SC I ENCE S I GNAL ING | R E S EARCH ART I C L E

GPCR S IGNAL ING

1Department of Biochemistry, Duke University, Durham, NC 27710, USA. 2Departmentof Medicine, Duke University, Durham, NC 27710, USA. 3Department of Dermatology,Duke University, Durham, NC 27710, USA. 4Primity Bio, Fremont, CA 94538, USA. 5De-partment of Immunology, Duke University, Durham, NC 27710, USA.*Corresponding author. Email: sudarshan.rajagopal@dm.duke.edu

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Biased agonists of the chemokine receptor CXCR3differentially control chemotaxis and inflammationJeffrey S. Smith1,2, Lowell T. Nicholson2, Jutamas Suwanpradid3, Rachel A. Glenn1,Nicole M. Knape1, Priya Alagesan1, Jaimee N. Gundry1, Thomas S. Wehrman4,Amber Reck Atwater3, Michael D. Gunn2,5, Amanda S. MacLeod3,5, Sudarshan Rajagopal1,2*

The chemokine receptor CXCR3 plays a central role in inflammation bymediating effector/memory T cell migration invarious diseases; however, drugs targeting CXCR3 and other chemokine receptors are largely ineffective in treatinginflammation. Chemokines, the endogenous peptide ligands of chemokine receptors, can exhibit so-called biasedagonism by selectively activating either G protein– or b-arrestin–mediated signaling after receptor binding. Biasedagonists might be used as more targeted therapeutics to differentially regulate physiological responses, such as im-mune cellmigration. To testwhether CXCR3-mediatedphysiological responses couldbe segregatedbyGprotein– andb-arrestin–mediated signaling, we identified and characterized small-molecule biased agonists of the receptor. In amouse model of T cell–mediated allergic contact hypersensitivity (CHS), topical application of a b-arrestin–biased,but not a G protein–biased, agonist potentiated inflammation. T cell recruitment was increased by the b-arrestin–biased agonist, and biopsies of patients with allergic CHS demonstrated coexpression of CXCR3 and b-arrestin inT cells. In mouse and human T cells, the b-arrestin–biased agonist was the most efficient at stimulating chemotaxis.Analysis of phosphorylated proteins in human lymphocytes showed that b-arrestin–biased signaling activated thekinase Akt, which promoted T cell migration. This study demonstrates that biased agonists of CXCR3 produce dis-tinct physiological effects, suggesting discrete roles for different endogenous CXCR3 ligands and providing evi-dence that biased signaling can affect the clinical utility of drugs targeting CXCR3 and other chemokine receptors.

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INTRODUCTIONThe chemokine receptor CXCR3 is a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor (GPCR) that is expressedprimarily on activated effector/memory T cells and plays an importantrole in atherosclerosis, cancer, and inflammatory disease. Activationof CXCR3 by chemokines causes the migration of activated T cells ina concentration-dependent manner. Increased tissue concentrations ofactivated T cells initiate inflammatory responses, and the ability tomodulate T cell chemotaxis would likely be therapeutically useful inmany disease processes. Despite the importance of the more than 20chemokine receptors in various disease states, there are currently onlythree U.S. Food and Drug Administration (FDA)–approved drugs thattarget chemokine receptor family members (1–3). This is somewhatsurprising, because GPCRs constitute the plurality of FDA-approvedmedications, with >30% of therapeutics targeting this class of receptors(4). The difficulty in successfully targeting chemokine receptors wasoriginally thought to be due to redundancy across themultiple chemokineligands and chemokine receptors that bind to one another (5). However,this presumed redundancy appears to be more granular than was initiallyappreciated. Similar to most other chemokine receptors, CXCR3 signalsthrough both Gai family G proteins and b-arrestins. GPCR signalingdeviates at critical junctions, including G protein and b-arrestins, whichsignal through distinct intracellular pathways. For example, b-arrestinspromote interactions with kinases independently from their interactionswith G proteins to induce downstream signaling (6).

It is now appreciated that many chemokines that bind to the samechemokine receptor can selectively activate such distinct signalingpathways downstream of the receptor (7–9). This phenomenon is re-

ferred to as biased agonism (10, 11). Biased ligands at other GPCRs,such as the m-opioid receptor (MOR) (12, 13), the k-opioid receptor(14), and the type 1 angiotensin II receptor (AT1R) (15), have shownpromise in improving efficacy while reducing side effects through dif-ferential activationofGprotein– andb-arrestin–mediated signalingpath-ways (16). Animal and human studies suggest that G protein–mediatedsignaling by the MOR primarily mediates analgesic efficacy, whereasb-arrestin–mediated signaling causes many adverse effects, such as res-piratory depression, constipation, tolerance, and dependence (12, 13).Furthermore, relative degrees of G protein and b-arrestin bias can predictsafer m-opioid analgesics (17). At the AT1R, biased and balanced AT1Ragonists have distinct physiologic responses: Gaq-dependent signalingmediates vasoconstriction and cardiac hypertrophy, whereas b-arrestin–mediated signaling activates antiapoptotic signals and promotes cal-cium sensitization (15). At chemokine receptors, both pertussis toxin(PTX)–sensitive G protein signaling and b-arrestin–mediated signalingcontribute to chemotaxis (18–23). However, chemokines with distinctG protein– and b-arrestin–biased signaling properties often inducechemotaxis to similar degrees (9). The relative contribution of b-arrestin–or G protein–mediated signaling to chemotaxis and inflammation isunclear, and it is experimentally challenging to discern the physiologicalrelevance of biased signaling with peptide agonists in many assays be-cause of the high molecular weight and short half-life of chemokinesrelative to those of small molecules. It is unknown whether endogenousor synthetic chemokine receptor ligands that preferentially targetGpro-tein or b-arrestin pathways would result in different physiological out-comes in models of disease and inflammation. If such differences inselective pathway activation result in distinct physiological outcomes,then biased agonism could be used to develop new insights into chemo-kine biology that could be harnessed to increase the therapeutic utility ofdrugs targeting chemokine receptorswhile reducing on-target side effects.

Given its prominent role in effector T cell function, we focused onbiased signaling at CXCR3-A, the dominantly expressed CXCR3 isoform

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on T cells in humans andmice. CXCR3 signaling is implicated in variousdisease processes, including cancer (24), atherosclerosis (25), vitiligo(26, 27), and allergic contact dermatitis (ACD) (28). The chemokinesCXCL9, CXCL10, and CXCL11, the endogenous ligands of CXCR3,stimulate the chemotaxis of CXCR3-expressing T cells (29). Thesechemokines are secreted in response to interferon-g (IFN-g) by variouscell types, including monocytes, endothelial cells, keratinocytes, andfibroblasts.Wepreviously demonstrated that the three ligands ofCXCR3act as biased agonists, displaying different efficacies at signaling throughG proteins and b-arrestins and stimulating receptor internalization(8, 30), suggesting that biased agonists targeting CXCR3may have dis-tinct physiological effects. Given the importance of CXCR3 signaling indisease, we tested whether small-molecule biased agonists of CXCR3could elicit biochemically and physiologically distinct effects in miceand humans.

