inhibition of diacylglycerol kinase a restores...

R E S EARCH ART I C L E

IMMUNODEF I C I ENCY

httD

ownloaded from

Inhibition of diacylglycerol kinase a restoresrestimulation-induced cell death and reducesimmunopathology in XLP-1Elisa Ruffo,1* Valeria Malacarne,1* Sasha E. Larsen,2* Rupali Das,3* Laura Patrussi,4

Christoph Wülfing,5 Christoph Biskup,6 Senta M. Kapnick,7 Katherine Verbist,8 Paige Tedrick,8

Pamela L. Schwartzberg,7 Cosima T. Baldari,4 Ignacio Rubio,9 Kim E. Nichols,8† Andrew L. Snow,2†

Gianluca Baldanzi,1† Andrea Graziani1,10†‡

X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with theexuberant expansion of activated CD8+ T cells after Epstein-Barr virus (EBV) infection. XLP-1 is caused by defectsin signaling lymphocytic activation molecule (SLAM)–associated protein (SAP), an adaptor protein that modulates Tcell receptor (TCR)–induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death(RICD) and diminished TCR-induced inhibition of diacylglycerol kinase a (DGKa), leading to increased diacylglycerolmetabolism and decreased signaling through Ras and PKCq (protein kinase Cq). We show that down-regulation ofDGKa activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD viainduction of the proapoptotic proteins NUR77 and NOR1. Pharmacological inhibition of DGKa prevents the exces-sive CD8+ T cell expansion and interferon-g production that occur in SAP-deficient mice after lymphocytic chorio-meningitis virus infection without impairing lytic activity. Collectively, these data highlight DGKa as a viabletherapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients.

p:/
by guest on M
ay 1, 2019/stm

.sciencemag.org/

INTRODUCTION

X-linked lymphoproliferative disease (XLP-1) is a heritable im-mune disorder caused by germline mutations in the SH2D1A gene,which encodes the signaling lymphocytic activation molecule(SLAM)–associated protein (SAP) (1). SAP is a small SH2 domain–containing adaptor primarily expressed in T, natural killer (NK), andinvariant NKT (iNKT) cells (1). XLP-1 is best recognized for theincreased susceptibility of affected males to develop overwhelminglymphoproliferation after primary Epstein-Barr virus (EBV) infection(2). Also known as fulminant infectious mononucleosis (FIM), thislymphoproliferative process is characterized by the massive accumu-lation of activated CD8+ T cells, which infiltrate multiple organs andinflict severe tissue damage. FIM is the most common and clinicallychallenging manifestation of XLP-1, with up to 65% of patients dyingdespite the use of chemoimmunotherapy (3). Accordingly, alternativeandmore effective treatment strategies are sorely needed for XLP-1 pa-tients who develop FIM.

T lymphocytes derived from XLP-1 patients exhibit multiple func-tional defects, including reduced cytotoxic activity (4) and impairedrestimulation-induced cell death (RICD) (5). RICD is a self-regulatory

1Department of TranslationalMedicine and Institute for Research and Cure of AutoimmuneDiseases, University of Piemonte Orientale, 28100 Novara, Italy. 2Department of Pharmacologyand Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda,MD 20814, USA. 3Department of Physiology, Michigan State University, East Lansing,MI 48824,USA. 4Department of Life Sciences, University of Siena, 53100 Siena, Italy. 5School of Cellularand Molecular Medicine, University of Bristol, BS8 1TH Bristol, UK. 6Biomolecular PhotonicsGroup, Jena University Hospital, D 07740 Jena, Germany. 7Genetic Disease Research Branch,NationalHumanGenomeResearch Institute, National InstitutesofHealth, Bethesda,MD20892,USA. 8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN 38105,USA. 9Integrated Research and Treatment Center, Center for Sepsis Control and Care andInstitute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital,D-07745 Jena,Germany. 10School ofMedicine,University Vita e Salute SanRaffaele, 20132Milan, Italy.*These authors contributed equally to this work.†These authors contributed equally to this work.‡Corresponding author. Email: [email protected]

www.Scien

apoptosis program triggered by repeated T cell receptor (TCR) stim-ulation that maintains peripheral immune homeostasis by constrain-ing the accumulation of activated T cells (6). A similar death defect ispresent in the activated T cells of Sh2d1a−⁄− mice (7). It is proposedthat defective RICD, combined with impaired clearance of EBV-infected B cells, sustains and amplifies the expansion of activated Tcells that typifies FIM (5, 6).

SAP binds to immunotyrosine-based switch motifs within the cyto-plasmic domains of the SLAM family receptors (SLAM-Rs) (8), thuscompeting with the binding of SH2 domain–containing inhibitory lipidand tyrosine phosphatases such as SH2-containing inositol poly-phosphate 5-phosphatase (SHIP) and SH2-containing protein tyrosinephosphatase 1 and 2 (SHP-1)/SHP-2 (9). In addition, SAP facilitatesrecruitment of kinases such as FynT and Lck to SLAM-Rs to promoteoptimal signaling within T, NK, and NKT cells (10, 11). Indeed, RICDresistance inXLPpatient T cells results in part fromweakTCR signalingassociated with excess SHP-1 activity and defective recruitment of Lckto theNTB-A receptor, which colocalizes with the TCR (5, 11). AlthoughSAP links SLAM-R signaling to several downstream functions via activa-tion of Src-family kinases [for example, interleukin-4 (IL-4) secretion(12), iNKT cell development (13)], this signaling axis is not the onlypathway in which SAP is involved for signal regulation. For example,the requirement for SAP in the provision ofCD4+T cell–mediated “help”for B cell differentiation is Fyn-independent (14). To fully understandXLP-1pathogenesis anddevelopmore effective therapeutic interventions,the mechanistic characterization of signaling molecules involved in these“alternative” SAP-dependent signaling pathways is imperative.

We recently observed that after TCR stimulation, SAP selectivelyinhibits diacylglycerol kinase a (DGKa) without requiring FynT orLck (15). DGKa and DGKz phosphorylate diacylglycerol (DAG) togenerate phosphatidic acid, thereby modulating TCR signal strength byregulating DAG levels and downstream biochemical events (16, 17). Inactivated T cells, silencing SAP expression results in persistently active

ceTranslationalMedicine.org 13 January 2016 Vol 8 Issue 321 321ra7 1

http://stm.sciencemag.org/


http://stm.scie

Dow

nloaded from

DGKa and thus impaired DAG signaling, leading to reduced proteinkinase Cq (PKCq) membrane recruitment, NFAT (nuclear factor ofactivated T cells) and ERK1/2 (extracellular signal–regulated kinase1/2) activation, and IL-2 production (15). These data collectivelysuggest that upon antigen stimulation, SAP inhibits DGKa activityto facilitate optimal DAG accumulation and full TCR signal strength,ultimately leading to cell activation.

Because TCR signal strength directly correlates with RICD sensitiv-ity (18), we hypothesized that the reduced RICD of XLP-1 T cells mightbe linked to deregulation of DGKa in the absence of functional SAP.Consistent with this notion, we show herein that the loss of SAP in Tcells results in reduced DAG polarization to the immune synapse (IS)and impaired TCR-induced DAG-dependent TCR signaling. Both ofthese events are due to persistent DGKa activity and contribute toRICD resistance. Consequently, the inhibition of DGKa in XLP-1 Tcells restored DAG signaling and RICD by rescuing IS architectureand triggering a specific DAG-dependent apoptotic process mediatedby the orphan nuclear receptors NR4A1 (NUR77) and NR4A3(NOR1). Strikingly, in vivo inhibition of DGKa activity reduced theexcessive CD8+ T cell accumulation and interferon-g (IFNg) productionthat occur in Sh2d1a−⁄−mice infected with lymphocytic choriomeningitisvirus (LCMV), amurinemodel of FIM.Our findings illuminate the SAP/DGKa signaling axis as a key regulator of TCR-induced apoptosis. Theseresults highlight DGKa as a novel, druggable target for treating FIM bypromoting RICD, reducing the accumulation of pathogenic, activatedCD8+ T cells, and thusmitigating the life-threatening immunopathologythat often occurs in EBV-infected XLP-1 patients.

by guest on May 1, 2019

ncemag.org/

RESULTS

DGKa inhibition rescues RICD in SAP-deficient T cellsTo investigate whether reduced DAG signaling contributes to the Tcell–driven pathologic manifestations of XLP-1, we examined whethersilencing or inhibition of DGKa could restore the sensitivity of XLP-1 T cells to RICD. SAP-deficient XLP-1 T cells exhibit reduced RICDrelative to control T cells after stimulation with increasing concentra-tions of the agonistic anti-CD3 antibody (Ab) OKT3 (5) (Fig. 1, A andB). Remarkably, this defect in RICD was substantially rescued by thesmall interfering RNA (siRNA)–mediated silencing of DGKa (Fig. 1,A to C) or by pretreatment with the DGKa inhibitor R59949 (Fig. 1,D and E) or R59022 (Fig. 1F) (19). The rescue in RICD obtained uponDGKa inhibition was likely due to the induction of apoptosis, as in-dicated by an increased percentage of AnnexinV+ cells (Fig. 1G). Con-versely, the inhibition or silencing of DGKa had little effect on RICDin activated T cells from healthy subjects (Figs. 1 and 2).

