 Th17cells or a diminished conversion rate could also be contributorsresponsible for a smaller number of Th1 effectors. By contrast, thefrequencies of donor WT and Sh2d1a2/2 CD4 T cells in uninfected

recipient mice at 15 d posttransfer were found to be similar relativeto one another, suggesting that SAP is not critical for the mainte-nance of naive CD4 T cells (WT/Sh2d1a2/2 CD4 T cell ratio inspleens: 1.1; MLN: 1.0; colons: 1.1). Together, these findings in-dicate that SAP within naive CD4 T cells regulates Th17 cell dif-ferentiation in vivo.

SAP exacerbates the development of EAE

Given the well-described pathogenic role of Th17/Tc17 effectors inEAE (8, 18, 23) and our findings that SLAM-SAP signaling acts topromote generation of IL-17–secreting CD4 and CD8 T cellsin vitro (Figs. 3–6), we investigated how SAP deficiency wouldaffect disease progression (Fig. 7). WT and Sh2d1a2/2 mice wereinjected with MOG35–55 in CFA, and disease course was moni-tored in a blinded fashion for 26 d postimmunization. Cohorts ofSh2d1a2/2 mice showed delayed disease and decreased diseaseseverity relative to cohorts of WT mice (Fig. 7A; mean clinicalscores at day 15 postimmunization: Sh2d1a2/2 mice 0.9 6 0.3,WT mice 2.3 6 0.1, p , 0.001). Moreover, groups of Sh2d1a2/2

mice had a reduced incidence of disease as compared with WT

FIGURE 4. SAP–SLAM controls the differentiation of naive CD4 and CD8 T cells into Th17 and Tc17 effectors. (A) Expression of CD25 and CD44 on

bulk and sorted naive (CD252CD44lo) CD4+ and CD8+ T cells from WT and Sh2d1a2/2 mice are shown (left side). Sorted WT and Sh2d1a2/2 naive

(CD252CD44lo) CD4 and CD8 T cells were treated with 1, 3, or 10 mg/ml of plate-bound CD3 Ab alone (TCR) or CD3 plus SLAM Abs under IL-17–

polarizing conditions (right side). After 4 d, cells were restimulated for 4 h with PMA plus ionomycin and IL-17 and IFN-g cytokine profiles measured for

WT and Sh2d1a2/2 CD4 and CD8 T cells. (B) Cumulative data are presented as bar graphs, representing the frequencies of IL-17A–producing CD4 (left

bar graph) and CD8 T cells (right bar graph). Error bars represent the SEM. Statistical significance was calculated using unpaired, two-tailed t tests. *p ,0.05, **p , 0.01, ***p , 0.001.

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(Fig. 7B; 0 versus 60% between days 6 and 8, p , 0.05; 40 versus100% at day 10, p , 0.05). Collectively, these experiments dem-onstrate that SAP contributes to pathogenic immune responses thatoccur in EAE model of autoimmunity.

Sh2d1a2/2 mice show defective CD4 T cell priming anddecreased numbers of CNS-infiltrating Th17 and Th1/Th17effectors upon MOG immunization

Next, we investigated whether SAP expression affected the de-velopment of Th1 and Th17 effectors, two T cell lineages impli-cated in the autoimmune pathogenesis of EAE CNS pathology. Toassess whether SAP affected Ag priming of naive CD4 and CD8T cells, WT and Sh2d1a2/2 mice were treated with MOG35–55 asdescribed above, and splenic T cell numbers and their profiles ofcytokine secretion were determined on day 14 postimmunizationat which time WT mice had reached a mean clinical score of 2.Previous work has shown that naive Sh2d1a2/2 mice have similarsplenic CD4 and CD8 T cell numbers as WT mice (42), and ourfindings support those observations (data not shown). By contrast,Sh2d1a2/2 mice immunized with MOG35–55 had reduced numbersof both splenic CD4 and CD8 T cells relative to immunized WTmice (Fig. 8A; CD4 T cells, 1.4-fold: 7.5 6 0.33 106 versus 10.76