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RESULTSScreening enabled identification of biased agonists ofCXCR3 with distinct signaling profilesTo explore the physiological relevance of biased signaling at CXCR3, wescreened a panel of publicly available small-molecule CXCR3 ligandsand identified two agonists with biased signaling properties: VUF10661(31) and VUF11418 (Fig. 1, A and B). These two small molecules werepreviously shown to have similar affinities for CXCR3 (table S1) (32).Wefirst confirmed previous reports that both compounds signal throughGaiG proteins (Fig. 1A) (33, 34), displaying similar potencies. Because the Gprotein–dependent signal did not saturate at high concentrations ofagonist, differences in the efficacies of both compounds to activate Gaisignaling could not be completely assessed. However, VUF10661increased b-arrestin2 recruitment to CXCR3 compared to VUF11418(Fig. 1B). These small-molecule CXCR3 agonists also displayed dif-ferential abilities to recruit b-arrestin1, recruit GPCR kinases (GRKs),and stimulate CXCR3 internalization (fig. S1, A to G), consistent withtheir biased signaling properties.

Because bias is a relative measurement, an appropriate referencemust be used. Here, we used the endogenous agonist CXCL11 as areference, which is a full agonist of CXCR3 for both G protein– andb-arrestin–mediated pathways (30, 35). As expected, CXCL11 displayedincreased potency for CXCR3 relative to the small-molecule agonists(fig. S1, H and I). Because it exhibited increased efficacy in b-arrestinrecruitment but a similar degree of efficacy in G protein activation,VUF10661 was considered to be a b-arrestin–biased agonist relativeto VUF11418 and CXCL11. Similarly, the preserved G protein activa-tion property of VUF11418 together with its reduced ability to induceb-arrestin recruitment demonstrated that it is relativelyGprotein biasedcompared to VUF10661 and CXCL11 (Fig. 1, A and B, and fig. S1, Hand I). Consistent with its ability to stimulate increased b-arrestin re-cruitment, VUF10661 also induced a distinct b-arrestin2 conformation,as assessed with an intramolecular biosensor (Fig. 1C), increased theb-arrestin–dependent internalization of CXCR3 (Fig. 1D), and pro-moted serum response element (SRE) response factor–mediatedtranscription [whichwas previously correlatedwith increased b-arrestinsignaling (30)] relative to VUF11418 (Fig. 1E).

A b-arrestin–biased agonist of CXCR3 potentiatesT cell–mediated inflammation and chemotaxisAfter confirming that ligand bias was conserved at the murine CXCR3receptor, which has 86% sequence homology to humanCXCR3 (fig. S2,

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Fig. 1. VUF10661 and VUF11418 are biased agonists of CXCR3. (A) Transfectedhuman embryonic kidney (HEK) 293T “DG six” cells expressing CXCR3 and the interro-gatedGa subunit were analyzed for Gai activation by the transforming growth factor–a(TGF-a)–shedding assay as described in Materials and Methods. The cells were treatedfor 1 hour with vehicle or the indicated concentrations of G protein–biased CXCR3agonist VUF11418 or b-arrestin–biased CXCR3 agonist VUF10661. The extent of G pro-tein signal is expressed as a percentage of that induced by the CXCR3 agonist CXCL11.Data aremeans ± SEM of eight or nine experiments. n.s., not significant. (B) TransfectedHEK 293T cells expressing CXCR3-Rluc and b-arrestin2–yellow fluorescent protein (YFP)were analyzed for b-arrestin2 recruitment by bioluminescence resonance energy trans-fer (BRET) as described in Materials and Methods. The cells were treated for 5 min withvehicle or the indicated concentrations of VUF11418 or VUF10661. The extent ofb-arrestin2 recruitment is expressed as a percentage of that induced by the CXCR3agonistCXCL11,whichwasusedas a control. Data aremeans±SEMof four experiments.(C) Top: Schematic of the detection of a conformational change in b-arrestin by mea-suring BRET between a nanoLuc donor and a YFP acceptor. Bottom: Transfected HEK293T cells expressing CXCR3 and the b-arrestin biosensor were analyzed by BRET todetect changes in the conformation of b-arrestin. The cells were treated for 5 min withvehicle (Veh), 1 mMVUF11418 (11418), 1 mMVUF10661 (10661), or 250 nM CXCL11 as apositive control, and the net BRET ratio was calculated by subtracting the vehicle signalfrom the drug signal. Data are from 15 to 26 wells from four independent experiments.(D) U2OS cells stably expressing b-arrestin2–dependent internalization assay compo-nents (enzyme fragments tagged to b-arrestin2 and endosomes) and transientlyexpressing CXCR3 were analyzed for CXCR3 internalization as described in Materialsand Methods. The cells were treated for 90 min with vehicle or the indicated con-centrations of VUF11418 or VUF10661. As a control, the cells were treatedwith CXCL11(1 mM). The percentage of CXCR3 internalizationwas calculated relative to that inducedby CXCL11. Data are means ± SEM of three to five experiments. (E) Transfected HEK293T cells expressing CXCR3 and an SRE reporter were analyzed by luminescence tomeasure changes in SRE transcriptional activation. The cells were treated for 5 hourswith vehicle, 10 mMVUF11418, 10 mMVUF10661, 10% fetal bovine serum (FBS), or 1 mMCXCL11. The SRE signal was normalized to that of the vehicle control. Data aremeans ±SEM of five to eight experiments. For (A), (B), and (D) data were analyzed by two-wayanalysis of variance (ANOVA). *P < 0.05 when comparing VUF10661 to VUF11418. For(C) and (E) data were analyzed by one-way ANOVA and Tukey post hoc analysis. *P <0.05 when comparing VUF10661 to VUF11418; #P < 0.05 when compared to vehicle.

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A to D), we next studied how these smallmolecules affected a mouse model of al-lergic contact hypersensitivity (CHS),an inflammatory condition that is de-pendent on effector memory T cells(Fig. 2A).TheCHS-delayed type IVhyper-sensitivity reaction enables an assessmentof T cell–mediated inflammation and therecruitment of effector T cells to the sen-sitizer dinitrofluorobenzene (DNFB).Topical applicationof theb-arrestin–biasedagonist, but not the G protein–biasedagonist, potentiated the inflammatoryresponse (Fig. 2B). The potentiation in-duced by VUF10661 was not observed ineither b-arrestin2 knockout (KO) mice(Fig. 2C) or CXCR3 KO mice (fig. S3),consistent with the effects of VUF10661requiring both CXCR3 and b-arrestin2,the predominant arrestin isoform inT cells(23). Furthermore, the smallmolecules didnot cause inflammation in the absence ofDNFB-induced T cell allergy and there-fore did not act as irritants or haptens(Fig. 2D).

Given the established role of CXCR3in T cell chemotaxis (2) and the increasedabundance of CXCR3 on CD8+ T cellsrelative to that on CD4+ T cells in ACD(36), we hypothesized that the increasedinflammation induced by the b-arrestin–biased agonist was due to increased effectormemory CD8+ T cell chemotaxis com-pared to that induced by the G protein–biased agonist. Supporting this, we foundthat the b-arrestin–biased agonist inducedmore chemotaxis of effector memoryT cells from wild-type (WT) mice in aTranswell migration assay than did theG protein–biased agonist (Fig. 2E). Thisdifference in migration was not observedfor activated CD8+ T cells from b-arrestin2KO mice (Fig. 2E) and was reduced inactivated CD4+ T cells (fig. S4A). Con-sistentwith a role for b-arrestin–mediatingchemotaxis stimulated by other chemo-kine receptors (7, 37, 38), migration ofeffector memory T cells to the endoge-nousmurine chemokineCXCL10was alsomarkedly attenuated in b-arrestin2 KOmice (fig. S4, B to E). T cells from CXCR3KO mice failed to migrate to either theb-arrestin–biased agonist or the G protein–biased agonist (Fig. 2E).