Because patient-derived cells were limited, we repeated these as-says using siRNA to knock down SAP expression in activated Tcells from healthy donors (Fig. 2) (5). In agreement with our pre-vious findings, SAP-silenced cells exhibited defective RICD thatwas rescued by concomitant silencing of DGKa (Fig. 2, A and B)or by treatment with the DGKa inhibitor R59949 (Fig. 2C) orR59022 (Fig. 2D). This restoration of RICD in SAP-silenced T cellswas associated with enhanced apoptosis, as indicated by increasedAnnexinV staining (Fig. 2E). For other isoforms expressed in Tcells, silencing of DGKz, but not DGKd, also partially rescuedRICD in SAP-silenced cells (fig. S1, A to D). Conversely, overex-pression of DGKa or DGKz conferred partial resistance to RICD in

www.Scien

normal T cells (fig. S1, E and F). These findings suggest a link be-tween the RICD resistance of SAP-deficient lymphocytes and un-restrained DAG depletion caused by enhanced DGK activity. Toexplore this further, we supplemented cultures with the DAG ana-log 1,2-dioctanoyl-sn-glycerol (C8-DAG), which is rapidlyincorporated into the cell membrane and triggers DAG-dependentsignaling (20). Indeed, C8-DAG treatment markedly enhancedRICD in SAP-silenced but not control T cells (Fig. 2F). Collectively,these data demonstrate that excessive DGKa activity contributesto RICD resistance in SAP-deficient T cells and that this processcan be reversed by inhibition of DGKa. These data suggest thatSAP promotes TCR signal strength and RICD sensitivity by at-tenuating DAG metabolism carried out by DGKa in activated Tcells (Fig. 2G).

Inhibition of DGKa rescues defective DAG polarization andsignaling at the IS in SAP-deficient cellsDAG generation and polarization at the IS are required for TCR-induced cellular responses (21). To investigate whether the deregulatedDGKa activity caused by SAP deficiency affects DAG polarizationtoward the IS, we imaged DAG localization using a PKCq–cysteine-rich domain (CRD)–based biosensor (22). After activation bysuperantigen-loaded Raji B cells, we observed that PKCq-CRD po-larization to the IS was strongly reduced in SAP-silenced versus con-trol Jurkat cells (Fig. 3, A to D). In contrast, co-silencing of DGKa(Fig. 3, A and B) or pretreatment with the DGK inhibitor R59949(Fig. 3, C and D) restored PKCq-CRD polarization in SAP-silencedT cells. Consistent with the finding that DGKa shapes the DAG gra-dient at the IS (23), DAG polarization was also reduced in SAP-expressing, DGKa-silenced Jurkat cells (Fig. 3, A and B).

Polarized DAG signaling triggers F-actin polymerization and mi-crotubule organizing center (MTOC) orientation (24, 25). Consistentwith reduced DAG polarization, SAP-silenced T cells exhibited astrong defect in F-actin accumulation at, and MTOC orientationtoward, the IS upon contact with superantigen-loaded Raji cells(Fig. 3, E to J). Again, silencing or inhibition of DGKa partially re-stored these processes (Fig. 3, E to J). These findings indicate thatSAP regulates the architecture of the IS by inhibiting DGKa, therebylimiting DAG metabolism locally.

We next investigated whether inhibition of DGKa restores DAG-mediated signaling downstream of the TCR in SAP-silenced primaryhuman T cells. PKCq and RasGRP1 are recruited to the IS in a DAG-dependent manner (26, 27) and are required for induction of RICD(28, 29). Consistent with our hypothesis, SAP-silenced primary T cellsexhibited defective recruitment of PKCq and RasGRP1 to the IS,which was fully restored upon DGKa silencing (Fig. 4, A, B, E, andF) or pharmacological inhibition (Fig. 4, C, D, G, and H). Consideringinhibition of DGKa also rescues defective ERK1/2 activation in SAP-deficient T cells (15), these data underscore the importance of theSAP/DGKa axis in regulating DAG-dependent signaling.

To test if inhibition of DGKa rescues RICD in SAP-deficient Tcells by restoring specific DAG-mediated signaling pathways, weexamined whether the rescue of RICD requires the activity of PKCqor RasGRP1. Silencing of PKCq (Fig. 5A) or RasGRP1 (Fig. 5B) re-duced RICD in control siRNA–transfected T cells and completely ab-rogated the rescue of RICD in SAP and DGKa siRNA–transfectedcells. Moreover, pharmacological inhibition of PKC or MEK (mitogen-activated protein kinase)/ERK enzymatic activity also prevented the





http://stm.sciencem

ag.org/D

ownloaded from

restoration of RICD noted after DGKa silencing in SAP-deficient Tcells (fig. S2).

TCR activation stimulates DAG-dependent induction of IL-2 andthe high-affinity IL-2 receptor CD25 (30, 31), which are both required

www.Scien

for RICD (32). Indeed, inhibition or silencing of DGKa restored in-duction of CD25 in SAP-silenced T cells after TCR restimulation, anda similar trend was observed with IL-2 expression (Fig. 5, C to E).These findings further establish the SAP/DGKa axis as a critical

Fig. 1. DGKa silencing or inhibition restores RICD in XLP-1 pa-tient T cells. (A and B) Activated T cells from normal donors (Ctrl)or indicated XLP-1 patients were transfectedwith control (Cntrl) orDGKa-specific siRNA, and then restimulated 4 days later withOKT3Ab. After 24 hours, % cell loss was evaluated by propidium iodide
(PI) staining. Data are means ± SD of two experiments (A) or one experi-ment (B) performed in triplicate, representative of two independentexperiments using different control donors. (C) DGKa relative expression(rel exp) in siRNA-transfected cells from (A) measured by quantitative re-verse transcription polymerase chain reaction (qRT-PCR) (upper panel,mean ± SEM, n = 4) or byWestern blotting, with tubulin as loading control(lower panel). (D to F) Ctrl or XLP patient T cells were restimulated withOKT3 Ab after pretreatment with DGK inhibitor R59949 or R59022 (5 to10 mM) or dimethyl sulfoxide (DMSO). After 24 hours, % cell loss was evalu-ated by PI staining. Data are means ± SD of three experiments (E) or one
experiment (D and F) performed in triplicate representative of twoindependent experiments using different control donors. (G) Cells used in(D) were pretreated with R59949 (10 mM) or DMSO and restimulated withOKT3 (100 ng/ml) for 0, 6, and 12 hours. The % of apoptotic cells wasmeasured by AnnexinV staining. Representative histograms are shown;marker numbers denote % AnnexinV+ cells. The net increase in AnnexinV+

cells at 12 hours is shownat the right. Data aremeans± SDof six independentexperiments using four separate controls and two XLP patients. Asterisksdenote statistical significance by two-way analysis of variance (ANOVA)with Sidak correction (A, B, D, and F) or paired t test (C and G).





http://stm.sciencem

ag.org/D

ownloaded from

Fig. 2. DGKa silencing or inhibition restoresRICD in SAP-silenced T cells. (A) Activated
normal donor T cells were transfected with con-trol or SAP siRNA and restimulated 4 days laterwith OKT3 Ab. After 24 hours, % cell loss wasevaluated by PI staining. Data are means ±SEM of three experiments performed in tripli-cate. (B) SAP expression in siRNA-transfected Tcells from (A) was measured by qRT-PCR (upperpanel, mean ± SEM of four experiments) or byWestern blotting, with actin as a loading control(lower panel). (C and D) siRNA-transfected cells(A) were restimulated with OKT3 Ab after pre-treatment with DMSO and DGK inhibitorR59949 or R59022 (5 to 10 mM). After 24 hours,the% cell loss was evaluated by PI staining. Dataare means ± SEM of five experiments (C) or five(control) and eight (SAP siRNA) independentexperiments (D) performed in triplicate. (E)siRNA-transfected cells as in (A) were pretreatedwith DMSO or R59022 (10 mM) and restimulatedwith OKT3 (10 ng/ml). After 12 hours, the % ap-optotic cells was evaluated by AnnexinV stain-ing. Representative histograms are shown;marker numbers denote % AnnexinV+ cells.The net increase in AnnexinV+ cells at 12 hoursis shown at the right. Data are means ± SD offour experiments. (F) siRNA-transfected cells(A) were treated with C8-DAG (50 mM) and re-stimulated with OKT3 Ab. After 24 hours, % cellloss was evaluated by PI staining. Data aremeans ± SEM of five experiments performed intriplicate. Asterisks denote statistical significanceby two-way ANOVA with Sidak correction (A andC to F) or paired t test (B and E). (G) Schematiccartoon: Proapoptotic TCR signaling is governedby DGKa inhibition in activated T cells.
www.ScienceTranslationalMedicine.org 13 January 2016 Vol 8 Issue 321 321ra7 4




