S1PR4 Signaling Attenuates ILT 7 Internalization to Limit IFN- Α Production by Human Plasmacytoid Dendritic Cells

S1PR4 Signaling Attenuates ILT 7 Internalization To Limit IFN- α Production by Human Plasmacytoid Dendritic Cells

This information is current as Christina Dillmann, Christian Ringel, Julia Ringleb, Javier of September 24, 2021. Mora, Catherine Olesch, Annika F. Fink, Edward Roberts, Bernhard Brüne and Andreas Weigert J Immunol published online 18 January 2016 http://www.jimmunol.org/content/early/2016/01/15/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 18, 2016, doi:10.4049/jimmunol.1403168 The Journal of Immunology

S1PR4 Signaling Attenuates ILT 7 Internalization To Limit IFN-a Production by Human Plasmacytoid Dendritic Cells

Christina Dillmann,* Christian Ringel,* Julia Ringleb,* Javier Mora,* Catherine Olesch,* Annika F. Fink,* Edward Roberts,† Bernhard Brune,*€ and Andreas Weigert*

Plasmacytoid dendritic cells (pDCs) produce large amounts of type I IFN in response to TLR7/9 ligands. This conveys antiviral effects, activates other immune cells (NK cells, conventional DCs, B, and T cells), and causes the induction and expansion of a strong inﬂammatory response. pDCs are key players in various type I IFN–driven autoimmune diseases such as systemic lupus erythematosus or psoriasis, but pDCs are also involved in (anti-)tumor immunity. The sphingolipid sphingosine-1-phosphate (S1P) signals through ﬁve G-protein–coupled receptors (S1PR1–5) to regulate, among other activities, immune cell migration and activation. The present study shows that S1P stimulation of human, primary pDCs substantially decreases IFN-a produc-

tion after TLR7/9 activation with different types of CpG oligodeoxynucleotides or tick-borne encephalitis vaccine, which Downloaded from occurred in an S1PR4-dependent manner. Mechanistically, S1PR4 activation preserves the surface expression of the human pDC-speciﬁc inhibitory receptor Ig-like transcript 7. We provide novel information that Ig-like transcript 7 is rapidly internalized upon receptor-mediated endocytosis of TLR7/9 ligands to allow high IFN-a production. This is antagonized by S1PR4 signaling, thus decreasing TLR-induced IFN-a secretion. At a functional level, attenuated IFN-a production failed to alter Ag-driven T cell proliferation in pDC-dependent T cell activation assays, but shifted cytokine production of T cells from a Th1

(IFN-g) to a regulatory (IL-10) proﬁle. In conclusion, S1PR4 agonists block human pDC activation and may therefore be a promising http://www.jimmunol.org/ tool to restrict pathogenic IFN-a production. The Journal of Immunology, 2016, 196: 000–000.