With the relative differences in biasedagonist–induced chemotaxis, we then in-vestigated whether topical application ofthe biased agonists caused an increase inthe number of effector memory T cells in

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Fig. 2. A b-arrestin–biased, but not G protein–biased, CXCR3 agonist increases inflammation and the chemotaxisof effector/memory T cells. (A) Experimental design of the DNFB CHS model of inflammation. DNFB sensitization wasinduced with 0.5% DNFB, and contact allergy was elicited 5 days later with 0.3% DNFB. (B) Ear thickness after topicalapplication of vehicle, the b-arrestin–biased agonist VUF10661 (50 mM), or the G protein–biased agonist VUF11418(50 mM) on the ear ofWTmice after DNFB elicitation. Data aremeans ± SEMof 7 to 11mice per treatment group. (C) Earthickness after topical application of the b-arrestin–biased agonist VUF10661 or vehicle on the ear of b-arrestin2 KOmice. Data are means ± SEM of eight or nine mice per treatment group. (D) As a negative control, in the absence ofDNFB treatment, VUF10661 or VUF11418 was applied to the ears. Data are means ± SEM of 7 to 10 mice per treatmentgroup. (E) Measurement of the chemotaxis of CD8+CD44+ T cells isolated from the indicated mice toward the indicatedconcentrations of the b-arrestin–biased agonist VUF10661or theGprotein–biased agonist VUF11418. VUF11418 (1mM)didnot cause statistically significant chemotaxis compared to the 0nM treatment (P<0.05by two-tailed t test). Data aremeans±SEMof three or fourmice per treatment group. (F) Skin infiltration by effector T cells in either vehicle or DNFB allergen–elicited WT mouse ears induced by topical application of vehicle, the b-arrestin–biased agonist VUF10661 (50 mM), orthe G protein–biased agonist VUF11418 (50 mM). Data are means ± SEM of six to nine mice per treatment group. (G) Skininfiltration by effector T cells in DNFB allergen–elicited b-arrestin2 KO mouse ears induced by topical application ofVUF10661 (50 mM) or VUF11418 (50 mM). Data are means ± SEM of six to eight mice per treatment group. For (B), *P <0.05 by two-way ANOVA analysis. For (E), *P < 0.05 by two-way ANOVA analysis, showing statistically significant effects ofdrug for WT VUF10661 versus WT VUF11418 (Tukey post hoc analysis for 1 mM; also, P < 0.05 corrected for multiple com-parisons), of genotype forWT VUF10661 versus b-arrestin2 KO VUF10661, and of genotype forWT VUF10661 versus CXCR3KO VUF10661.

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the skin. The b-arrestin–biased agonist in-creased the number of effector memorycytotoxic T cells in the ear relative to thenumbers induced by either vehicle or Gprotein–biased agonist after treatment ofWTmice with DNFB (Fig. 2F). Treatmentwith theb-arrestin–biasedagonistVUF10661also increased the number of effectormemory T helper cells in the ear relativeto the number of cells induced by treatmentwith the G protein–biased agonist (fig. S5,A and B). The ears of b-arrestin2 KOmiceaccumulated fewer effectormemory T cellsthan did those of WT mice in response toVUF10661 (Fig. 2G and fig. S5C). No dif-ference in the number of CD4+ regulatoryT cells was observed (fig. S5, D and E).Together, these findings are consistentwiththe b-arrestin–biased agonist increasingskin inflammation by promoting the che-motaxis and recruitment of effectormemoryT cells.

Patients with CHS coexpress CXCR3and b-arrestin in T cells in the skinA major confounder in studies of chemo-kine biology is differences in expressionpatterns and function between species(39). To correlate CXCR3 signaling withCHS in human patients, we sampled skinfrom patch-tested patients (Fig. 3A). Skinpatch testing involves the placement ofcontact allergens directly on the skin. Posi-tive patch test reactions include erythematicinduration, with severe reactions causingulceration. Patch testing is the gold stan-dard for the diagnosis of ACD, and the roleof T cells in the pathophysiology of allergicCHS that underlies this disease is wellestablished (40). Ninety-six to 120 hoursafter allergen application, the patch testwas read, and biopsies of positive and neg-ative siteswere collected and stained for theT cell marker CD3, CXCR3, and b-arrestin(Fig. 3B).More T cells andmore T cells co-expressingCXCR3 and b-arrestinwere ob-served inACDbiopsies than in nonlesional(negative) biopsies at the epidermal-dermal junction (Fig. 3, C and D). In addi-tion, the percentage of T cells coexpressingthese markers was increased in ACD bi-opsies (92 of 189, or 49%) relative to thatin nonlesional biopsies (8 of 23, or 35%).To test whether b-arrestin signaling andG protein–biased signaling resulted in asimilar effect on human T cell migration,we isolated leukocytes fromperipheral bloodand tested their chemotaxis to the biasedagonists of CXCR3. Consistent with our

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Fig. 3. HumanCXCR3+T cells displaydifferential responses tobiasedagonists of CXCR3, andCXCR3andb-arrestinare coexpressed in T cell clusters within lesions from human allergen patch-tested skin. (A) Diagram of ACD

patch testing. Colored regions on day 5 signify a positive response. (B) Representative immunohistochemistry of CXCR3(green), CD3 (red), b-arrestin (purple), and Hoechst (blue) in skin from human allergen patch-tested skin (+) andmatchednonlesional (NL; −) controls from the same patient. Data are representative of three patient samples with similar results.Original magnifications were ×200 (left) and ×400 (right), with scale bars of 100 mmand 50 mm, respectively. White boxesin ×200 images indicate the area of magnified images in subsequent pictures and are located at the epidermal (Epi)–dermal junction. In allergenpatch-tested skin, this areawaswheremost T cell clusterswere found, as previously described(66), whereas the same area in nonlesional skinwas devoid of such immune cell conglomerates and served as a control.(C and D) Quantitative analysis of the number of dermal CD3+ T cells (C) and coexpression of CD3 with CXCR3 andb-arrestin (D) in skin from allergen patch-tested skin (ACD) andmatched nonlesional controls from the same patient.(E and F) T cells isolated from patch-tested patients were tested for chemotaxis toward the indicated concentrations ofthe b-arrestin–biased agonist VUF10661 or the G protein–biased agonist VUF11418. (E) CXCR3+CD8+ T cells (n= 3) and (F)CD44+CD8+ cells (n = 5). Patch test quantitative analyses are expressed as positive cells ± SEM from a microscopic fieldwith six views at ×400, from a total of three patients. For (C) and (D), *P < 0.05 by unpaired two-tailed t test. For (E) and (F),*P < 0.05 by two-way ANOVA.

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observations inmice, the b-arrestin–biased agonist caused increased hu-man effector memory CD8+ T cell migration relative to the G protein–biased agonist (Fig. 3, E and F), but not in the case of CXCR3+CD4+T cells(fig. S6). These findings are consistent with a similar role for CXCR3-mediated signaling through b-arrestin in T cells in humanCHS responses.