0.9 3 106, p , 0.05; CD8 T cells, 1.6-fold: 4.7 6 0.2 3 106 versus7.7 6 0.5 3 106, p , 0.01). To assess their cytokine-secretingpotential, splenocytes from MOG35–55-immunized mice were stim-ulated ex vivo with PMA/ionomycin for 4 h and stained for intra-cellular cytokine expression (Fig. 8B). Sh2d1a2/2 mice displayed asubstantial decrease in Th17 (IL-17+IFN-g2), Th1/Th17 (IL-17+

IFN-g+), and Th1 (IL-172IFN-g+) effectors compared with WTmice (Fig. 8C; Th17, 4.3-fold: 4.46 1.83 104 versus 19.06 4.63104, p , 0.05; Th1/Th17, 14.7-fold: 0.1 6 0.1 3 104 versus 2.1 60.53 104, p, 0.05; Th1, 7.9-fold: 14.26 4.83 104 versus 112.4613.9 3 104, p , 0.01). By contrast, WT and Sh2d1a2/2 mice pos-sessed similar numbers of Tc1 cells and only scant numbers ofIL-17–secreting CD8 T cells. These findings show that SAP pro-motes the priming of Ag-specific CD4 T cells to differentiate intoTh17, Th1/Th17, and Th1 effectors.To determine whether differences in disease severity between

WT and Sh2d1a2/2 mice was associated with changes in the de-gree of lymphocytic infiltration within the CNS, mononuclearcells isolated from brains and spinal cords of MOG35–55-immu-nized mice were enumerated, surface stained, and their cytokine-secreting profiles determined as described above. Sh2d1a2/2 miceexhibited markedly decreased numbers of infiltrating CD4 and

FIGURE 5. SAP–SLAM signal-

ing promotes Th17 cell differentia-

tion through an IFN-g– and IL-4–

independent mechanism. WT and

Sh2d1a2/2 splenocytes were treated

with 0.3 or 1 mg/ml of plate-bound

CD3 Ab alone (TCR), CD3 plus

SLAM Abs (+ SLAM), CD3 plus

CD28 Abs (+ CD28), or CD3, SLAM,

and CD28 Abs (+ SLAM/CD28) under

IL-17–polarizing conditions (TGF-b,

IL-6, and IL-23) in the presence (A)

or absence (B) of neutralizing anti-

cytokine Abs (anti–IL-4, anti–IFN-g,

and anti–IL-2 Abs; seeMaterials and

Methods). After 4 d, cells were re-

stimulated with PMA/ionomycin and

IL-17A secretion and FOXP3 ex-

pression measured in CD4 T (CD4+

TCRb+) cells by intracellular flow

cytometry. Cumulative data are pre-

sented as bar graphs, and error bars

represent the SEM. Statistical sig-

nificance was calculated using un-

paired, two-tailed t tests. *p , 0.05,

**p , 0.01, ***p , 0.001.

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CD8 T cells relative to WT mice (Fig. 8D; CD4 T cells, 5.3-fold:1.6 6 0.4 3 104 versus 7.9 6 0.5 3 104, p , 0.001; CD8 T cells,4.2-fold: 1.9 6 0.5 3 104 versus 8.0 6 0.5 3 104, p , 0.001).Moreover, CNS-infiltrates of Sh2d1a2/2 mice consisted of greatlydiminished frequencies (Fig. 8E) and absolute numbers of Th17,Th1/Th17, and Th1 effectors as compared with WT mice (Fig. 8F;Th17, 21.5-fold: 0.36 0.13 103 versus 6.06 0.43 103, p, 0.001;Th1/Th17, 63-fold: 0.03 6 0.01 3 103 versus 1.9 6 0.1 3 103, p ,0.001; Th1, 11.8-fold: 1.96 0.53 103 versus 23.46 1.23 103, p,0.001). Additionally, Sh2d1a2/2 mice had fewer CNS-infiltratingT cell effectors of the Tc17, Tc1/Tc17, and Tc1 phenotype relativeto WT animals (Tc17, 33.3-fold: 0.04 6 0.01 3 103 versus. 1.0 60.1 3 103, p , 0.001; Tc1/Tc17, 62.4-fold: 0.02 6 0.01 3 103

versus 1.06 0.13 103, p, 0.001; Tc1, 7.4-fold: 3.26 0.83 103

versus 23.6 6 1.4 3 103, p , 0.001). Together, these findingsshow that SAP contributes to the generation of IL-17–secretingCD4 and CD8 T cell effectors and autoimmune pathology of EAE.