lasmacytoid dendritic cells (pDCs) are a rare DC sub- the transcription factor IFN regulatory factor 7 (IRF7) (3). TLR7 population, characterized by their potent ability to pro- and -9 sense viral ssRNA and unmethylated CpG oligodeoxy- P duce large amounts of type I IFN (IFN-a/b; IFN-I), a nucleotides (ODNs), respectively, and are located within endo- plasma cell-like morphology, and a unique set of cell-surface somes. The subcellular localization of TLR7/9 prevents the host markers. IFN-a plays a crucial role in antiviral immunity by to respond to self-DNA, which might induce autoreactive im- augmenting the expression of a broad repertoire of antiviral mune responses. The uptake of physiological TLR7/9 ligands by guest on September 24, 2021 molecules that dampen viral spread and promote apoptosis of and their shuttling to the endosomal compartment is a con- infected cells (reviewed in Ref. 1). Furthermore, IFN-a was trolled, receptor-mediated process. However, detailed mecha- shown to inhibit the growth of cells undergoing malignant nisms of TLR7/9 ligand uptake are currently unclear and require transformation (reviewed in Ref. 2). In pDCs, IFN-a is produced further investigation (4). mainly via the TLR7/9–MyD88 pathway and the rapid produc- Controlling pDC activation is indispensable to avoid an tion of high levels IFN-a is due to the constitutive expression of overshooting immune response that can harm the host. There- fore, pDCs express a set of regulatory surface receptors that *Faculty of Medicine, Institute of Biochemistry I, Goethe-University Frankfurt, regulate IFN-a production. Among these, Ig-like transcript 7 60590 Frankfurt, Germany; and †Department of Chemistry, The Scripps Research (ILT7), a surface marker exclusively expressed on human pDCs, Institute, La Jolla, CA 92037 was shown to associate with the adapter protein FcεRIg. Acti- ORCID: 0000-0002-7276-3838 (J.M.). vation of this receptor complex causes ITAM-mediated signal- Received for publication December 19, 2014. Accepted for publication December 9, ing to restrict IFN-a production (5–7). The only known ligand 2015. for ILT7 is bone marrow stromal Ag 2 (BST2), which is This work is supported by the Else Kro¨ner-Fresenius Foundation Research Training expressedonimmunecellssuchaspDCs,butalsooncancer Group Translational Research Innovation–Pharma, the Sander Foundation (2013.036. 01), the Deutsche Krebshilfe (110637), and by the Deutsche Forschungsgemeinschaft cells, including melanoma and breast cancer (8). The current (SFB 1039 TP B04 and SFB 1039 TP B06). model of ILT7/BST2 interaction suggests that BST2 expression Address correspondence and reprint requests to Dr. Andreas Weigert, Faculty of is upregulated in an inflammatory environment to interact with Medicine, Institute of Biochemistry I–Pathobiochemistry, Goethe-University ILT7, serving as a negative-feedback loop to prevent an un- Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail address: [email protected] controlled and overshooting IFN-a response. In contrast, con- The online version of this article contains supplemental material. stitutive expression of BST2 by various cancer types inhibits IFN-a production of tumor-infiltrating pDCs. As IFN-a has Abbreviations used in this article: BST2, bone marrow stromal Ag 2; CBA, cytometric bead array; cDC, conventional DC; DC, dendritic cell; FSME, tick-borne antiangiogenic and proapoptotic effects on cancer cells, this encephalitis vaccine; GrB, granzyme B; IFN-I, type I IFN; IMQ, imiquimod; mechanism allows tumor-associated immunosuppression. How- IRF7, IFN regulatory factor 7; ISG, IFN-I–stimulated gene; LPI, Lyn peptide inhibitor; MHC I, MHC class I; MHC II, MHC class II; ODN, oligodeoxynucleotide; pDC, ever, leukemic pDCs tend to downregulate ILT7 although the plasmacytoid dendritic cell; qPCR, quantitative PCR; ROCK, Rho-associated pro- underlying reason is unclear (9). pDCs were shown to infiltrate hu- tein kinase; siRNA, small interfering RNA; S1P, sphingosine-1-phosphate; S1PR, man solid tumors without secreting adequate levels of IFN-a after sphingosine-1-phosphate receptor; Treg, regulatory T cell; TTX, tetanus toxoid. TLR9 activation (10). Tumors orchestrate an immunosuppressive Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 microenvironment, which is characterized by specific mediators

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403168 2 S1P INHIBITS IFN-a FROM pDCs