Akt is activated by b-arrestin and promotesT cell chemotaxisGiven the overlap of signaling pathways mediated by both b-arrestinand G proteins, it was unclear which specific signaling effectorsdownstream of b-arrestin were responsible for differences in chemo-taxis and inflammation induced by b-arrestin– and G protein–biasedagonists. To identify potential pathways, we performed a targeted flowcytometry–based analysis of intracellular phosphorylation in humanperipheral blood mononuclear cells (PBMCs) after incubation with ve-hicle, VUF10661, or VUF11418. The Akt pathway, which can be acti-vated by b-arrestin, displayed qualitative differences in these targetedanalyses. The b-arrestin–biased agonist increased the phosphorylationof Akt residues Thr308 and Ser473, which are necessary for full-efficacykinase activity, relative to the G protein–biased agonist (Fig. 4A). Dif-ferences in the phosphorylation of Ser9 of glycogen synthase kinase 3b(GSK-3b), where phosphorylation at this site reduces the kinase activityof GSK-3b and is regulated in part by Akt, were also observed (Fig. 4Aand fig. S7). We verified these differences in Akt phosphorylation in aT cell line (Jurkat cells) stably expressing CXCR3, observing increasedAkt Thr308 phosphorylation by the b-arrestin–biased ligand VUF10661relative to that induced by the G protein–biased ligand VUF11418 (Fig.4B). Greater amounts of phosphorylated Akt (pAkt)–Thr308 werecoimmunoprecipitated with b-arrestin2 after CXCR3 activation withVUF10661 than after incubationwith eitherVUF11418 or vehicle (Fig. 4,C and D, and fig. S8). The small interfering RNA (siRNA)–mediatedknockdownofb-arrestin2 abolished theVUF10661-stimulated phospho-rylation of Akt Thr308 (Fig. 4, E and F) and attenuated Akt Ser473 phos-phorylation (fig. S9). VUF10661 and VUF11418 did not stimulatedifferential phosphorylation of extracellular signal–regulated kinase(ERK; fig. S9), a kinase whose activity is increased by both G proteinsand b-arrestins (41, 42). Consistent with the contribution of b-arrestin–mediated Akt activation to chemotaxis, pretreatment of lymphocyteswith the Akt inhibitor AZD5363 decreased effector memory T cellchemotaxis toward VUF10661 (Fig. 4G). T cell chemotaxis towardVUF10661 was also inhibited by either the phosphatidylinositol 3-kinase(PI3K) inhibitor LY20042 (fig. S10, A and B) or PTX (fig. S10, C and D).These findings are consistentwith bothPTX-sensitiveGprotein signalingand b-arrestin–mediated Akt signaling contributing to CXCR3-dependentchemotaxis.

DISCUSSIONIn this study, we demonstrated that small-molecule biased agonists ofCXCR3 with similar receptor affinity but divergent G protein andb-arrestin efficacies caused distinct physiological effects on T cell–mediated skin inflammation through differential T cell chemotaxis. Wefound that a small-molecule b-arrestin–biased agonist (VUF10661) wassuperior in promoting T cell chemotaxis compared to aGprotein–biasedagonist (VUF11418). Similar to previous studies, we found that both Gprotein– and b-arrestin–mediated signaling were necessary for chemo-kine receptor–mediated chemotaxis (7, 23). These findings suggest thatbiased endogenous chemokines might promote distinct physiologicaleffects, with clear relevance for drug discovery at CXCR3 and other

Smith et al., Sci. Signal. 11, eaaq1075 (2018) 6 November 2018

chemokine receptors. For example, drugs designed to block the mi-gration of CXCR3+ T cells to reduce inflammation should impairb-arrestin–mediated signaling, whereas drugs intended to increase therecruitment of CXCR3+ T cells, for example, in cancer immunotherapy,should promote signaling through both b-arrestin andGai. The distinctphysiological effects caused by biased agonists offer a plausible reasonfor the difficulties in drugging chemokine receptors and also for biasedsignaling patterns encoded by different endogenous chemokines at thesame receptor.

Given the roles of b-arrestin in the negative feedback of receptorsignaling (desensitization and internalization), it was unclear how smallmolecules regulating complex G protein– and b-arrestin–mediatedsignaling pathways would interact in physiologically relevant modelsof chemotaxis and inflammation. Our findings show that biased ago-nists of CXCR3 can differentially affect inflammatory processes.b-Arrestins scaffold a number of effectors that control cell polarityand influence cellular migration (43–46); however, it was unclear whichof these effectors were relevant to chemotaxis. Using targeted analysis ofintracellular phosphorylation followed by hypothesis-driven experiments,we identified phosphorylation of Akt, which is promoted by b-arrestinsignaling, as being necessary for full chemotactic function. AlthoughPTX eliminated T cell chemotaxis, Akt inhibition did not fully inhibitmigration, suggesting that multiple CXCR3-mediated pathways me-diate T cell chemotaxis. Note that Akt has a well-established role inmediating cell polarity andmigration. Akt translocates to the leadingedge of migrating cells (47, 48), and this pathway is known to be redstimulated by endogenous CXCR3 chemokines (49, 50). Moreover,b-arrestin alters Akt activation through the formation of signalingcomplexes, such as with protein phosphatase 2A, leading to Akt de-phosphorylation downstream of the D2 dopamine receptor (51, 52),or with Src, in which both are phosphorylated and activated downstreamof the insulin receptor (53).We found that the G protein–biased agonistfailed to induce substantial chemotaxis above baseline, whereas at highconcentrations it reduced migration below basal levels, which could bedue to reduced Akt phosphorylation or off-target effects at high drugconcentrations.

Despite previous studies that implicated both b-arrestin– and Gprotein–mediated signaling as being critical to cell function and mi-gration downstream of other chemokine receptors, such as CXCR4(7, 23, 54, 55), it was unclear how biased agonists could differentiallyaffect physiological models of T cell movement and disease, if at all. De-spite the fact that the b-arrestin–biased agonist caused greater CXCR3internalization thandid theGprotein–biased agonist, theb-arrestin–biasedagonist caused increased chemotaxis and potentiated a T cell–mediatedinflammatory response. Consistent with studies of other chemokine re-ceptors (18, 56), the fact that PTX eliminatedCXCR3-mediated chemo-taxis toward the b-arrestin–biased agonist implicates both G proteinsand b-arrestins in chemotaxis. Studies classify CXCL11 as a b-arrestin–biased ligand relative to the other two CXCR3 endogenous ligandsCXCL9 and CXCL10 (8, 30). CXCL10 and CXCL11 bind to nonover-lapping region(s) of CXCR3. In addition, CXCL11 binds to both Gprotein–coupled and PTX-uncoupled forms of CXCR3. In contrast,the relatively G protein–biased endogenous ligand CXCL10 has noaffinity for the PTX-uncoupled form of CXCR3 (57), providing anillustrative example ofmultisite chemokine binding observed at CXCR3(58). These data suggest that a different transducer, such as b-arrestin,may stabilize a distinct active receptor conformation when bound toCXCL11. Functionally, CXCL11 induces a greater chemotactic re-sponse than those induced by CXCL9 and CXCL10 (29, 59), which

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suggests that CXCL11 may be the most proinflammatory of the en-dogenous CXCR3 ligands. Consistent with b-arrestin playing a centralrole in T cell migration, we found that a b-arrestin–biased agonist wasmore effective than a G protein–biased agonist at increasing CXCR3-mediated T cell chemotaxis and enhancing a T cell–mediated inflam-matory response. In addition, T cells isolated from b-arrestin2 KOmicedisplayed defective chemotaxis to an endogenousCXCR3 chemokine asevidenced by a ~10-fold decrease in potency, consistent with previousobservations (23). Our data are consistent with a model in which theactivation of b-arrestin–mediated signaling can increase T cell migra-tion and function, whereas inhibiting either b-arrestin–mediatedsignaling or the association between b-arrestin and Akt would opposemigration and inflammation.