DiscussionSLAM/SAP pathways are known to regulate the immune systemthrough control of lymphocyte/lymphocyte interactions necessaryfor the differentiation of T follicular helper, B, and NKT cells (25)

and for optimal effector functions of NK and CD8 T cells (32, 33).In this study, we show that a consequence of SAP deficiency isimpaired differentiation of Th17 and Tc17 effectors and reducedsusceptibility to T cell–mediated autoimmunity, raising the pos-sibility that IL-17 deficits might also contribute to the phenotypeof XLP and in particular to the antiviral immunity to EBV. IL-17–secreting effectors have been strongly implicated in the clearanceof bacterial and fungal infections (8, 12), and interestingly, someevidence suggests that IL-17–secreting T cell effectors may havea role in the control of viral infections (11, 13, 19). Experimentalinfections of mice with influenza have shown that Th17 and Tc17cells are generated upon repeat challenge and that Tc17 cells canprotect against a lethal viral dose (13). In addition, Tc17 cells havebeen found to be expanded by vaccinia virus and to mediate viralclearance through the expression of molecules associated withcytotoxicity (11). Furthermore, patients with chronic hepatitis Cvirus (HCV) were found to have HCV-specific IL-17–producingCD8 T cells, and the cell frequencies were highest among indi-viduals with low inflammatory activity, suggesting a protectiverole for Tc17 cells in chronic HCV infection (19). These obser-vations along with our results suggest the possibility that dimin-ished Th17/Tc17-mediated immunity within XLP patients may

FIGURE 6. SAP plays a CD4 T cell–intrinsic role in regulating Th17 cell differentiation. Expression of CD25 and CD44 on CD4+TCRb+ T cells from

WT and Sh2d1a2/2 mice is shown before (A) and after (B) CD25/CD44 Ab-mediated depletion (see Materials and Methods) of Tregs and memory/effector

T cells. (C) Naive (CD252CD44lo) CD4 T cells from WT (Thy1.1+CD45.2+) and Sh2d1a2/2 (Thy1.1+CD45.2+) mice were mixed at a 1:1 ratio and injected

i.v. into host (CD45.1+) mice. (D) At day 10 postinfection with C. rodentium, mononuclear single-cell suspensions from spleens, MLNs, and colons were

restimulated prior to detection of intracellular IFN-g and IL-17 expression by donor CD4 T cells. (E) The representation of WT and Sh2d1a2/2 donor CD4

T cells relative to total donor CD4 T cells is shown within the indicated tissue. The proportion of WT and Sh2d1a2/2 donor CD4 T cells expressing IL-17+

IFN-g2 (F), IL-17+IFN-g+ (G), and IL-172IFN-g+ (H) relative to the total donor CD4 T cells producing the indicated cytokine. Error bars represent the

SEM. Statistical significance was calculated using unpaired, two-tailed t tests. *p , 0.05, **p , 0.01, ***p , 0.001.

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contribute to the inability to control EBV and virus-induced im-mune pathology.Sh2d1a2/2 CD4 and CD8 T cells displayed decreased differ-

entiation into Th17 and Tc17 effectors relative to WT when cul-tured with TCR and stimulating SLAM Abs (Figs. 3–5). However,Sh2d1a2/2 CD4 and CD8 T cells also exhibited marked deficitswhen stimulations were performed in the absence of SLAM Abs,with TCR plus CD28 Abs or TCR Abs alone, demonstrating thatSAP influences TCR signaling even when SLAM receptors are notapparently engaged (Figs. 2, 3). This phenomenon might reflectthe recent finding that SAP associates with ITAM sequences ofCD3z (51, 52) and TCR activation results in the SLAM familyreceptors and SAP being recruited to the immunological synapse(27, 53). SAP has also been found to sustain TCR signaling throughinhibition of diacylglycerol (DAG) kinase a, a negative regulator ofthe DAG-responsive RasGRP1–Ras–ERK pathway via its conver-sion of DAG to phosphatidic acid (54). These studies argue thatSAP regulates cognate Ag-specific T cell responses directly bybinding the TCR or by bringing together SLAM family receptorsand SAP with the TCR signalosome.The influence of SLAM–SAP signaling pathway on the gen-