(e.g., sphingosine-1-phosphate [S1P], PGE2, IL-10, vascular en- RNA isolation and quantitative RT-PCR dothelial growth factor, and TGF-b), released from tumor- RNA from PBMCs was isolated using peqGold RNA Pure (Peqlab associated immune or cancer cells, favoring regulatory immune Biotechnologie, Erlangen, Germany) followed by cDNA transcription € cell phenotypes (reviewed in Ref. 11). Furthermore, pDCs, like all with the iScript cDNA synthesis kit (Bio-Rad, Munchen, Germany). pDC RNA (#1 3 106 cells/sample) was isolated using the RNeasy Micro kit DCs, are able to present Ags to T cells and therefore induce, (Qiagen, Hilden, Germany) and quantitated using the Bioanalyzer depending on the microenvironment, variable adaptive immune (Agilent Technologies, Bo¨blingen, Germany) followed by transcription responses. There is evidence that pDCs promote regulatory T cell with sensiscript RT kits (Qiagen). Real-time quantitative PCR (qPCR) (Treg) expansion within the tumor by presentation of self-Ags to was performed using the MyIQ real-time PCR system (Bio-Rad) and naive T cells. However, other studies show that fully activated Absolute Blue qPCR SYBR Green fluorescein mix (Thermo Scientific, Karlsruhe, Germany). Primers against all S1P receptors (S1PR1–5), IFN-a2, pDCs can have a cytotoxic, tumoricidal function (12). and IRF7 were from Qiagen. Additional primers (Biomers, Ulm, Germany) S1P is a bioactive sphingolipid mediator that is produced by were: Mx1 sense, 59-CACCGTGACACTGGGATTC-39 and antisense, 59- various (immune) cell types (e.g., RBCs, platelets, macrophages, ATAGCGAGGAGGTGCTGAAG-39; and dsRNA-activated protein kinase and DCs) (reviewed in Ref. 13). S1P acts as a specific ligand for sense, 59-CTAATTTGGCTGCGGCATT-39 and antisense, 59-CCGTCA- GAAGCAGGGAGTAG-39. Actin expression was used for normalization. five receptors (S1PR1–5), which differ in tissue distribution. For RT-qPCR results were quantified using Gene Expression Macro (Bio-Rad). instance, S1PR1 is ubiquitously expressed, whereas S1PR4 is largely restricted to lymphoid tissue (14). Due to the fact that Small interfering RNA transfection of primary human pDC S1PRs couple to diverse G-proteins, S1P is able to affect multiple S1PR4 silencing in primary human pDCs was performed using the Accell cellular and physiological functions (e.g., immune cell trafficking, siRNA system (Dharmacon, Lafayette, CO) essentially following the 5 angiogenesis, vascular maturation, cardiac development, and im- manufacturer’s instructions. Briefly, 1 3 10 pDCs were incubated with Downloaded from mune cell activation) (reviewed in Ref. 15). Currently, S1PR4 1 mmol pooled small interfering RNA (siRNA; S1PR4 or nontargeting) in 100 ml serum-free medium containing IL-3 for 48 h, followed by addition signaling is ill defined. However, its activation might alter immune of 100 ml medium containing human serum for another 24 h. cell phenotypes and activation (16–19). In the current study, we investigated the influence of S1P on Cytokine quantification pDC activation and function. Administration of S1P decreases CCL5, CXCL10, IL-2, IL-6, IL-8, IL-10, IFN-a, INF-g, or TNF-a in cell-