http://stm.sciencem

ag.org/D

ownloaded from

Fig. 3. DGKa silencing orinhibition restores synapse
formation in SAP-deficientT cells. ShCNTRL or shSAPJurkat T cells were transient-ly transfected as indicated.(A to J) After 48 hours (C, D,and G to J) or 96 hours (A, B,E, and F), T cells were chal-lenged with SEE-loaded RajiB cells, and confocal live-cellimages were captured duringT cell–antigen presenting cell(APC) conjugation. In (C), (D),(G), (H), (I), and (J), T cells werepretreated for 30min with 10mM R59949 or DMSO. (A andC) Top row: Enhanced greenfluorescent protein (EGFP)–taggedPKCq-CRD(pseudoco-lor) together with the perime-ter of the APC (dotted line).Bottom row: Phase-contrastimages with APC denotedby *. Scale bar, 10 mm. (Band D) Quantification ofEGFP-PKCq-CRD accumula-tion at the IS. Mean ± SEMof >20 conjugates per condi-tion from three experiments.(E and G) Top row: LifeAct–GFP (green fluorescent pro-tein) (green). Bottom row alsoshows Raji B cells stainedwith CellTracker Red CMTPX(red). Scale bar, 10 mm. (Fand H) Quantification ofLifeAct-GFP accumulationat the IS. Mean ± SEM of>20 conjugates per condi-tion from three experiments.(I) Top row:GFP-tubulin (green).Bottom row also shows Raji Bcells stained with CellTrackerRed CMTPX (red). Scale bar,10 mm. (J) Quantification ofMTOC polarization index.Mean ± SEM of >35 conju-gates per condition from twoexperiments. Asterisks in allpanels denote statistical signif-icance by one-way ANOVAwith Sidak correction.
www.ScienceTranslationalMedicine.org 13 January 2016 Vol 8 Issue 321 321ra7 5



www.ScienceTranslationalMedicine.org


http://stm.sciencem

ag.org/D

ownloaded from

regulator of DAG signaling potency.Collectively, these findings underscorethe vital role of SAP-dependent inhibitionof DGKa in sustaining DAG signaling,leading to the activation of PKCq andRas-ERK and RICD (Fig. 5F).

NUR77 and NOR1 mediatethe rescue of RICD that is inducedby DGKa inhibition in SAP-deficientT cellsWe next investigated the mechanism bywhich the enhancement of DAG sig-naling obtained after inhibition of DGKarestores RICD sensitivity in SAP-deficientT cells. We previously showed that inSAP-deficient T cells, TCR-induced ex-pression of key proapoptotic genes suchas FASLG and BCL2L11 is impaired (5).We observed that silencing or inhibitionof DGKa failed to rescue FASLG orBCL2L11 expression after TCR restimula-tion of SAP-silenced T cells (fig. S3, Aand B). Similarly, DGKa blockade failedto restore the induction of all three majorisoforms of BIM protein (extra long, EL;long, L; and short, S), as well as full-lengthand soluble FASL protein in SAP-silencedand XLP-1 patient T cells after restimula-tion (fig. S3, C to E). These observationsimply that DGKa inhibition does not re-store all SAP-dependent, proapoptotic ef-fector functions that contribute to RICDsensitivity.

Instead, we found that SAP-deficientT cells exhibit a previously unrecognizeddefect in TCR restimulation–induced up-regulation of NR4A1 (NUR77) andNR4A3 (NOR1), two nuclear receptorsinvolved in negative selection of thymo-cytes and RICD of mature T cells (33).DGKa silencing or inhibition selectivelyrestored TCR-dependent induction ofboth NR4A1 and NR4A3 in SAP-silencedactivated T cells (Fig. 6, A to D). DGKainhibition also partially rescued NUR77and NOR1 protein induction in XLP-1 Tcells after TCR restimulation (Fig. 6E).Upon TCR engagement, NUR77 andNOR1 proteins are phosphorylated bythe ERK1/2-regulated 90-kD ribosomalS6 kinase (RSK), triggering the intrinsicapoptosis pathway (34). Indeed, the RSK-specific inhibitor SL0101 (35) significantlyreduced RICD in control T cells, confirmingthat phosphorylation of NUR77 and NOR1is an important component of RICD ex-ecution (Fig. 6, F to H). SL0101 significantly

Fig. 4. DGKa silencing or inhibition restores PKCq and RasGRP1 recruitment to the IS in SAP-deficient cells. (A, C, E, and G) Activated T cells were transfected with the indicated siRNA and after
72 hours were incubated with SEE-loaded Raji B cells (denoted with *) for 15 min and fixed and stainedfor PKCq (A and C) or RasGRP1 (E and G). Top rows: Target protein (green); bottom rows also show phasecontrast. Scale bar, 5 mm. (B) Percentage of cells displaying PKCq at the IS. Data are means ± SEM of sixreplicates from two independent experiments. (D) Percentage of cells displaying PKCq at the IS. Data aremeans ± SEM of three experiments. (F) Percentage of cells with RasGRP1 at the IS. Data are means ± SD ofone representative experiment performed in quadruplicate. (H) Percentage of cells displaying RasGRP1 atthe IS. Data are means ± SEM of three experiments. Asterisks in all panels denote statistical significance bytwo-way ANOVA + Sidak correction.
13 January 2016 Vol 8 Issue 321 321ra7 6




http://stm.sciencem

ag.org/D

ownloaded from

blunted the RICD rescue triggered by DGKa inhibition in XLP-1 Tcells, as well as in SAP/DGKa-silenced T cells (Fig. 6, F to H). These dataindicate that the rescue of RICD afforded by DGKa blockade in SAP-deficient T cells is dependent on RSK activity. Moreover, concomitantknockdown of NUR77 and NOR1 reduced the rescue of RICD inducedby DGKa inhibition in XLP-1 T cells (Fig. 6, I to K). Together, theseobservations indicate that inhibition of DGKa boosts RICD in SAP-deficient T cells in part by selectively restoring TCR-induced up-regulationand RSK-dependent phosphorylation of NUR77 and NOR1 (Fig. 6L).

DGKa inhibition reduces CD8+ T cell accumulation andactivation in LCMV-infected Sh2d1a−⁄− miceDefective RICD is thought to contribute to the aberrant T cell activa-tion and accumulation that occur in EBV-infected XLP-1 patients(36). The demonstration that DGKa silencing or inhibition sensitizesSAP-deficient lymphocytes to RICD in vitro prompted us to assesswhether DGKa inhibition might influence T cell–mediated immuno-pathology in vivo. Toward this end, we used a murine model in whichSh2d1a−⁄− mice are infected with LCMV. In this model, Sh2d1a−⁄−

mice develop many of the cardinal manifestations of FIM, includingCD8+ T cell expansion, proinflammatory cytokine production, andtissue infiltration (37, 38). For these experiments, wild-type (Sh2d1a+/+)or Sh2d1a−⁄− mice were infected with LCMV Armstrong and 4 dayslater were treated with vehicle or R59022 (39). On day 8, the peakof the antiviral T cell response, mice were euthanized and evaluatedfor hyperinflammation.