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On the basis of our findings, it is perhaps unsurprising that small-molecule screens of CXCR3 ligands that focus primarily on receptoraffinity or G protein–mediated signaling could potentially under-perform in identifying promising preclinical agonists or antagonists.The role of b-arrestin in other chemokine receptor signaling path-ways suggests that an expanded screen to include the b-arrestinpathway (and potentially Akt) would improve candidate selection.In summary, targeting different CXCR3 pathways with biased ago-nists that have similar receptor affinities but different efficacies for Gprotein– and b-arrestin–mediated signaling pathways produces dis-tinct physiological differences in chemotaxis and CHS reactions,which has implications for drug development within the chemokinereceptor family.

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-Arrestin2/pAkt association -Arrestin2 siRNA knockdown

IB: pAkt T308

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siRNA: Cntrl arr2 Cntrl arr2Vehicle VUF10661

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IB: pAkt T308Lysate

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10661 11418VehNTTreatment:

Fig. 4. Akt activation is dependent onb-arrestin2 andpro-motes CXCR3-mediated chemotaxis. (A) Selected heat mapof the targeted analysis of intracellular phosphorylation events(log2 of the fold change relative to vehicle) of human CD8+

T cells after stimulationwith either VUF10661 (1 mM)or VUF11418(1 mM) for 15 min and normalized to vehicle-treated cells. In-creased phosphorylation of Akt-activating sites Thr308 andSer473 and decreased phosphorylation of the GSK-3b–inhibitingsite Ser9 were observed after stimulation with VUF10661 (see fig.S7 for the full heat map). (B) Jurkat cells stably expressing CXCR3were analyzed for the relative abundance of pAkt-Thr308 relativeto that of total Akt after stimulation with the b-arrestin–biasedagonist VUF10661 (1 mM) or the G protein–biased agonistVUF11418 (1 mM) for 60 min. Data are from six experimentsper condition. (C) HEK 293T cells expressing CXCR3 andFLAG–b-arrestin2 were analyzed for co-immunoprecipitation ofpAkt-Thr308 with FLAG–b-arrestin2 after 60 min of treatmentwith vehicle, VUF10661 (1 mM), or VUF11418 (1 mM). Data arefrom three experiments per condition. (D) Representative co-immunoprecipitation (IP) and Western blots (IB) from theexperiments described in (C). Immunoprecipitation ofFLAG–b-arrestin2 was followed by Western blotting analysisof pAkt-Thr308 after 60 min of treatment with vehicle of theindicated agonist (1 mM). Blots are representative of threeexperiments. (E) HEK 293T cells expressing CXCR3 and treatedwith siRNA-targeting b-arrestin2 or control siRNA were ana-lyzed for the relative abundance of pAkt-Thr308 after 60minof stimulation with VUF10661 (1 mM). Data are from four orfive experiments per condition. (F) Representative Westernblotting analysis of the siRNA-treated cells shown in (E). (G) WTmouse leukocytes were pretreated with the selective Akt in-hibitor AZD5363 (100 nM) or vehicle, and then the chemotaxisof CD8+ T cells to VUF10661 (1 mM)wasmeasured. Inset: Pairedcomparison between vehicle and AZD5363 conditions. Dataare from eight mice. For (B), *P < 0.05 by unpaired two-tailedt test. For (C), *P < 0.05 by one-way ANOVA followed by Tukeypost hoc comparison; #P < 0.05 compared to vehicle by one-way ANOVA followed by Tukey post hoc comparison. For (E),*P < 0.05 by two-way ANOVA showing a statistically significantinteraction of siRNA and ligand treatment followed by Tukeypost hoc comparison of control siRNA-VUF10661 versusb-arrestin2 siRNA-VUF10661. For (G), *P < 0.05 by repeated-measures two-way ANOVA showing a statistically significantinteraction of AZD5363 and VUF10661 followed by Tukey posthoc comparison of vehicle-VUF10661 versus AZD5363-VUF10661; P < 0.05. For the inset in (G), P < 0.05 by pairedtwo-tailed t test.

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MATERIALS AND METHODSStudy designThe main research objectives of these studies were to investigate thephysiological effects of biased agonists of CXCR3 and determine themechanisms bywhich engagement of theb-arrestin–mediated signalingpathway potentiates chemotaxis and inflammation. These studies wereperformedwith a combination of patient leukocytes, patient skin, genet-ically modified mice, and pharmacologic treatments supported by invitro mechanistic data. The number of patients and animals used ineach group for each experiment is reported in the figure legends.Animals were randomly assigned to treatment groups once their geno-types were confirmed, and investigators were blinded to mouse phar-macological treatments. Statistical details are provided at the end of thissection and within the figure legends.

Small molecules and peptidesVUF10661 (Sigma-Aldrich), VUF11418 (Aobious), LY294002 (Sigma-Aldrich), and AZD5363 (Axon Medchem) were dissolved in dimethylsulfoxide (DMSO) to make stock solutions and were stored at −20°C ina desiccator cabinet. Recombinant human CXCL11 and murineCXCL11 proteins (PeproTech) were diluted according to the manufac-turer’s specifications, and aliquots were stored at −80°C until neededfor use.

BRET assaysIntermolecular and intramolecular b-arrestin–based biosensor BRETexperimentswere performed as previously described (30) andwere sim-ilar to those outlined originally by the Bouvier laboratory (60, 61). Briefly,HEK 293T cells were transiently transfected with CXCR3-Rluc togetherwith plasmids encoding b-arrestin–YFP, GRK2-YFP, or GRK6-YFP andplated on a 96-well plate at ~50,000 cells per well (Corning) or with un-tagged CXCR3 and 50 ng of the Nanoluc–b-arrestin2–YFP biosensor,which was previously determined to be the optimal amount of biosensorexpression vector (30). Forty-eight hours after transfection, the cells wereincubated with the compounds indicated in the figure legends in assaybuffer consisting of Hanks’ balanced salt solution (HBSS) supplementedwith 20mMHepes and 3 mMcoelenterazine-h (Promega). The plate wasread by a Mithras LB940 instrument (Berthold, Germany), and the netBRET ratio was calculated by subtracting the YFP:Rluc ratio in vehicle-treated wells from the YFP:Rluc ratio in the ligand-stimulated wells. Cellstested negative for mycoplasma contamination.