IFN-a production via S1PR4. Mechanistically, S1PR4 signaling culture supernatants were quantified using a cytometric bead array (CBA) http://www.jimmunol.org/ preserves ILT7 surface expression that normally decreases upon Th1/Th2/Th17 Kit or CBA Flex Sets (BD Biosciences, Heidelberg, Ger- pDC activation. This enables ILT7 to bind to its ligand and to de- many). Samples were acquired by flow cytometry and processed with FCAP software V1.0.1 (BD Biosciences). crease IFN-a production. As a consequence of reduced IFN-a production, S1PR4 signaling in pDCs shifts cytokine production of PGE2 ELISA cocultured T cells from a Th1 (IFN-g) to a regulatory (IL-10) For quantification of PGE2 levels in pDC supernatants, a commercial profile. S1PR4 signaling inhibits human pDC activation and ELISA (Biomol) was used as described (20). might therefore be a promising target to restrict pathogenic IFN-a production in several IFN-I–induced autoimmune diseases or to Flow cytometry increase IFN-I production in tumors. For detection of surface markers, cells were harvested and washed in PBS, by guest on September 24, 2021 followed by incubation with Fc-blocking reagent (Miltenyi Biotec) in 0.5% BSA/PBS for 15 min and Ab staining for 30 min on ice. The following Abs Materials and Methods were used: anti-CD123–allophycocyanin or anti-CD123–PE-Cy5, anti- Primary human immune cell isolation and cell culture blood DC Ag 4–allophycocyanin, anti-ILT7–biotin (all from Miltenyi Biotec); anti–MHC II–PE-Cy7, anti-ILT7–PE, anti-CD86–FITC, streptavidin- PBMCs were obtained from Buffy Coats (DRK-Blutspendedienst Baden- PE–CF594 (all from BD Biosciences); anti-CD11c–FITC (Immuno- Wurttemberg-Hessen,€ Frankfurt, Germany) using Ficoll–Isopaque (PAA tools), anti-CD40–FITC, anti-CD80–allophycocyanin, anti-CD83–PE, Laboratories, Co¨lbe, Germany) gradient centrifugation; RBCs were re- anti-OX40L–Alexa Fluor647 (all from BioLegend, San Diego, CA); moved by KCl lysis. PBMCs were cultured in six-well plates in and anti-CD3–eFluor 605NC (eBioscience, San Diego, CA), or anti- RPMI 1640 (PAA Laboratories) containing 2% FCS. Primary pDCs TLR9–PE Ab (clone 26C593.2; Novus Biologicals, Wiesbaden, Ger- were magnetically purified from PBMCs using a pDC purification kit many). To detect intracellular protein levels, pDCs were resuspended in Fix/ (negative selection), according to the manufacturer’s instructions, and the Perm solution (BD Biosciences) and incubated for 20 min on ice. Cells were autoMACS Separator (Miltenyi Biotec, Bergisch Gladbach, Germany). washed twice in Perm/Wash solution (BD Biosciences), followed by anti- + + pDCs were characterized as blood DC Ag 4 ,CD123,MHCclassII S1PR4–FITC (Biozol, Eching, Germany), anti–IFN-a–PE (BD Biosciences), or int 2 (MHC II) ,andCD11c . The purity was $93%. Cells were cultured in anti-granzyme B (GrB)–PE Ab (Immunotools) staining for 30 min on ice. To 96-well plates in X-Vivo (Lonza, Verviers, Belgium) supplemented with analyze accumulation of pDCs in cell clusters, PBMCs were stimulated as in- 2.5% human serum containing 20 ng/ml IL-3 (Immunotools, Friesoythe, dicated and immediately fixed in paraformaldehyde (1% in PBS), followed by Germany). T cells were isolated from PBMCs using the Pan T cell Ab staining. Samples were analyzed on an LSR II/Fortessa flow cytometer (BD isolation kit, according to the manufacturer’s instructions, and the Biosciences). autoMACS Separator (Miltenyi Biotec) and cultured in RPMI 1640 (PAA Laboratories) supplemented with 10% heat-inactivated FCS, 1% nones- Immunofluorescence staining sential amino acids, 1% essential amino acids, 1% sodium pyruvate, 1% HEPES buffer (all from PAA Laboratories), 2-ME, and 10 ng/ml IL-2 Human primary pDCs were isolated as described above and plated in (Immunotools). 96-well plates with IL-3 (20 ng/ml) overnight. pDCs were treated with FITC-labeled CpG-A (10 mg/ml) for 5 min and prestimulated with an Reagents S1PR4 agonist (Cym50138; 200 nmol) for 30 min. Cells were then harvested and centrifuged on glass slides using cytospin. pDCs were S1P and VPC23019 (both 1 mmol; Avanti Polar Lipids, Alabaster, AL) were fixed with 1% paraformaldehyde for 30 min at room temperature fol- dissolved following the manufacturers’ instructions. JTE-013 (100 nmol; lowed by permeabilization in 2% Triton X-100/PBS for 10 min. For Biomol, Hamburg, Germany), Cym50358 (200 nmol), and Cym50138 (200 analyzing ILT7 internalization, biotinylated ILT7 Abs (Miltenyi Biotec) nmol) were dissolved in DMSO. CpG-A (2336) ODN (5 mg/ml), CpG-A and streptavidin-PE–CF594 (BD Biosciences) were added for 1 h each. (2336) FITC, CpG-B (2006) ODN (1 mmol), imiquimod (IMQ; 2.5 mg/ml) Nuclei were counterstained with DAPI (1 mg/ml) for 20 min at room (all from Invivogen, San Diego, CA), 10% v/v FSME-Immun (Baxter, temperature. Cover slips were mounted on microscopy slides using Unterschleißheim, Germany), Lyn peptide inhibitor (LPI; 10 mmol), RhoA Vectashield H 1400 mounting medium (Vector Laboratories, Burlingame, inhibitor Rhosin (10 mmol), and Rho-associated protein kinase (ROCK) CA) and analyzed with the AxioVert 200M fluorescence microscope inhibitor Y-27632 (10 mmol) (Tocris Bioscience, Bristol, U.K.) were and the AxioVision software (all from Carl Zeiss Microimaging, Jena, dissolved following the manufacturers’ instructions. Germany). The Journal of Immunology 3