eration of Th17 and Tc17 cells is unclear. Engagement of SLAMfamily receptors through their ligand interactions causes SAP torecruit the Src family tyrosine kinase FYN and possibly othereffectors to mediate downstream signaling (25). One plausibleconnection comes from an investigation suggesting that a SAP/FYN/protein kinase Cu signaling network modulates NF-kB sig-naling (55). NF-kB activation induces the basic leucine zippertranscription factor ATF-like (56), an inhibitor of AP-1 activitythat is itself essential for RORgt expression and Th17 differenti-ation (57). In addition, SLAM costimulation has been proposed todrive IL-17 transcription in human T cells through the activationof NFAT (58). Consequently, SAP may regulate the formation of

Th17 and Tc17 lineages through its control of the NF-kB or NFATpathways. Recently, a study has found that the SAP downstreameffector FYN promotes the differentiation of Th17 cells throughinfluences on RORgt expression (59). However, it remains unclearwhether FYN’s effects on Th17 differentiation are dependent onSAP or its functions are mediated through a SAP-independentroute downstream of the TCR (59).Previous work has demonstrated that SAP can play a pivotal role

in the development of T cell–dependent humoral autoimmunity(60, 61). The loss of SAP expression was found to result in de-creases in Ab levels, anti-nuclear Abs, renal pathology, and ame-lioration of disease in experimental and spontaneous mouse modelsof SLE (60, 61), fitting with the critical functions that SAP mediatesin humoral immunity (25). Moreover, Hron et al. (61) have reportedthat a deficiency of SAP exacerbated EAE, contrasting theirobservations with lupus and contradicting our findings documentedin this study (Fig. 7). It remains unclear whether the discrepantresults are a consequence of variation in genetic background het-erogeneity, SAP mutagenesis, method used to induce EAE, orenvironmental factors. With respect to genetic background, Sh2d1a-targeted mouse strains used in our study (42) and the one employedby Hron et al. (61) were both derived using 129 embryonic stemcells and subsequently bred with the C57BL/6 strain. Moreover,129:C57BL/6 hybrid mice have been shown to be especially auto-immune prone and thus may confound experiments assessing au-toimmune susceptibility (62). Importantly, our Sh2d1a2/2 mouseline has been backcrossed to C57BL/6J mouse line (.10 gen-erations), minimizing the number of potentially meddling genesdescended from the 129 strain, whereas Sh2d1a2/2 mice used byHron et al. (61) appear to be of mixed 129:C57BL/6 background.Regardless of backcrossing, both gene-targeted mouse lines retainsome adjacent 129 DNA tightly linked to the mutated Sh2d1alocus on the X chromosome. With respect to methodologicaldifferences, we used a different Sh2d1a-targeted mouse strain(42 versus 45), MOG peptide (MOG35–55 versus MOG38–55), andamount of peptide (200 versus 300 mg), and we did not administersupplemental heat-killed M. tuberculosis H37RA (0 versus 1.2 mg)besides what is present within CFA. Finally, cohorts of WT andSh2d1a2/2 mice used in our study were derived from intercrosseswithin our animal facility, likely limiting variances in gut micro-biota, another factor that influences autoimmunity (63).NKT cells have been implicated in acting as important im-

munoregulatory cells necessary to suppress autoimmune T cellresponses (64). Given that a number of studies have demonstratedthat NKT cell activation via aGalCer administration protects miceagainst EAE (65–67), the lack of NKT cells in Sh2d1a2/2 micemight be expected to result in more severe EAE than WT mice.However, NKT cell–deficient mice (CD1d2/2 or Ja182/2) ona C57BL/6 background have been shown to exhibit an EAE courseof disease closely resembling WT mice (65–67), suggesting thatNKT cell deficiency in Sh2d1a2/2 mice likely has little impact onEAE disease course. Collectively, our findings allude to the possi-bility that diminished Th17 differentiation is responsible for thereduced severity of EAE in Sh2d1a2/2 mice. However, Sh2d1a2/2

mice also exhibit greatly decreased numbers of IFN-g–producingCD4 and CD8 T cells relative to WT mice (Fig. 8). Moreover,decreased EAE disease in Sh2d1a2/2 mice may be a consequenceof defective Ag priming or homing, leading to reduced numbers ofpathogenic Th1 or Th17 cells at the target site.T–B cell cooperation likely promotes pathogenesis of MS as the

depletion of B cells from MS patients has been found to reduceinflammation and myelin loss (68, 69). Given the establishedimportance of SAP functioning in lymphocyte–lymphocytecommunication (25), our findings documenting roles for SAP in