After LCMV infection, both wild-type and Sh2d1a−⁄− mice devel-oped marked and comparable splenomegaly (Fig. 7, A and B) that wasassociated with an increase in the absolute number of total splenocytes(Fig. 7C). Examination of splenocyte immunophenotype revealed asignificant increase in the percentage and absolute number of totalas well as LCMV-specific (gp33+) CD8+ T cells, most of which exhib-ited an activated CD44+ phenotype (Fig. 7, D to I). Treatment ofLCMV-infected wild-type mice with R59022 did not significantly af-fect any of these parameters (Fig. 7). Conversely, R59022 treatment ofLCMV-infected Sh2d1a−⁄− mice appeared to lessen organomegaly (Fig.7, A and B) and decrease the total splenic lymphocyte count (Fig. 7C).Although R59022 treatment did not affect the percentage of activatedsplenic CD8+ T cells in either mouse strain, it did induce a significant

www.Scien

Fig. 5. DGKa silencing restores TCR-induced PKCq and Ras-mediatedsignalingpathways todriveRICD inSAP-deficient cells. (AandB) Activated
normal donor T cells were transfected with the indicated siRNA and re-stimulated 4days laterwithOKT3Ab (10ng/ml). After 24 hours,% cell losswasevaluated by PI staining. Data are means ± SEM of seven (A) or six (B)experiments performed in triplicate. Right panels: Expression of PKCq (A)or RasGRP1 (B) was measured by Western blotting, with actin as a loadingcontrol. (C andD) qRT-PCR for IL2mRNA in T cells pretreated with R59949(10 mM) (C) or transfected with DGKa siRNA (D) after restimulation withOKT3 (10 mg/ml, 4 hours). GUSB served as the reference gene. Graphs rep-resentmean ± SEM of six (C) or seven (D) experiments. (E) Left: Represent-ative flow cytometric histograms showing CD25 surface expression onsiRNA-transfected T cells from (A) ±OKT3 restimulation (24 hours). Right:Graph depicts mean fluorescence intensity (MFI) of CD25 expression. Dataare means ± SEM of four experiments. Asterisks in all panels denote statis-tical significance by two-way ANOVA with Sidak correction. IgG, immuno-globulin G. (F) Schematic cartoon: SAP-mediated inhibition of DGKaactivity ensures a sufficient pool of DAG required for proper IS organizationand recruitment of PKCq and RasGRP, which mediates downstreamsignaling for RICD.



www.ScienceTranslationalMedicine.org 13 January 2016 Vo


http://stm.sciencem

ag.org/D

ownloaded from

FinrtiToeNPBfrdwssoDwfoaDo(Cimc(C73Rre(1weNtetiefeGnXtrDRfowAloPmp

using different donors. (H) Activated donor T cells were transfecteand treated 4 days later with SL0101 (50 mM) for 30 min, followed24 hours, % cell loss was evaluated by PI staining. Data are meansperformed in triplicate. (I and J) Activated T cells from normal donwere transfected control or NUR77 + NOR1 siRNA and treated 4R59022 (10 mM) for 30min, followed by OKT3 (100 ng/ml). After 24uated by PI staining. Data are means ± SD of one experiment eausingdifferent donors. Asterisks inall panels denote statistical signiwith Sidak correction. (K)Western blot for NUR77 andNOR1 expressiRNA-transfected T cells from (I). Actin served as a loading contMechanism of proapoptotic TCR signaling governed by SAP-depactivated T cells. n.s., not significant.

l

ig. 6. Silencing orhibition of DGKa

estores RICD sensi-vity in SAP-deficientcells via inductionf proapoptotic mol-cules NUR77 andOR1. (A to D) qRT-CR for NR4A1 (A and) or NR4A3 (C and D)om activated normalonor T cells transfectedith control or SAP-pecific siRNA ± DGKa-pecific siRNA (A and C)r 5 mM R59949 (B and) and restimulatedith OKT3 (10 mg/ml)r 4hours.GUSB serveds the reference gene.ata are means ± SEMfeight (A), five (B), seven), or six (D) exper-ents. (E) Activated T

ells from normal donortrl) or XLP-1 patientwere pretreated for0 min with R59022 or59949 (10 mM), thenstimulated with OKT300 ng/ml). Cell lysatesere analyzed byWest-rn blotting for NUR77,OR1, and b-actin con-nt.Dataare representa-ve of two independentxperiments using dif-rent donors. (F and)Activated T cells fromormal donors (Ctrl) orLP-1 patients were pre-eated for 30 min withMSO, SL0101 (90 mM),59949 (10 mM), or both,llowedby restimulationith OKT3 (100 ng/ml).fter 24 hours, % cellss was evaluated byI staining. Data areeans ± SD of one ex-eriment each per-

formed in triplicate
d with the indicated siRNAby OKT3 (10 ng/ml). After± SEM of five experimentsors (Ctrl) or XLP-1 patientsdays later with DMSO orhours, % cell loss was eval-ch, performed in triplicateficanceby two-wayANOVAsion inOKT3-restimulated,rol. (L) Schematic cartoon:endent DGKa inhibition in
8 Issue 321 321ra7 8




http://stm.sciencem

ag.org/D

ownloaded from

www.ScienceTranslationalMedicine.org 13 January 2016 Vo

Fig. 7. In vivo DGKainhibition reduces the
number of activatedvirus-specific CD8+ Tcells in LCMV-infectedSh2d1a−⁄− mice. (A)Images of spleens fromuninfected [phosphate-buffered saline (PBS),“P”] and LCMV-infectedmice without (LCMV,“L”) or with R59022 treat-ment (LCMV + R59022,“L + R”). Representativespleens from each co-hort from B6 wild-type(WT) (top panel) andSh2d1a−⁄− mice (lowerpanel) are shown. (Band C) Ratio of spleenover body weight (B)and total splenocytecount (C) for animalsin each group are pre-sented. B6 WT mice,red symbols; Sh2d1a−⁄−
mice, blue symbols. (Dto I) Representativeflow cytometric (density)plots showing the per-centages of CD8+ CD44+

(top) and LCMV-specificCD8+ gp33+ (bottom) inthe spleens (D) and livers(G) of B6WTand Sh2d1a−⁄−

mice. Percentages (E andH) and absolute numbers(F and I) of CD8+ CD44+

and CD8+ CD44+ gp33+

cells in the spleens (Eand F) and livers (H andI) of B6 WT (red symbols)andSh2d1a−⁄− (blue sym-bols) mice. Data are fromone of two experimentsin which a total of 6 to10 mice in each cohortwere examined. Errorbars represent SD. As-terisks denote statisti-cal significance thatwas determined bytwo-way ANOVA withSidak correction.

l 8 Issue 321 321ra7 9




http://stm.sciencem

ag.org/D

ownloaded from

decrease in the number of total as well as LCMV-specific CD8+ T cellsselectively in the Sh2d1a−⁄− animals (Fig. 7, D to I).

Compared to wild-type animals, LCMV-infected Sh2d1a−⁄− micealso exhibited a trend toward higher serum IFNg levels (Fig. 8A)and greater degrees of tissue inflammation (Fig. 8, B to D). To evaluatewhether DGKa inhibition affected CD8+ T cell functions such as cy-tokine production or degranulation, splenocytes from LCMV-infectedor uninfected, R59022 treated or untreated mice were cultured directly exvivo with the major histocompatibility complex (MHC) class I–restrictedLCMV peptide gp33 and examined for expression of intracellular TNFa(tumor necrosis factor–a), IFNg, and surface CD107a. R59022 treatmentdid not affect the percentage of CD8+ T cells that secreted cytokines(TNFa or IFNg) or degranulated (CD107a exposure) in LCMV-infectedwild-type or Sh2d1a−⁄− mice (Fig. 8, E and F). R59022 treatment ac-tually enhanced the cytolytic activity of Sh2d1a−⁄− CD8+ T cells againstautologous B cell targets in vitro (fig. S5). However, such treatmentdid reduce the absolute number of cytokine-producing and degranu-lating cells only in the Sh2d1a−⁄− animals (Fig. 8G). Consistent withthese findings, only R59022-treated Sh2d1a−⁄− mice exhibited a signif-icant reduction in the serum IFNg levels (Fig. 8A). Finally, R59022treatment significantly reduced the number and size of hepatic inflam-matory infiltrates in LCMV-infected Sh2d1a−⁄− but not wild-type mice(Fig. 8, B to D). These findings collectively indicate that inhibition ofDGKa selectively decreases the magnitude of the CD8+ T cell effectorpool in LCMV-infected Sh2d1a−⁄− mice. DGKa inhibition had no adverseeffects on viral clearance, as LCMV was efficiently cleared from wild-typeand Sh2d1a−⁄− mice by day 8 with or without R59022 treatment (fig. S6).These preclinical data suggest that pharmacologic inhibition of DGKamight reduce the accumulation of aberrantly activated CD8+ T cells andsubsequent hypercytokinemia and tissue inflammation that occur inEBV-infected XLP-1 patients, without impairing CD8+ T cell activityor viral clearance.

One major limitation of this study is its focus on the role of theSAP/DGKa axis strictly in T cells. It does not address the putative roleof DGKa in other SAP-deficient immune cells such as NK or NKTcells, which also likely contribute to the development of various XLP-1 manifestations. In addition, although LCMV infection of Sh2d1a−⁄−

mice is a widely used murine model of FIM, it does not fully recapi-tulate the pathogenesis of EBV infection in humans. Finally, thetranslation of these findings to the clinic will require the developmentand characterization of novel, clinical-grade DGKa-specific inhibitors.Nonetheless, our data clearly provide proof of concept that DGKamay be a novel drug target for treating XLP-1–associated FIM.

DISCUSSION

Our results demonstrate that inhibition of DGKa restores sensitivityto RICD in SAP-deficient T cells and reduces hyperinflammation inLCMV-infected Sh2d1a−⁄− mice. These data support the hypothesisthat in SAP-deficient T cells, persistent DGKa activity increasesDAG metabolism at the IS, thus reducing DAG signaling and RICDsensitivity, and underscore the role of SAP in modulating DGKa ac-tivity (15). In SAP-expressing T cells, further inhibition of DGKa onlymarginally influenced sensitivity to RICD in vitro and did not dampenthe LCMV-induced CD8+ T cell response of wild-type mice in vivo.