T cell assays dium containing IL-3 (20 ng/ml) and IL-2 (10 ng/ml) with or without the addition of CpG-A (5 mg/ml). T cells were stained with the proliferation For direct coculture assays, T cells and pDCs were isolated from human dye eFluor 670 (5 mmol; eBioscience), washed twice in PBS, and mixed 3 5 blood of the same donor. A total of 5 10 T cells/ml was seeded in a 96- with pDCs at a ratio of 1:5 (pDC/T cells). For indirect coculture assays, 5 well plate and cultured with IL-2 (10 ng/ml) for 24 h. A total of 1 3 10 primary human T cells were stained with eFluor670 and preactivated pDCs/ml was seeded in a 96-well plate and cultured with IL-3 (20 ng/ml) using ImmunoCult Human CD3/CD28/CD2 T Cell Activator (Stemcell and incubated with tetanus toxoid (TTX; 2 mg/ml) for 24 h. To investigate Technologies, Cologne, Germany), seeded in 96-well plates (5 3 105 the inﬂuence of pDC S1PR4 signaling on T cell proliferation and cytokine T cells in 100 ml T cell medium), and daily pulsed with 50 ml primary production, pDCs were stimulated with Cym50138 (200 nml) for 30 min. pDC supernatants for 5 d. Cells and supernatants were harvested after 5 d Afterwards, pDCs were washed with PBS and resuspended in T cell me- of culturing. Supernatants were measured for T cell cytokine secretion Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. S1PR4-dependent reduction of IFN-a production in human pDCs. (A–J) Human pDCs were isolated from buffy coats and cultured in X-Vivo medium containing IL-3 (20 ng/ml). (A) pDCs were controls or activated with the TLR9 agonist CpG-A (5 mg/ml) for 16 h and prestimulated with 0.1– 1 mmol S1P for 30 min. IFN-a secretion was measured by CBA. Data are means 6 SEM of five independent experiments. (B) S1PR expression on freshly isolated human pDCs and 4 or 16 h after activation with CpG-A was determined by qPCR. Data are means 6 SEM of five independent experiments. (C) pDCs were controls or activated with the TLR9 agonist CpG-A (5 mg/ml) for 16 h. Expression of S1PR4 and IFN-a were measured by intracellular Ab staining and FACS. Representative FACS plots of three independent experiments are shown. (D) pDCs were activated with CpG-A (CpG2336) for 16 h with or without 30 min S1P (1 mmol) prestimulation and/or the S1PR antagonists JTE-013 (S1PR2/4 antagonist; 100 nmol), VPC23019 (S1PR1/3 antagonist, 1 mmol), and Cym50358 (S1PR4 antagonist; 200 nmol). The CpG-A–activated group was set to 1. Data are means 6 SEM of three independent experiments. (E) Cells were activated with CpG-A for 4 or 16 h, respectively, with or without preaddition of the specific S1PR4 agonist Cym50138 (200 nmol) for 30 min. IFN-a secretion was measured by CBA. The control group was set to 1. Data are means 6 SEM of four independent experiments. pDCs were transfected with nontargeting siRNA (ctrl siRNA) or S1PR4 siRNA for 72 h, activated with CpG-A for 16 h with or without preaddition of the specific S1PR4 agonist Cym50138 (200 nmol) for 30 min, and expression of IFN-a (F) and S1PR4 (G) compared with actin was quantified by quantitative RT-PCR. Data are means 6 SEM of five independent experiments. pDCs were activated with CpG-A for 16 h with or without preaddition of the specific S1PR4 agonist Cym50138 (200 nmol) for 30 min, and IL-6, IL-8, and TNF-a (H), CXCL10 and CCL5 (I), and PGE2 (J) levels in supernatants were quantified by CBA (H and I) or ELISA (J). Data are means 6 SEM of six independent experiments. p values were calculated using one-sample t test (G), Student t test (D and F), one-way ANOVA with Bonferroni correction (A and E), or two-way ANOVA with Bonferroni correction (B). *p # 0.05, **p # 0.01. 4 S1P INHIBITS IFN-a FROM pDCs as described above. Cells were stained for CD3 expression, and prolif- growth inhibitory effects through expression of IFN-I–stimulated + eration of CD3 T cells was measured using flow cytometry by analyzing genes (ISGs) (reviewed in Ref. 24). To investigate the relevance eFluor 670 dilution. of the reduced IFN-a levels after S1PR4 activation on antiviral properties of pDCs, whole PBMC cultures were prestimulated Statistical analysis with Cym50138 and activated with CpG-A, followed by IFN-a Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, San measurement and elucidation of specific ISGs at mRNA level. As Diego, CA). The p values were calculated using one-sample t test, PBMCs depleted of pDCs only produce minor amounts of IFN-a Student t test, or ANOVA with Bonferroni correctionasindicatedinthe , figure legends. ( 2 pg/ml) in response to CpG-A compared with whole PBMC cultures containing pDCs (.10,000 pg/ml) (Fig. 2A), we pre- sume that pDCs are the main source of IFN-a. TLR9-induced Results cytokine secretion was also significantly reduced in whole PBMC Activation of S1PR4 blocks IFN-a secretion by pDCs cultures upon Cym50138 addition (Fig. 2B). Next, we ana- Taking immunomodulatory effects of S1P on various cell types lyzed three ISGs for their expression after CpG-A treatment, into account, we asked whether stimulation with S1P affects pDC with or without Cym50138 prestimulation. The expression of the activation. We stimulated human primary pDCs with the TLR9 ISGs Mx1, IFN-induced dsRNA-activated protein kinase, and agonist CpG-A to induce IFN-a secretion. Supplying S1P, fol- IRF7 were upregulated after CpG-A addition. Preadministration lowed by CpG-A addition for additional 16 h, concentration- of Cym50138 did not reduce their expression, rather inducing a dependently reduced IFN-a secretion (Fig. 1A). Next, we asked slight but insignificant upregulation (Fig. 2C). Therefore, we for the S1P receptor suppressing IFN-a secretion. We first ana- conclude that reduced IFN-a levels did not translate into reduced Downloaded from lyzed the S1PR expression profile on human primary pDCs antiviral activity in our experimental setup. before and after activation with CpG-A by RT-qPCR. Unstimu- APC function of pDCs is unaffected by S1PR4 signaling lated human pDCs dominantly expressed S1PR1 as well as S1PR4 and, to a lesser extent, S1PR5 (Fig. 1B). Interestingly, Besides conventional DCs (cDCs), also pDCs are considered as S1PR4 was strongly downregulated 16 h after pDC activation APCs that can induce T cell proliferation/activation (12, 25). The both on mRNA (Fig. 1B) and protein level (Fig. 1C), sug- quality of the resulting T cell response depends on the pDC http://www.jimmunol.org/ gesting a regulatory role in pDC activation. To identify the S1PR activation state and microenvironment (12, 25–27). We asked transmitting the S1P-dependent IFN-a inhibition, we prestimu- whether S1P alters surface expression of costimulatory mole- lated human primary pDCs with S1P in combination with an- cules on pDCs and concomitant T cell activation. Activation of tagonists of different S1PRs for 30 min, followed by TLR9 human primary pDCs with CpG-A for 16 h increased MHC class activation with CpG-A for an additional 16 h. We used I (MHC I) but not MHC II surface expression (Fig. 3A). Neither VPC23019, a selective antagonist of S1PR1/3 (21), JTE-013, a MHC I nor MHC II expression was altered upon S1P treatment S1PR2 inhibitor when used at 100 nmol (18), and Cym50358, (Fig. 3A). Furthermore, we analyzed costimulatory molecules on a potent and selective S1PR4 antagonist (22). Neither VPC23019 pDCs (Fig. 3B). The unaltered expression of CD80, CD83, nor JTE-013 prevented the S1P-triggered decrease in IFN-a, by guest on September 24, 2021 indicating that S1PR1/2/3 signaling were dispensable for IFN-a suppression by S1P. However, blocking S1PR4 with Cym50358 significantly abolished the S1P-dependent IFN-a decrease (Fig. 1D). Thus, S1P inhibits IFN-a secretion exclusively via S1PR4. Because IFN-a production by pDCs is amplified in a positive feed-forward loop, we asked whether the S1P-triggered reduction was due to a blockade of IFN-a/b receptor signaling or whether it affected early IFN-a production. We used the selective pharmacological S1PR4 agonist Cym50138 (23) to mimic S1P effects. S1PR4-dependent reduction of TLR9-induced IFN-a secretion was obvious 4 h after TLR9 activation (Fig. 1E). Therefore, S1PR4 signaling influences early IFN-a production by pDCs. To validate the specificity of the S1PR4 agonist, we performed siRNA-mediated knockdown of S1PR4 in primary human pDC and analyzed IFN-a2 expression by qPCR after activation with CpG-A with or without addition of Cym50138. In control siRNA–transfected pDCs, the S1PR4 agonist reduced CpG-A–dependent IFN-a expression, which was reversed in S1PR4 knockdown pDCs (Fig. 1F, 1G). The effect of S1PR4 signaling on soluble mediator production seemed to be rather specific for IFN-a,as FIGURE 2. Attenuated IFN-a secretion does not influence the expres- A 2 CpG-A–dependent expression of the immune mediators IL-6, IL-8, sion of IFN-inducible genes. ( ) PBMCs with ( pDCs) or without TNF-a, CXCL10, CCL5, and PGE was not affected by pre- (+ pDCs) pDC depletion were activated with CpG-A for 16 h, and IFN-a 2 secretion was measured using CBA Flex Sets. Data are means 6 SEM of incubation with the S1PR4 agonist (Fig. 1H–J). In conclusion, S1P seven independent experiments. (B) Whole PBMC cultures were controls selectively reduced IFN-a expression by primary human pDC via or treated with CpG-A (2.5 mg/ml) for 16 h with or without prestimulation S1PR4. with Cym50318 for 30 min. IFN-a secretion in all samples was measured 6 The S1PR4-triggered decrease of IFN-a does not affect using CBA Flex Sets. Data are means SEM of 13 independent experi- C antiviral properties of human primary pDCs ments. ( ) Expression of IFN-a–inducible genes and actin were quantified by quantitative RT-PCR. Data are means 6 SEM of seven independent Next, we asked for consequences of S1PR4-dependent IFN-a experiments. p values were calculated using one-sample t test (B and C)or suppression. PDC-derived IFN-a mediates its antiviral and Student t test (A). *p # 0.05, **p # 0.01. The Journal of Immunology 5 Downloaded from