FIGURE 7. SAP exacerbates the development of experimental autoim-

mune encephalitis. Mice (n = 8/group) were immunized with MOG35–55 in

CFA and pertussis toxin to induce EAE. Mice were monitored for disease

development and severity using the following scale for clinical scoring: 0,

no overt disease; 1, tail or hind limb weakness; 2, tail and hind limb

weakness; 3, partial hind limb paralysis; and 4, complete paralysis. Re-

gardless of genotype, mice were not found in a moribund state (clinical

score of 5) during the course of these experiments. (A) Data are presented

as the mean clinical scores 6 SEM for each group. Mann–Whitney U tests

were used to calculate statistical significance. (B) Data are presented in-

dicating the disease incidence for each group. Statistical significance was

determined using Fisher exact tests. *p, 0.05, **p, 0.01, ***p, 0.001.

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potentiating Th17/Tc17 differentiation and the development ofT cell–mediated autoimmunity lend additional support to the hy-pothesis that SAP contributes to the pathogenesis of MS. More-over, elevated levels of Th17 cells and IL-17 have been found incerebrospinal fluid of MS patients (70), and disease susceptibilityis linked to IL-23R and STAT3 genes, known regulators of IL-17expression (71). Increased SLAM (CD150) and SLAMF3 (CD229or Ly9)/SLAMF6 (CD352 or NK–T–B Ag) expression has beendescribed for T cells of MS (72) and SLE patients (73), respec-tively, suggesting the possibility that the heightened SLAM familyreceptor–SAP signaling promotes inflammation by amplifying IL-17 expression. The observations reported in this study suggest thatattenuation of SAP signaling through application of SLAM familyreceptor Abs or SLAM family receptor/Ig fusion proteins mayprove valuable as novel therapeutics for MS and other forms ofautoimmunity or inflammatory disease.

AcknowledgmentsWe thankXiaoxiaWang andHuilian Qin for technical assistance, Drs. Kenneth

W. Harder (University of British Columbia) and Jacqueline Quandt (Uni-

versity of British Columbia) for discussion, Drs. Andre Veillette (Clin-

ical Research Institute of Montreal) and Cox Terhorst (Harvard Medical

School) for providing the anti-SAP Ab and breeders of the Sh2d1a2/2

mouse line, respectively. We also thank Lisa Xu for excellent technical

assistance with cell sorting and the Child and Family Research Institute

animal facility for mouse husbandry.

DisclosuresThe authors have no financial conflicts of interest.

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FIGURE 8. Sh2d1a2/2 mice show defective CD4 T cell priming and decreased numbers of CNS-infiltrating Th17 and Th1/Th17 effectors upon MOG

immunization. On day 14 post–EAE induction (WT mice reached a clinical score of 2), splenic and CNS-infiltrating T cells were counted and analyzed for

cytokine production. (A) Absolute numbers of splenic T cells from WT and Sh2d1a2/2 mice are shown at day 14 post–EAE induction. (B) Representative

cytokine production by WT and Sh2d1a2/2 splenic T cells are shown. (C) Cumulative data on numbers of cytokine-secreting WT and Sh2d1a2/2 splenic

CD4 and CD8 T cells. (D) Absolute numbers of CNS-infiltrating CD4 and CD8 T cells from WT and Sh2d1a2/2 mice. (E) Representative cytokine profiles

are shown for CNS-CD4 and CD8 T cells. (F) Cumulative data on numbers of cytokine-producing T cells within the CNS. Data are represented from three

independent experiments. Error bars represent the SEM. Statistical significance was calculated using unpaired, two-tailed t tests. *p , 0.05, **p , 0.01,

***p , 0.001.

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