Our finding that inhibition of DGKa restores proper IS organiza-tion in SAP-deficient T cells indicates that SAP, through regulation of

www.Scienc

DGKa, controls DAG polarization at the IS, thus promoting F-actinpolymerization and MTOC orientation. Indeed, DGKa is recruited tothe peripheral SMAC (pSMAC), where it shapes the DAG gradientresponsible for the recruitment of novel PKC isoforms (e, h, andq), which control MTOC polarization and actin polymerization(Fig. 3) (23, 25). DGKa inhibition also partially rescued impairedcytolytic activity in SAP-deficient cytotoxic T lymphocytes (CTLs)(fig. S5), further highlighting the link between SAP-dependent in-hibition of DGKa and IS function (4). Together, these data indicatethat SAP-mediated negative regulation of DGKa controls IS struc-tural organization and function by regulating the TCR-inducedgradient of DAG.

Both DGKa and DGKz regulate TCR-induced DAG signaling(16, 17). Consistently, inhibition or overexpression of either isoformrendered T cells more or less sensitive to RICD, respectively,confirming that both isoforms regulate DAG signalling in this context(fig. S1). Accordingly, administration of exogenous DAG partially res-cued the defective RICD in SAP-deficient T cells (Fig. 2F). However,only DGKa is regulated by SAP and shapes the DAG gradient at theIS (15, 23). We speculate that DGKa, which colocalizes with F-actin atthe pSMAC, regulates the DAG gradient and F-actin polymerizationat the IS, whereas DGKz, which is more evenly distributed in the IS,metabolizes most of the DAG generated there, consistent with pro-posed models (23, 40). Notably, silencing of DGKd, which is highlyexpressed in T cells, did not affect RICD, underscoring the specificroles of DGKa and DGKz in regulating the DAG pool relevant forsignaling and RICD onset.

Our finding that inhibition of DGKa restored RasGRP1 and PKCqrecruitment to the IS in SAP-deficient T cells, and subsequent DAG-dependent induction of IL-2 and CD25, illuminates a biochemical linkbetween the SAP/DGKa axis, IS restoration, and downstreamsignaling events required for RICD (28–31). Indeed, activation ofRasGRP1 and PKCq was required to rescue RICD in SAP-deficientT cells upon DGKa blockade (Fig. 5, A and B, and fig. S2). DGKainhibition cannot recapitulate all SAP-dependent signaling functions,such as TCR-induced expression of FASLG or BCL2L11, genes previ-ously implicated in SAP-associated induction of RICD (5). This obser-vation suggests that rescue of DAG-mediated signaling activates otherproapoptotic pathways that partially compensate to boost RICD sen-sitivity. Here, we show that the induction of NUR77 and NOR1 isdefective in TCR-restimulated SAP-deficient T cells, and that the ex-pression of these genes is restored by inhibition of DGKa. NUR77 andNOR1 are orphan nuclear receptors known to trigger thymocyte ap-optosis during negative selection and mediate RICD (33, 41). Theproapoptotic activity of NUR77 and NOR1 depends on their phos-phorylation by RSK, an ERK1/2-dependent kinase, and subsequenttranslocation to the mitochondria to promote mitochondrial de-polarization and apoptosis (33, 34). We observed that ERK andRSK activity, as well as NUR77 and NOR1, were required for RICDrescue triggered by DGKa inhibition in XLP-1 T cells. These observa-tions provide a mechanistic connection between the rescue of DAGsignaling and the execution of RICD.

EBV-induced FIM is proposed to result from defective RICD ofCD8+ T cells and impaired cytotoxic elimination of EBV-infected Bcells by CD8+ T cells and NK cells. These events contribute to theexcessive accumulation of activated effector CD8+ T cells and life-threatening damage to the liver, the bone marrow, and other organs(2, 5). Using a murine model of FIM (37, 38), we showed that DGKa

eTranslationalMedicine.org 13 January 2016 Vol 8 Issue 321 321ra7 10




http://stm.sciencem

ag.org/D

ownloaded from

www.ScienceTranslationalMedicine.org 13 January

Fig. 8. In vivo DGKa inhibition re-duces the number but not inci-
dence of virus-specific CD8+
cytokine-producing cells of LCMVinfected Sh2d1a−⁄− mice. B6 WT(red symbols) and Sh2d1a−⁄− mice(blue symbols) were either un-infected (PBS, “P”) or infected withLCMV without (LCMV, “L”) or withR59022 treatment (LCMV + R59022,“L+R”). (A) Serum IFNg levelswereas-sayed on day 8 after infection byenzyme-linked immunosorbentassay(ELISA). Data are compiled from twoexperiments in which a total of 6 to10 mice in each cohort wereexamined. Error bars represent SEM.(B to D) Hematoxylin and eosin–stained liver sections from mice ineach group were analyzed for thenumber of inflammatory foci (B) andarea of the inflammatory infiltrate(C). For each sample, five randomfields were captured at ×20 magnifi-cation and scored. Histology of thelivers from representative mice ineach group under ×20 magnification(top row) is shown (D). Arrows pointto the inflammatory foci. Micrographsin the bottom row are the respectivecomputer analyzed images shown inthe top row. (E toG) Splenocytes (2 ×106) from PBS (P), LCMV-infected (L),and LCMV-infected mice withR59022 treatment (L+R)groupswereleft unstimulated or stimulated withgp33 peptide (0.4 ng/ml) in the pres-ence of monensin (1000 mg/ml) for5hours. Cellswere thenanalyzed for in-tracellular cytokine production anddegranulation. Representative flowcytometric (density) plots gated onCD8+ CD44+ splenocytes showingthe percentages of CD8+ IFNg+

(top), IFNg+ TNFa+ (middle), andIFNg+ CD107a+ (bottom) cells fromB6 WT and Sh2d1a−⁄− mice (E). Per-centages (F) andabsolutenumbers (G)of CD8+ IFNg+, IFNg+ TNFa+, and IFNg+

CD107a+ cells. Absolute numberswere calculated by multiplying thepercentageswith the respective abso-lute numbers of CD8+ gp33+ cells. Er-ror bars in (B), (C), (F), and (G) representSD. Asterisks denote statistical signifi-cance that was determined by two-way ANOVA with Sidak correction.

2016 Vol 8 Issue 321 321ra7 11



hD

ownloaded from

inhibition had no significant effect on reducing the activation status orfunction (for example, cytokine secretion or degranulation) of effectorCD8+ T cells in either wild-type or Sh2d1a−⁄− mice. These data suggestthat treatment with R59022 after LCMV infection does not impairinitial lymphocyte activation. However, this treatment did significantlydecrease the absolute number of activated CD8+ T cells in Sh2d1a−⁄−

mice, leading to fewer and smaller lymphocytic infiltrates within theliver and marked reductions in the level of IFNg in the serum. Theseresults suggest that RICD resistance is connected to aberrant DGKaactivity and serves as a key driver of virus-induced immunopathologyin Sh2d1a−⁄− mice. Remarkably, the apoptosis resistance of activated Tcells in LCMV-infected Sh2d1a−⁄− mice can be overcome via DGKainhibition, even when such inhibition is initiated well after infection isestablished. These data are relevant to the clinical setting, where patientsoften present with FIM days to weeks after primary EBV infection.

In conclusion, our findings underscore the importance of SAP-mediated DGKa inhibition in maintaining lymphocyte homeostasisby ensuring sufficient TCR-induced DAG signaling strength for apop-tosis. These data provide proof of principle that treatment with aDGKa inhibitor could serve as a novel, reasonable strategy to coun-teract pathological EBV-driven lymphohistiocytosis that occurs inEBV-infected XLP-1 patients by restoring the RICD sensitivity of ac-tivated CD8+ T cells.


ttp://stm.sciencem

ag.org/

MATERIALS AND METHODS

Study designThis was a preclinical study to (i) determine if DGKa inhibition couldrescue RICD in SAP-deficient T cells and (ii) assess the efficacy of aDGKa inhibitor in attenuating CD8+ T cell lymphocytosis and immu-nopathology in LCMV-infected SAP-deficient mice, a model of FIM.Although in vitro experiments utilizing XLP-1 patient T cells were of-ten constrained by limited sample availability, each RICD experimentwas performed with at least two separate XLP patients and differentcontrol donors (for example, Fig. 1, A and B). We also generatedrobust corroborating data using siRNA-mediated SAP knockdownin T cells from multiple human donors (n ≥ 3 experiments each).Once we established that DGKa blockade restored RICD sensitivityin SAP-deficient T cells, we focused on delineating the biochemicalmechanism that explains this phenomenon. For all in vitro data, thenumber of experiments (including technical replicates) is defined ineach figure legend. For in vivo experiments, numbers of mice are out-lined in each figure legend. All statistical analyses described below wereverified by consultation with an experienced biostatistician [C. Olsen,Uniformed Services University of the Health Sciences (USUHS)].