FIGURE 3. S1PR4 signaling has no impact on APC properties of pDCs. (A–E) Human pDCs were isolated from buffy coats and cultured in X-Vivo medium containing IL-3 (20 ng/ml). (A and B) Human pDCs were controls or cultured with CpG-A (5 mg/ml) for 16 h, with or without Cym50138 (1 mmol) prestimulation http://www.jimmunol.org/ for 30 min. Surface expression of the indicated proteins was measured by FACS. CpG-A–treated group set to 1. Data are means 6 SEM of three independent experiments for each protein. (C) Expression of GrB by pDCs was measured by intracellular Ab staining and FACS. The IL-3–treated (20 ng/ml) group was used as control and set to 1. Data are means 6 SEM of four independent experiments. (D and E) Human pDCs were controls or cultured with CpG-B (1 mmol) for 16 h, with or without Cym50138 (200 nmol) prestimulation for 30 min. Surface expression of the indicated proteins was measured by FACS. (D) Representative FACS plots of resting versus CpG-B–activated pDCs are shown. (E) Quantiﬁcation of FACS data. CpG-B–treated group set to 1. Data are means 6 SEM of six independent experiments. The p values were calculated using one-sample t test. *p # 0.05, **p # 0.01, ***p # 0.001. MFI, mean ﬂuorescence intensity.