The DiscoveRx b-arrestin–dependent internalization assayThis assay was conducted as previously described (8) and in accordancewith themanufacturer’s protocols. Briefly, an EnzymeAcceptor–taggedb-arrestin and a ProLink tag localized to endosomes were stablyexpressed in U2OS cells. The cells were transiently transfected withplasmid encoding untagged CXCR3. b-Arrestin–mediated internaliza-tion resulted in the complementation of the two b-galactosidase enzymefragments that hydrolyzed a substrate (DiscoveRx) to produce a chemi-luminescent signal.

SRE/serum response factor pathway assaySRE/serum response factor (SRF) experiments were performed as pre-viously described (30). Briefly, HEK 293T cells were transiently trans-fected with plasmid encoding CXCR3 and either the SRE or SRFluciferase reporters. Four hours later, the cells were plated on a 96-wellplate at a concentration of ~25,000 cells per well. The next day, the cellswere serum starved overnight, incubatedwith the compounds indicated

Smith et al., Sci. Signal. 11, eaaq1075 (2018) 6 November 2018

in the figure legends for 5 hours, and subsequently lysed with passivelysis buffer (Promega). Luciferinwas added to the lysate, and the resultingluminescence was quantified using a Mithras LB940 instrument.

TGF-a–shedding assayThe ability of CXCR3 to stimulateGai activity was assessed by the TGF-a–shedding assay as previously described (62). Briefly, HEK 293 cellslacking Gaq, Gai, Gas, and Ga12/13 were transiently transfected withplasmids encoding CXCR3, a modified TGF-a–containing alkalinephosphatase (AP-TGF-a), and either theGaq/i1,2 assay subunit [becauseGai2 is observed to be indispensable in CXCR3 signaling in T cells (63)]or, as a negative control, the DC subunit (which lacks the distal aminoacid residues of G protein a subunits required for receptor interaction)and were reseeded 24 hours later in HBSS (Gibco, Gaithersburg, MD)supplementedwith 5mMHepes in aCostar 96-well plate (Corning Inc.,Corning, NY). Cells were then stimulated with the indicated concentra-tion of ligand for 1 hour. Conditioned medium (CM) containing theshed AP-TGF-a was transferred to a new 96-well plate. Both the celland CM plates were treated with para-nitrophenylphosphate (p-NPP;100 mM) (Sigma-Aldrich, St. Louis, MO) substrate for 1 hour, which isconverted to para-nitrophenol (p-NP) by AP-TGF-a. This activity wasmeasured at OD405 (optical density at 405 nm) in a Synergy Neo2Hybrid Multi-Mode (BioTek) plate reader immediately after the addi-tion of p-NPP and a 1-hour incubation. Gai activity was calculated byfirst determining the amount of p-NP by absorbance through thefollowing equation:

100*DOD 405 CM

DOD 405 CMþ DOD 405 cell

� �

where DOD 405 = OD 405 1 hour −OD 405 0 hour, and DOD 405 celland DOD 405 CM represent the changes in absorbance after 1 hour inthe cell andCMplates, respectively.Datawere normalized to those fromthe vehicle-treated sample, and the nonspecific DC signal was sub-tracted from Gai signal percentage AP-TGF-a release as follows:

Gai AP activityligand� vehicle

vehicle

� �� �� DC

AP activityligand� vehicle

vehicle

� �� �

Only the two highest concentrations of VUF11418 ligand (8 and16 mM) resulted in consistent appreciable background DC signals,which resulted in larger errors relative to those from all other conditions(fig. S1G).

Generation of CXCR3+ Jurkat cellsJurkat cells (an immortalized humanCD4+ T cell line) stably expressingCXCR3 were generated by transfecting a linearized pcDNA3.1 ex-pression vector encoding geneticin (G-418) resistance, selecting fortransfected cells with geneticin (1000 mg/ml), and collecting cells thathighly expressed CXCR3 by fluorescence-activated cell sorting (FACS).Cells were maintained in RPMI 1640 medium (Sigma-Aldrich)supplemented with 10% FBS, 1% penicillin/streptomycin, 0.23%glucose, 10 mM Hepes, 1 mM sodium pyruvate, and geneticin(250 mg/ml).

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Western blottingCells were serum starved for at least 4 hours, incubated with the ligandsindicated in the figure legends, subsequently washed once with ice-coldphosphate-buffered saline (PBS), lysed in ice-cold radioimmunoprecip-itation assay buffer containing phosphatase and protease inhibitors[PhosSTOP (Roche) and cOmplete EDTA-free (Sigma-Aldrich)] for15 min, sonicated, and cleared of insoluble debris by centrifugation at>12,000g at 4°C for 15 min, after which the supernatant was collected.Proteins were resolved by 10% SDS–polyacrylamide gel electrophoresis(PAGE), transferred to nitrocellulose membranes, and analyzed byWestern blotting at 4°C overnight with the indicated primary antibody.Antibodies against phosphorylated ERK (pERK) (#9106, Cell SignalingTechnology) and total ERK (#06-182, Millipore) were used to assessERK activation. Antibodies against pAkt-Thr308 (#13038, Cell SignalingTechnology), pAkt-Ser473 (#9271, Cell Signaling Technology), and totalAkt (#4691, Cell Signaling Technology) were used to assess Akt phos-phorylation. The A1-CT antibody, which recognizes both isoforms ofb-arrestin, was supplied by the laboratory of R. J. Lefkowitz. Proteinloading was assessed with an antibody against a-tubulin (#T6074,Sigma-Aldrich). Horseradish peroxidase–conjugated polyclonal mouseanti-rabbit immunoglobulin G (IgG) or anti-mouse IgG was used assecondary antibodies. Immune complexes onnitrocellulosemembraneswere imaged by SuperSignal enhanced chemiluminescent substrate(Thermo Fisher Scientific). After the detection of phosphorylated ki-nases, the nitrocellulose membranes were stripped and reblotted fortotal kinases. For quantification, band intensities corresponding tophosphorylated proteins were normalized to signals corresponding tothe appropriate total protein on the same membrane with Image Labsoftware (Bio-Rad).

ImmunoprecipitationHEK293N cells were cultured in 100-mm tissue culture plates and tran-siently transfected with 5 mg of CXCR3-encoding plasmid and 2.5 mg ofplasmid encoding FLAG-tagged b-arrestin2 or were cultured in six-welltissue culture plates and transfectedwith 1 mg of CXCR3-encoding plas-mid and 0.5 mg of plasmid encoding FLAG-tagged b-arrestin2. The cellswere lysed as described earlier, and the lysates were incubated for4 hours at 4°Cwith anti-FLAGmagnetic beads (ThermoFisher Scientific)and washed according to the manufacturer’s protocol. Samples werethen immediately eluted with 2× SDS, resolved by 10% SDS-PAGE,and analyzed by Western blotting as described earlier.

siRNA-mediated knockdownHEK 293N cells in six-well tissue culture plates were transiently trans-fectedwith 1 mg of plasmid encoding CXCR3A and 3.5 mg of either con-trol siRNA or with previously validated b-arrestin2–specific siRNA(“wem2”) (64) with Lipofectamine 3000 (Thermo Fisher Scientific) asper the manufacturer’s specifications. Seventy-two hours later, the cellswere stimulated with 1 mM VUF10661 for 60 min and the cells werethen lysed and analyzed by Western blotting as described earlier.