Cell culturePeripheral blood mononuclear cells (PBMCs) were isolated fromnormal controls or XLP-1 patients by Ficoll-Paque PLUS (GEHealthcare) density gradient centrifugation, washed, and resuspendedat 2 × 106 cell/ml in complete media (cRPMI): RPMI-GlutaMAX (LifeTechnologies) containing 10% heat-inactivated fetal calf serum (FCS)(Lonza), 2 mM glutamine, and penicillin and streptomycin (100 U/ml)(Life Technologies). T cells were activated with anti-CD3 (1 mg/ml)(clone UCHT1) and anti-CD28 (clone CD28.2) antibodies. After3 days, activated T cells were washed and cultured in cRPMI plus re-combinant human IL-2 (rhIL-2) (100 IU/ml) (PeproTech) at 1.2 × 106

www.Scienc

cells/ml for ≥7 days before apoptosis assays were conducted (mediachanged every 2 to 3 days).

Jurkat A3 cells were from American Type Culture Collection, and293FT from Life Technologies. Cells were cultured in RPMI or Dul-becco’s modified Eagle’s medium (Life Technologies) with 10% FCSand antibiotics/antimycotics (Sigma-Aldrich).

DGK inhibitors R59949 and R59022 (Sigma-Aldrich) were dis-solved in DMSO. All reagents and antibodies used are listed intable S1.

siRNA transfectionsPBMCs were transfected with 200 pmol of Stealth Select siRNA orStealth RNAi Negative Control Duplexes (Life Technologies). siRNAsequences are listed in table S2. Transient transfections were per-formed using Amaxa Nucleofector Kits for human T cells (Lonza)and the Amaxa Nucleofector II or 4D Systems (program T-20 orEI-115). Cells were cultured in IL-2 (100 IU/ml) for 4 days to allowtarget gene knockdown. Knockdown efficiency was periodically eval-uated by RT-PCR and Western blotting.

Conjugation and live-cell imaging of Jurkat T cellsJurkat T cells and Raji B cells were labeled, imaged, and analyzed asdescribed in the Supplementary Methods.

Immunofluorescence experiments with primary T cellsHuman T cells were stimulated with soluble anti-CD3 and anti-CD28(1 mg/ml) for at least 7 days and transfected with Amaxa NucleofectorKit for human T cells (Lonza) with control, SAP-specific, and/orDGKa-specific siRNA. After 72 hours, T cells were incubated withRaji B cells loaded with mixed SEE (staphylococcal enterotoxin E)and SEB (staphylococcal enterotoxin B) superantigens (1 mg/ml) for15 min, fixed, and stained for either PKCq or RasGRP1. For someexperiments, transfected T cells were pretreated with R59949 (10 mM,30 min, 37°C) or DMSO before conjugation.

CytofluorimetryTo examine RICD, activated T cells (105 cells per well) were plated intriplicate in 96-well round-bottom plates and treated with anti-CD3emAb (monoclonal antibody) OKT3 (1 to 100 ng/ml) in cRPMI + rhIL-2(100 IU/ml) for 24 hours. R59949 (5 to 10 mM), R59022 (5 to 10 mM),DAG (50 mM), U0126 (5 mM), FR180204 (10 mM), or Rottlerin (6 mM)inhibitors were added 30 min before restimulation. At 24 hours afterrestimulation, cells were stained with PI (1 mg/ml) and collected for30 s per sample on FACScan or Accuri C6 flow cytometers (BD). Celldeath was analyzed with CellQuest/CFlow software (BD) or Flowingsoftware (Turku Bioimaging) as percentage of cell loss = (1 − [numberof viable cells (treated)/number of viable cells (untreated)]) × 100 (5).

For AnnexinV assays, ~1 × 106 cells were treated with OKT3(10 ng/ml) as above. Cells were stained 6 to 12 hours later withAnnexinV–phycoerythrin (BioLegend) and analyzed on Accuri C6.

To evaluate CD25 expression, 1 × 106 cells were stimulated withOKT3 (100 ng/ml) for 24 hours and fixed and stained with anti-CD25plus anti-mouse Alexa Flour 488. Stained cells were collected on aFACSCalibur.

Western blottingLymphocytes (1 × 106 to 10 × 106 cells) were stimulated, lysed, andsubjected to SDS–polyacrylamide gel electrophoresis and immunoblotting





http://stm.sciencem

ag.org/D

ownloaded from

as described (5, 15). Immunoblot images were acquired and quantifiedusing a Versadoc Model 4000 Imaging System (Bio-Rad) or ImageJsoftware (for film). Spot densitometry analyses are summarized intable S4.

Quantitative RT-PCRActivated lymphocytes (30 × 106 cell/ml) were stimulated in cRPMIwith OKT3 (10 mg/ml) for 4 hours. R59949 (5 mM) was added 30 minbefore restimulation. Cells were washed with cold PBS, and mRNAwas extracted using a ChargeSwitch Total RNA Cell Kit (Life Tech-nologies). RNA was reverse-transcribed using High-Capacity cDNAReverse Transcription Kits (Life Technologies), and cDNA targetswere quantified by RT-PCR (C1000 Thermal Cycler CFX96, Bio-Rad) using TaqMan gene expression assays (see table S3), with GUSBas the housekeeping control (Life Technologies).

Mice and in vivo experimentsSh2d1a−/− mice were as described (38). C57BL/6 (B6) mice were pur-chased from Jackson Laboratories. To establish LCMV infection, micereceived 2 × 105 plaque-forming units of LCMV Armstrong by intra-peritoneal injection on day 0, and experiments were carried out untilday +8 after infection. Beginning at day 4, mice were given twice dailyintraperitoneal injections of R59022 at a dose of 2 mg/kg body weight,dissolved in DMSO. Mice in all groups were sex- and age-matched.Stimulation of mouse splenocytes in vitro, assessment of liver his-tology, and quantification of viral titers were performed as de-scribed in the Supplementary Methods.

Statistical analysisEvaluation of in vitro assays across multiple treatments (RICD, RT-PCR, and ELISA), and in vivo experiments was analyzed using two-way ANOVA (a = 0.05) with Sidak’s multiple comparisons correctionusing GraphPad PRISM software. When comparing two groups (RT-PCR, AnnexinV+ cells), a two-tailed paired Student’s t test was per-formed in Microsoft Excel. Error bars are described in the figurelegends as ± SEM or ± SD where appropriate. Asterisks denote sig-nificance in all experiments; P values are included in table S5.

Study approvalBlood samples were obtained with informed consent underprotocols approved by the respective Institutional Review Boards(Cincinnati Children’s Hospital Medical Center, National Instituteof Allergy and Infectious Diseases, and University of Piemonte Orien-tale). Experimental procedures on animals were approved by the In-stitutional Animal Care and Use Committee at The Children’sHospital of Philadelphia and St. Jude Children’s Research Hospital.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/321/321ra7/DC1MethodsFig. S1. Activity of DGKa and DGKz contributes to RICD resistance in T cells.Fig. S2. DGKa silencing restores RICD in SAP-deficient cells through PKCq- and Ras-mediatedsignaling pathways.Fig. S3. DGKa blockade fails to rescue TCR-induced up-regulation of proapoptotic mediatorsFASL and BIM in SAP-deficient cells.Fig. S4. Major steps of the automated segmentation and fluorescence quantificationalgorithm.

www.Scienc

Fig. S5. DGKa inhibition enhances SAP-deficient CD8+ T cell cytotoxicity against autologous B celltargets.Fig. S6. R59022 DGKa inhibitor does not impair viral clearance in the livers and spleens ofLCMV infected Sh2d1a−⁄− mice.Fig. S7. Gating strategies used in Figs. 7 and 8.Table S1. Reagents.Table S2. siRNA sequences.Table S3. TaqMan gene expression arrays.Table S4. Spot densitometry analysis for Western blotting.Table S5. Statistical analyses.Unmodified Western blot images.Source data (excel).

REFERENCES AND NOTES

1. K. E. Nichols, D. P. Harkin, S. Levitz, M. Krainer, K. A. Kolquist, C. Genovese, A. Bernard,M. Ferguson, L. Zuo, E. Snyder, A. J. Buckler, C. Wise, J. Ashley, M. Lovett, M. B. Valentine,A. T. Look, W. Gerald, D. E. Housman, D. A. Haber, Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome. Proc. Natl. Acad. Sci. U.S.A. 95,13765–13770 (1998).

2. S. G. Tangye, XLP: Clinical features and molecular etiology due to mutations in SH2D1Aencoding SAP. J. Clin. Immunol. 34, 772–779 (2014).