CD86, CD40, and OX40L after S1P treatment suggested that S1P duced by CpG-A and modulated by the addition of Cym50138. did not affect APC potential of pDCs. GrB production by pDCs TTX served as a natural Ag, presented by pDCs via MHCs to by guest on September 24, 2021 is downregulated upon TLR9 activation to support T cell pro- induce T cell proliferation. As pDCs can induce T cell anergy in liferation (28, 29). Freshly isolated pDCs did not express coculture systems, which can be reversed by adding IL-2 (32), we GrB (not shown), whereas IL-3 induced strong GrB expression supplemented adequate IL-2 levels. pDCs were washed before (Fig. 3C). Administration of the S1PR4 agonist together with IL- adding them to T cells to prohibit a direct contact of T cells with 3 induced GrB expression slightly but not significantly. Sup- the Ag or Cym50138. Proliferation of CD3+ T cells was measured plying CpG-A to IL-3 and Cym50138-treated cells failed to alter 5 d after coculture by monitoring eFluor670 proliferation dye intracellular GrB expression compared with the corresponding dilution by FACS (Fig. 4A, 4B). Compared to the negative control, control (Fig. 3C). These data suggested that S1P, via S1PR4, did activated and Ag-loaded pDCs induced moderate T cell prolifer- not influence the Ag-presenting capacity of pDCs upon CpG-A ation, but Cym50138 prestimulation of pDCs failed to mediate a treatment. CpG-A strongly induces IFN-a production by pDCs, significant change in T cell proliferation. In general, APCs are but has only minor effects on costimulatory molecule expres- able to prime naive cells into different effector cells. For instance, sion, which is strongly induced by CpG-B oligonucleotides (30). IFN-a induces Th1 responses, which are characterized by IFN-g Therefore, we analyzed whether S1PR4 signaling would affect secretion (33–35). Moreover, priming of naive T cells into IL-10 the APC potential of CpG-B–activated pDCs. As expected, CpG- producing Tregs by alternatively activated pDCs is discussed. BinducedstrongupregulationofCD80,CD83andCD86com- Based on this discrepancy we asked, if altered IFN-a levels in pared with CpG-A (Fig. 3D, 3E). However, pretreatment with the pDC/T cell cocultures may alter the differentiation of cocultured S1PR4 agonist Cym50138 had no impact on CpG-B–induced ex- T cells. To address this question, we measured cytokine levels (IL- pression of these molecules (Fig. 3E). Taken together, S1P signaling 2, IL-10, and IFN-g) in coculture supernatants. Besides IL-2, as a through S1PR4 unlikely alters the APC capacity of pDCs. marker for T cell proliferation (which we supplied), all other cytokines were induced in cocultures with activated and Ag- S1PR4 signaling in pDCs alters the outcome of T cell loaded pDCs (Fig. 4C). Interestingly, IL-10 was strongly in- responses duced in the Cym50138-treated group, whereas IFN-g levels were Cytokines act in synergy with presented Ags and costimulatory diminished compared with CpG-A only–treated cells. To gain molecules to support T cell activation and proliferation. IFN-a further evidence that IFN-a production rather than Ag presenta- directly affects CD8+ T cell responses by maintaining their pro- tion or costimulation were involved in altered T cell differentia- liferation (31). Despite the fact that S1PR4 signaling had no in- tion by S1PR4-stimulated pDCs, we employed an approach in fluence on costimulatory molecule expression on pDCs, we asked which isolated T cells were preactivated by stimulating CD2, if altered IFN-a levels affected T cell activation. We used a T cell CD3, and CD28 and subsequently pulsed daily with pDC super- activation and proliferation assay in which cocultured pDCs were natants for 5 d. Also in this setting, supernatants of Cym50138 pulsed with TTX. Cytokine secretion (IFN-a) of pDCs was in- plus CpG-A–stimulated pDCs resulted in increased IL-10, but 6 S1P INHIBITS IFN-a FROM pDCs