Study subjects and skin samplesAll studies involving human subjects were approved by the Institu-tional Review Board of Duke University Health System. Study par-ticipation inclusion was offered to patients undergoing patchtesting in a specialty contact dermatitis clinic. Inclusion criteriawere ≥18 years of age and completion of patch testing. Exclusioncriteria were pregnancy, topical corticosteroids at patch site, oralcorticosteroids, systemic immunosuppressants, phototherapy,

Smith et al., Sci. Signal. 11, eaaq1075 (2018) 6 November 2018

known bleeding disorders, and allergy to lidocaine or epinephrine.Skin biopsies and venipunctures were obtained from male and femalevolunteers under a protocol approved by the Institutional ReviewBoard of Duke University. Patches containing test allergens were ap-plied to study participants on day 1, removed on day 3, and analyzedafter 96 to 120 hours. If a study participant had a positive patch test,then a 4-mm punch biopsy was obtained at the positive test site and a4-mm punch biopsy was obtained at a negative site (normal skin)from normal regions of the skin nearby. Immunofluorescence analysiswas performed as previously described (65). Preceding cell counting,skin images from allergen patch-tested skin and matched nonlesionalcontrols from the same patient were separated by color channel. Allcollected images were processed with ImageJ software. The ImageJ cellcounter tool recorded mouse clicks on cells that were labeled withcolored dots. Cell numbers were expressed as counts per view in amicroscopic field with six views.

MiceWTC57BL/6; C57BL/6 CXCR3−/0, CXCR3−/−; and C57BL/6ARRB2−/−

mice were bred and maintained under specific pathogen–freeconditions in accredited animal facilities at the Duke University.C57BL/6 CXCR3−/0, CXCR3−/− mice were acquired from the JacksonLaboratory (Bar Harbor, ME, USA; stock #005796), and C57BL/6ARRB2−/− were provided by R. J. Lefkowitz (Duke University, USA).Mice were between 6 and 15 weeks of age when first used.

Mouse allergic CHSMice were sensitized by topical application of 50 ml of 0.5% DNFB(Sigma-Aldrich) in 4:1 acetone/olive oil on their shaved back andwere challenged on their ears 4 days later with 10 ml of 0.3% DNFBor vehicle control. Then, 4, 24, and 48 hours later, 10 ml of eithervehicle, VUF10661 (50 mM), or VUF11418 (50 mM) dissolved in72:18:10 acetone:olive oil:DMSO was topically applied to the indi-cated ear by an investigator blinded to treatment. Ear thickness wasmeasured at different time points with an engineer’s micrometer(Standard Gage).

Chemotaxis assaysChemotaxis assays were conducted similarly to those previously de-scribed (56). Briefly,mouse leukocytes, obtained by passing cells isolatedfrom the spleen and subjected to erythrocyte lysis through a 70-mm filter,were suspended in RPMI 1640 medium containing 5% FBS. For theassay, 1 × 106 cells in 100 ml of mediumwere added to the top chamberof a 6.5-mm-diameter, 5-mm-pore polycarbonate Transwell insert(Costar) and incubated in duplicate with the indicated concentrationsof ligand suspended in 600 ml of themedium in the bottom chamber for2 hours at 37°C. Cells that migrated to the bottom chamber were resus-pended, washed, stainedwith a Live/Deadmarker (AquaDead, ThermoFisher Scientific) and antibodies to cell surface markers (CD3, CD4,CD8, CD44, and CD45), fixed with paraformaldehyde, and subjectedto cell counting flow cytometric analysis with a BDLSRII flow cytometer.Flow cytometry was performed in the Duke Human Vaccine InstituteResearch Flow Cytometry Facility (Durham, NC). We were unable toidentify a reliable anti-murine CXCR3 antibody for flow cytometry suit-able for surface staining. CountBright beads (Thermo Fisher Scientific)were added immediately after bottom chamber resuspension to correctfor differences in final volume and any sample loss during wash steps. A1:10 dilution of input cells was similarly analyzed. Migration wascalculated by dividing the number of migrated cells by the number of

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input cells. In some studies, cells were preincubated for 1 hour at 37°Cwith PTX (100 ng/ml; List Biological Laboratories), 100 mM LY294002(Sigma-Aldrich) to inhibit PI3K, or 100 nMAZD5363 (AxonMedchem)to inhibit Akt. Human peripheral blood leukocytes were obtained byvenipuncture in accordance with the Duke Institutional ReviewBoard, subjected to erythrocyte lysis, and assayed as described ear-lier, with the addition of an anti-human CXCR3 antibody. For somesamples, reliable anti-human CXCR3 staining was not obtained,which resulted in two fewer replicates in the CD8+CXCR3+ groupcompared to the CD8+CD44+ group. Analysis was conducted withFlowJo (Ashland, OR) version 10 software. A representative gatingtree is shown in fig. S11.

Assessment of T cells in the skinMice were subjected to the mouse allergic CHS assay as describedearlier, with the exception that both ears received 0.3%DNFB to induceinflammation. Two ears were pooled from a single mouse to produceone biological replicate. About 4 hours after the last topical drug treat-ment,mouse ears were dissected and transferred dorsal side up to a dishcontaining ice-cold PBS. Dorsal and ventral layers were separated andadded to 5ml of digestionmedia consisting ofHBSS supplementedwith5% FBS (Corning), 10mMHepes, liberase (0.04mg/ml; Sigma-Aldrich),and deoxyribonuclease (0.3 mg/ml; Sigma-Aldrich). Ears were incubatedat 37°C for 10 min, minced, and incubated for an additional 30 min at37°C, vortexing gently every 10 min. After 40 min, 25 ml of PBS wasadded and the samplewas vortexed and strained through a 70-mmmeshinto a fresh 50-ml conical tube. Cells were spun down at 200g at 4°C andresuspended in PBS supplemented with 10 mM Hepes, 5 mM EDTA,and 1% bovine serum albumin. To obtain leukocyte cell counts, cellswere manually counted with Turks solution by an investigator blindedto treatment group. Cells were transferred to round-bottom FACStubes, washed twice with 2 ml of ice-cold PBS, and stained with a Live/Deadmarker (AquaDead, #L34957,ThermoFisher Scientific).Cellswereblocked in PBS supplemented with 3% FBS and 10 mM EDTA (FACSbuffer) with anti-CD16/32 (Fcg block), 5% normal mouse serum(Thermo Fisher Scientific), and 5% normal rat serum (Thermo FisherScientific). Cells were then stained with antibodies to cell surfacemarkers(CD3, CD4, CD8, CD44, and CD45) for 30 min at 4°C, washed withFACS buffer, and fixed with 0.4% paraformaldehyde. Foxp3 intracellularstaining was performed with an anti-Foxp3 antibody using a Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to themanufacturer’s protocol. Flowcytometrywasperformed in theDukeHu-man Vaccine Institute Research Flow Cytometry Facility on a BD LSRIIflow cytometer (Durham, NC). Analysis was conducted with FlowJoversion 10 software. Total cells were calculated by multiplying therelative abundance of total live cells by the manual leukocyte count.A list of flow cytometry antibodies used is listed in table S2.