3. C. Booth, K. C. Gilmour, P. Veys, A. R. Gennery, M. A. Slatter, H. Chapel, P. T. Heath, C. G. Steward,O. Smith, A. O’Meara, H. Kerrigan, N. Mahlaoui, M. Cavazzana-Calvo, A. Fischer, D. Moshous,S. Blanche, J. Pachlopnik Schmid, S. Latour, G. de Saint-Basile, M. Albert, G. Notheis, N. Rieber,B. Strahm, H. Ritterbusch, A. Lankester, N. G. Hartwig, I. Meyts, A. Plebani, A. Soresina,A. Finocchi, C. Pignata, E. Cirillo, S. Bonanomi, C. Peters, K. Kalwak, S. Pasic, P. Sedlacek, J. Jazbec,H. Kanegane, K. E. Nichols, I. C. Hanson, N. Kapoor, E. Haddad, M. Cowan, S. Choo, J. Smart,P. D. Arkwright, H. B. Gaspar, X-linked lymphoproliferative disease due to SAP/SH2D1Adeficiency: A multicenter study on the manifestations, management and outcome ofthe disease. Blood 117, 53–62 (2011).

4. F. Zhao, J. L. Cannons, M. Dutta, G. M. Griffiths, P. L. Schwartzberg, Positive and negativesignaling through SLAM receptors regulate synapse organization and thresholds of cytolysis.Immunity 36, 1003–1016 (2012).

5. A. L. Snow, R. A. Marsh, S. M. Krummey, P. Roehrs, L. R. Young, K. Zhang, J. van Hoff, D. Dhar,K. E. Nichols, A. H. Filipovich, H. C. Su, J. J. Bleesing, M. J. Lenardo, Restimulation-inducedapoptosis of T cells is impaired in patients with X-linked lymphoproliferative disease causedby SAP deficiency. J. Clin. Invest. 119, 2976–2989 (2009).

6. A. L. Snow, P. Pandiyan, L. Zheng, S. M. Krummey, M. J. Lenardo, The power and the promise ofrestimulation-induced cell death in human immune diseases. Immunol. Rev. 236, 68–82 (2010).

7. G. Chen, A. K. Tai, M. Lin, F. Chang, C. Terhorst, B. T. Huber, Increased proliferation of CD8+

T cells in SAP-deficient mice is associated with impaired activation-induced cell death. Eur.J. Immunol. 37, 663–674 (2007).

8. F. Poy, M. B. Yaffe, J. Sayos, K. Saxena, M. Morra, J. Sumegi, L. C. Cantley, C. Terhorst, M. J. Eck,Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended,phosphotyrosine-independent sequence recognition. Mol. Cell 4, 555–561 (1999).

9. L. M. Shlapatska, S. V. Mikhalap, A. G. Berdova, O. M. Zelensky, T. J. Yun, K. E. Nichols,E. A. Clark, S. P. Sidorenko, CD150 association with either the SH2-containing inositolphosphatase or the SH2-containing protein tyrosine phosphatase is regulated by theadaptor protein SH2D1A. J. Immunol. 166, 5480–5487 (2001).

10. B. Chan, A. Lanyi, H. K. Song, J. Griesbach, M. Simarro-Grande, F. Poy, D. Howie, J. Sumegi,C. Terhorst, M. J. Eck, SAP couples Fyn to SLAM immune receptors. Nat. Cell Biol. 5, 155–160(2003).

11. G. Katz, S. M. Krummey, S. E. Larsen, J. R. Stinson, A. L. Snow, SAP facilitates recruitmentand activation of LCK at NTB-A receptors during restimulation-induced cell death. J. Immunol.192, 4202–4209 (2014).

12. J. L. Cannons, L. J. Yu, B. Hill, L. A. Mijares, D. Dombroski, K. E. Nichols, A. Antonellis, G. A. Koretzky,K. Gardner, P. L. Schwartzberg, SAP regulates TH2 differentiation and PKC-q-mediated activationof NF-kB1. Immunity 21, 693–706 (2004).

13. S. Nunez-Cruz, W. C. J. Yeo, J. Rothman, P. Ojha, H. Bassiri, M. Juntilla, D. Davidson, A. Veillette,G. A. Koretzky, K. E. Nichols, Differential requirement for the SAP-Fyn interaction during NKT cell development and function. J. Immunol. 181, 2311–2320 (2008).

14. H. Qi, J. L. Cannons, F. Klauschen, P. L. Schwartzberg, R. N. Germain, SAP-controlled T–B cellinteractions underlie germinal centre formation. Nature 455, 764–769 (2008).

15. G. Baldanzi, A. Pighini, V. Bettio, E. Rainero, S. Traini, F. Chianale, P. E. Porporato, N. Filigheddu,R. Mesturini, S. Song, T. Schweighoffer, L. Patrussi, C. T. Baldari, X.-P. Zhong, W. J. van Blitterswijk,F. Sinigaglia, K. E. Nichols, I. Rubio, O. Parolini, A. Graziani, SAP-mediated inhibition of diacyl-glycerol kinase a regulates TCR-induced diacylglycerol signaling. J. Immunol. 187, 5941–5951(2011).





http://stm.sciencem

ag.org/D

ownloaded from

16. B. A. Olenchock, R. Guo, J. H. Carpenter, M. Jordan, M. K. Topham, G. A. Koretzky, X.-P. Zhong,Disruption of diacylglycerol metabolism impairs the induction of T cell anergy. Nat. Immunol.7, 1174–1181 (2006).

17. X.-P. Zhong, E. A. Hainey, B. A. Olenchock, M. S. Jordan, J. S. Maltzman, K. E. Nichols, H. Shen,G. A. Koretzky, Enhanced T cell responses due to diacylglycerol kinase z deficiency. Nat.Immunol. 4, 882–890 (2003).

18. J. She, K. Matsui, C. Terhorst, S.-T. Ju, Activation-induced apoptosis of mature T cells isdependent upon the level of surface TCR but not on the presence of the CD3 z ITAM.Int. Immunol. 10, 1733–1740 (1998).

19. M. Sato, K. Liu, S. Sasaki, N. Kunii, H. Sakai, H. Mizuno, H. Saga, F. Sakane, Evaluations of theselectivities of the diacylglycerol kinase inhibitors r59022 and r59949 among diacylglycerolkinase isozymes using a new non-radioactive assay method. Pharmacology 92, 99–107(2013).

20. M. Issandou, F. Bayard, J.-M.Darbon, InhibitionofMCF-7 cell growthby12-O-tetradecanoylphorbol-13-acetate and 1,2-dioctanoyl-sn-glycerol: Distinct effects on protein kinase C activity. Cancer Res.48, 6943–6950 (1988).

21. S. Carrasco, I. Merida, Diacylglycerol-dependent binding recruits PKCq and RasGRP1 C1domains to specific subcellular localizations in living T lymphocytes. Mol. Biol. Cell 15,2932–2942 (2004).

22. M. Spitaler, E. Emslie, C. D. Wood, D. Cantrell, Diacylglycerol and protein kinase D localizationduring T lymphocyte activation. Immunity 24, 535–546 (2006).

23. A. Chauveau, A. Le Floc’h, N. S. Bantilan, G. A. Koretzky, M. Huse, Diacylglycerol kinase aestablishes T cell polarity by shaping diacylglycerol accumulation at the immunologicalsynapse. Sci. Signal. 7, ra82 (2014).

24. E. J. Quann, E. Merino, T. Furuta, M. Huse, Localized diacylglycerol drives the polarization ofthe microtubule-organizing center in T cells. Nat. Immunol. 10, 627–635 (2009).

25. E. J. Quann, X. Liu, G. Altan-Bonnet, M. Huse, A cascade of protein kinase C isozymes pro-motes cytoskeletal polarization in T cells. Nat. Immunol. 12, 647–654 (2011).

26. J. O. Ebinu, S. L. Stang, C. Teixeira, D. A. Bottorff, J. Hooton, P.M. Blumberg,M. Barry, R. C. Bleakley,H. L. Ostergaard, J. C. Stone, RasGRP links T-cell receptor signaling to Ras. Blood 95, 3199–3203(2000).

27. A. Altman, M. Villalba, Protein kinase C-q (PKCq): It’s all about location, location, location.Immunol. Rev. 192, 53–63 (2003).

28. S. Manicassamy, Z. Sun, The critical role of protein kinase C-q in Fas/Fas ligand-mediatedapoptosis. J. Immunol. 178, 312–319 (2007).

29. K. Layer, G. Lin, A. Nencioni, W. Hu, A. Schmucker, A. N. Antov, X. Li, S. Takamatsu, T. Chevassut,N. A. Dower, S. L. Stang, D. Beier, J. Buhlmann, R. T. Bronson, K. B. Elkon, J. C. Stone, L. Van Parijs,B. Lim, RETRACTED: Autoimmunity as the consequence of a spontaneousmutation in Rasgrp1.Immunity 19, 243–255 (2003).

30. M. I. Chuck, M. Zhu, S. Shen, W. Zhang, The role of the LAT–PLC-g1 interaction in T regulatorycell function. J. Immunol. 184, 2476–2486 (2010).

31. C. Springael, S. Thomas, S. Rahmouni, A. Vandamme, M. Goldman, F. Willems, O. Vosters,Rottlerin inhibits human T cell responses. Biochem. Pharmacol. 73, 515–525 (2007).

32. M. J. Lenardo, Interleukin-2 programs mouse ab T lymphocytes for apoptosis. Nature 353,858–861 (1991).

33. L. E.-C. Cheng, F. K.-M. Chan, D. Cado, A. Winoto, Functional redundancy of the Nur77 andNor-1 orphan steroid receptors in T-cell apoptosis. EMBO J. 16, 1865–1875 (1997).

34. A. Wang, J. Rud, C. M. Olson Jr., J. Anguita, B. A. Osborne, Phosphorylation of Nur77 by theMEK-ERK-RSK cascade induces mitochondrial translocation and apoptosis in T cells. J. Immunol.183, 3268–3277 (2009).

www.Scienc

35. J. A. Smith, C. E. Poteet-Smith, Y. Xu, T. M. Errington, S. M. Hecht, D. A. Lannigan, Identi-fication of the first specific inhibitor of p90 ribosomal S6 kinase (RSK) reveals an un-expected role for RSK in cancer cell proliferation. Cancer Res. 65, 1027–1034 (2005).

36. A. H. Filipovich, K. Zhang, A. L. Snow, R. A. Marsh, X-linked lymphoproliferative syndromes:Brothers or distant cousins? Blood 116, 3398–3408 (2010).

37. C. Wu, K. B. Nguyen, G. C. Pien, N. Wang, C. Gullo, D. Howie, M. R. Sosa, M. J. Edwards,P. Borrow, A. R. Satoskar, A. H. Sharpe, C. A. Biron, C. Terhorst, SAP controls T cell responses tovirus and terminal differentiation of TH2 cells. Nat. Immunol. 2, 410–414 (2001).

38. M. J. Czar, E. N. Kersh, L. A. Mijares, G. Lanier, J. Lewis, G. Yap, A. Chen, A. Sher, C. S. Duckett,R. Ahmed, P. L. Schwartzberg, Altered lymphocyte responses and cytokine production inmice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP. Proc.Natl. Acad. Sci. U.S.A. 98, 7449–7454 (2001).

39. C. L. Dominguez, D. H. Floyd, A. Xiao, G. R.Mullins, B. A. Kefas,W. Xin,M. N. Yacur, R. Abounader,J. K. Lee, G. M.Wilson, T. E. Harris, B. W. Purow, Diacylglycerol kinase a is a critical signaling nodeand novel therapeutic target in glioblastoma and other cancers. Cancer Discov. 3, 782–797 (2013).

40. S. I. Gharbi, E. Rincón, A. Avila-Flores, P. Torres-Ayuso,M.Almena,M.A. Cobos, J. P.Albar, I.Mérida,Diacylglycerol kinase z controls diacylglycerol metabolism at the immunological synapse.Mol. Biol. Cell 22, 4406–4414 (2011).

41. A. E. Moran, K. L. Holzapfel, Y. Xing, N. R. Cunningham, J. S. Maltzman, J. Punt, K. A. Hogquist,T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novelfluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011).

Acknowledgments: We thank the patients and their families for participating in our study.We alsothank biostatistician C. Olsen (USUHS) for consultation on statistical analysis, J. Wherry for providingLCMV and assistance with performing LCMV infections, D. Cantrell for supplying the PKCq-CRDconstruct, R. Wedlich-Soldner for supplying the LifeAct-GFP construct, and J. Downward for supply-ing GFP-tubulin. Funding: K.N. was supported by the XLP Research Trust and the Sean Fischel Fundfor HLH (hemophagocytic lymphohistiocytosis) research. A.L.S. and S.E.L. were supported by grantsfrom the NIH (1R01GM105821), XLP Research Trust, and USUHS. A.G., E.R., and V.M. were supportedby grants from Telethon (GGP10034 and GGP13254) and AIRC (Associazione Italiana per la Ricerca sulCancro) (IG13524 and IG5392). G.B. is supported by the University of Piemonte Orientale (YoungInvestigators). V.M. was supported by a grant from the Compagnia di San Paolo. R.D. was supportedby an NIH K22 grant (1 K22 CA188149-01). Author contributions: E.R., V.M., S.E.L., A.L.S., and G.B.designed and carried out in vitro experiments; R.D., K.V., P.T., and K.E.N. designed and carried outin vivo experiments; S.E.L. and A.L.S. designed and carried out experiments on XLP-1 patient lympho-cytes; V.M., I.R., and C.B. designed and performed imaging in Jurkat cells; L.P. and C.T.B. performedimaging on primary lymphocytes; C.W. performed imaging on mouse lymphocytes; S.M.K. and P.L.S.performed cytotoxicity assays; G.B., A.G., K.E.N., and A.L.S. designed the project and some of theexperiments, supervised research, and wrote the paper. Competing interests: The authors declarethat they have no competing interests.

Submitted 4 August 2015Accepted 3 December 2015Published 13 January 201610.1126/scitranslmed.aad1565

Citation: E. Ruffo, V. Malacarne, S. E. Larsen, R. Das, L. Patrussi, C. Wülfing, C. Biskup,S. M. Kapnick, K. Verbist, P. Tedrick, P. L. Schwartzberg, C. T. Baldari, I. Rubio, K. E. Nichols,A. L. Snow, G. Baldanzi, A. Graziani, Inhibition of diacylglycerol kinase a restoresrestimulation-induced cell death and reduces immunopathology in XLP-1. Sci. Transl. Med.8, 321ra7 (2016).



reduces immunopathology in XLP-1 restores restimulation-induced cell death andαInhibition of diacylglycerol kinase

Nichols, Andrew L. Snow, Gianluca Baldanzi and Andrea GrazianiSenta M. Kapnick, Katherine Verbist, Paige Tedrick, Pamela L. Schwartzberg, Cosima T. Baldari, Ignacio Rubio, Kim E. Elisa Ruffo, Valeria Malacarne, Sasha E. Larsen, Rupali Das, Laura Patrussi, Christoph Wülfing, Christoph Biskup,

DOI: 10.1126/scitranslmed.aad1565, 321ra7321ra7.8Sci Transl Med

XLP-1 patients. may prevent viral-induced immunopathology inαexpansion. If these data hold true in humans, targeting DGK

) in SAP-deficient T cells restores restimulation-induced cell death, preventing this excessα (DGKαkinase report that down-regulating diacylglycerolet al.expansion of activated T cell after viral infection. Now, Ruffo

is associated with−−associated protein]−adaptor protein SAP [signaling lymphocytic activation molecule (SLAM) an immunodeficiency caused by defects in the T cell receptor−−X-linked lymphoproliferative disease (XLP-1)

Individuals with deficient immune systems may also paradoxically experience hyperimmune side effects.SAPping immunopathology

ARTICLE TOOLS http://stm.sciencemag.org/content/8/321/321ra7

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2016/01/11/8.321.321ra7.DC1

CONTENTRELATED

http://stke.sciencemag.org/content/sigtrans/10/469/eaah3737.fullhttp://stke.sciencemag.org/content/sigtrans/9/459/ra127.fullhttp://stm.sciencemag.org/content/scitransmed/8/328/328ra30.fullhttp://stm.sciencemag.org/content/scitransmed/3/97/97ra79.fullhttp://stm.sciencemag.org/content/scitransmed/6/227/227ra33.full

REFERENCES

http://stm.sciencemag.org/content/8/321/321ra7#BIBLThis article cites 41 articles, 20 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.Science Translational Medicinetitle licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. TheScience, 1200 New York Avenue NW, Washington, DC 20005. 2017 © The Authors, some rights reserved; exclusive

(ISSN 1946-6242) is published by the American Association for the Advancement ofScience Translational Medicine


http://stm.sciencem

ag.org/D

ownloaded from

http://stm.sciencemag.org/content/8/321/321ra7

http://stm.sciencemag.org/content/suppl/2016/01/11/8.321.321ra7.DC1

http://stm.sciencemag.org/content/scitransmed/6/227/227ra33.full



http://stke.sciencemag.org/content/sigtrans/9/459/ra127.full

http://stke.sciencemag.org/content/sigtrans/10/469/eaah3737.full

http://stm.sciencemag.org/content/8/321/321ra7#BIBL

http://www.sciencemag.org/help/reprints-and-permissions

http://www.sciencemag.org/about/terms-service


inhibition of diacylglycerol kinase a restores...

Documents