Immunofluorescence analysis of human skinSections of frozen human specimens were incubated overnight at4°C with anti-human CD3 (OKT3, BioLegend) and anti-humanb-arrestin (provided by R. J. Lefkowitz), which was followed byreaction with Cy3- and Alexa Fluor 647–conjugated secondary anti-bodies (Thermo Fisher Scientific). The sections were then incubatedwith Alexa Fluor 488–conjugated mouse IgG1 antibodies against hu-manCXCR3 (R&D Systems Inc.) ormouse IgG1 isotype control (TonboBiosciences). Nuclei were counterstained with Hoechst 33342, washedin PBS, and mounted with Anti-fade Mounting Media (Thermo FisherScientific).

Smith et al., Sci. Signal. 11, eaaq1075 (2018) 6 November 2018

Targeted phosphoprotein analysisHuman PBMCs were stimulated with saturating concentrations of theligands indicated in the figure legends for 1, 2, 5, or 15 min; fixed with1% paraformaldehyde for 10 min at room temperature; and preparedfor antibody staining as previously described (66). Briefly, the fixed cellswere permeabilized in 90% methanol for 15 min. The cells were thenstained with a panel of antibodies specific to the markers indicated inthe figure (Primity Bio Pathway Phenotyping service) and analyzed onan LSRII flow cytometer (BD Biosciences). The log2 ratio of the meanfluorescence intensity (MFI) of the stimulated samples divided by thatof the unstimulated control samples was calculated as a measure of theresponse.

Statistical analysesDose-response curves were fitted to a log agonist versus stimulus withfour parameters [Span, Baseline, Hill coefficient, and EC50 (medianeffective concentration)] with the minimum baseline constrained tozero using Prism 7.0 software (GraphPad). To compare ligands inconcentration-response assays or time-response assays, a two-wayANOVA of ligand and concentration or ligand and time, respectively,was conducted. If a statistically significant interaction or main effect oftreatment, depending on the experiment, was observed (P < 0.05), thencomparative two-way ANOVAs between individual ligands were per-formed. Further details of statistical analysis and replicates are includedin the figure legends. For mouse ear T cell counts, two micecorresponding to the highest and lowest values in the WT VUF10661treatment group were excluded from analysis (CD8+CD44+ counts79,515 and 2709, respectively). Lines indicate the mean, and error barssignify the SEM throughout themanuscript, unless otherwise noted.As-terisk indicates P < 0.05 throughout the paper to indicate statistical sig-nificance from pertinent comparisons detailed in the figure legends,unless otherwise noted.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/11/555/eaaq1075/DC1Fig. S1. Additional signaling analyses of CXCR3 ligands.Fig. S2. Biased signaling is conserved at murine CXCR3.Fig. S3. The inflammatory effects of the b-arrestin–biased agonist VUF10661 are absent inCXCR3 KO mice.Fig. S4. Loss of b-arrestin2 attenuates chemotaxis to mCXCL10, and both VUF10661 andmCXCL10 induce chemotaxis of only CD44+ T cell populations.Fig. S5. Biased ligands of CXCR3 differentially increased the numbers of CD4+CD44+ T cells andtotal T cells in DNFB-treated ears.Fig. S6. Human T cell chemotaxis.Fig. S7. Targeted phosphoprotein data in T cells, monocytes, and natural killer cells.Fig. S8. Co-immunoprecipitation of pAkt-Thr308 with b-arrestin2.Fig. S9. Differential phosphorylation of Akt, but not ERK1/2, in a T cell line stably expressingCXCR3 after stimulation with VUF10661 or VUF11418.Fig. S10. Both PTX and a PI3K inhibitor eliminate effector T cell migration to VUF10661.Fig. S11. Flow cytometry gating strategy.Table S1. Pharmacological properties of the biased agonists of CXCR3.Table S2. Flow cytometry antibodies.

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Acknowledgments: We thank R. J. Lefkowitz (Duke University, USA) for guidance, mentorship,and thoughtful feedback throughout this work and for supplying C57BL/6 ARRB2−/− mice;R. Premont (Harrington Discovery Institute, USA) for providing the GRK-YFP constructs;A. Inoue (Tohoku University, Japan) for G protein KO cells; M. Caron, S. Shenoy, and N. Freedmanfor the use of laboratory equipment; T. Pack, A. Wisdom, and M.-N. Huang for many helpfuldiscussions; N. Nazo for laboratory assistance; and K. Hines and K. Scoggins for assistancein patient sample acquisition. Funding: This work was supported by T32GM7171 (J.S.S.), theDuke Medical Scientist Training Program (J.S.S.), 1R01GM122798-01A1 (S.R.), K08HL114643-01A1, (S.R.), Burroughs Wellcome Career Award for Medical Scientists (S.R.), R21AI28727(A.S.M.), R01AI39207 (A.S.M.), Duke Physician-Scientist Strong Start Award (A.S.M.), DermatologyFoundation Research Grant (A.S.M.), and the Duke Pinnell Center for InvestigativeDermatology (J.S.S., A.S.M., and S.R.). Author contributions: J.S.S. and S.R. conceived andplanned the study. J.S.S., L.T.N., T.S.W., A.R.A., M.D.G., A.S.M., and S.R. helped plan and reviewexperiments. J.S.S., L.T.N., J.S., R.A.G, N.M.K., P.A., J.N.G., T.S.W., and A.S.M. performedexperiments. J.S.S., L.T.N., and S.R. analyzed flow cytometry data. A.S.M. and J.S. performed andanalyzed immunohistochemistry data. J.S.S. and S.R. analyzed all other data. J.S.S. and S.R.wrote the paper. Competing interests: The authors declare that they have no competinginterests. Data and materials availability: All data needed to evaluate the conclusions in thepaper are present in the paper and/or the Supplementary Materials. Additional data related tothis paper may be requested from the authors.

Submitted 2 October 2017Resubmitted 23 February 2018Accepted 19 October 2018Published 6 November 201810.1126/scisignal.aaq1075

Citation: J. S. Smith, L. T. Nicholson, J. Suwanpradid, R. A. Glenn, N. M. Knape, P. Alagesan,J. N. Gundry, T. S. Wehrman, A. R. Atwater, M. D. Gunn, A. S. MacLeod, S. Rajagopal, Biasedagonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation.Sci. Signal. 11, eaaq1075 (2018).

11 of 11

inflammationBiased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and

Gundry, Thomas S. Wehrman, Amber Reck Atwater, Michael D. Gunn, Amanda S. MacLeod and Sudarshan RajagopalJeffrey S. Smith, Lowell T. Nicholson, Jutamas Suwanpradid, Rachel A. Glenn, Nicole M. Knape, Priya Alagesan, Jaimee N.

DOI: 10.1126/scisignal.aaq1075 (555), eaaq1075.11Sci. Signal. 

physiological outcomes.Together, these data suggest that biased agonists of CXCR3, and perhaps other chemokine receptors, result in differentat stimulating mouse and human T cell chemotaxis in vitro and activated the kinase Akt, which promoted migration.

biased agonist was more potent−-arrestinβexacerbated inflammation in a mouse model of contact hypersensitivity. The -arrestin biased, but not one that was G protein biased,βtopical application of a small-molecule agonist that was

biased signaling by the receptor CXCR3, which directs T cell migration to sites of inflammation. The authors found that . investigatedet aldependent signaling. Smith −-arrestinβ or −whereby different ligands can stimulate either G protein

coupled receptors (GPCRs), chemokine receptors exhibit so-called biased agonism,−Like many other G proteinBiased chemokine responses

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