FIGURE 4. Influence of pDC S1PR4 signaling on T cell proliferation and activation. (A–C) T cells and pDCs were isolated from buffy coats of the same Downloaded from donor. T cells were cultured overnight in media containing IL-2 (10 ng/ml). Untreated pDCs were controls, or pDCs were pulsed for 24 h with TTX (2 mg/ml). After pulsing and prestimulation with Cym50138 for 30 min, pDCs were washed with PBS and resuspended in T cell media containing IL-3 and IL-2, with or without the addition of CpG-A (5 mg/ml). (A and B) To measure T cell proliferation, T cells were prestained with a proliferation dye (eFluor 670; 5 mmol) and seeded on pDCs in a ratio of 1:5 (DC/T) for 5 d. Gray bars are controls; black bar is Cym50138-pretreated cells. Proliferation (eFluor 670 dilution) was measured by flow cytometry. (A) The gating strategy is displayed. Single and clustered lymphocytes were analyzed for CD3 (T cells) versus CD123 (pDCs, usually absent after 5 d coculture) expression. Dilution of eFluor 670 indicating proliferation of CD3+ T cells was analyzed to identify cells http://www.jimmunol.org/ that had undergone at least one cycle of proliferation. (B) Quantification of proliferating T cells. Data are means 6 SEM of five independent experiments. (C) Cocultures supernatants were harvested at day 5, and cytokine secretion was measured using CBA. Data are means 6 SEM of five independent experiments. (D) Primary human T cells were stained with eFluor 670 and preactivated using human CD3/CD28/CD2 T Cell Activator, seeded in 96-well plates (5 3 105 T cells in 100 ml T cell medium), and pulsed daily with 50 ml primary pDC supernatants for 5 d. Supernatants were harvested at day 5, and cytokine secretion was measured using CBA. Data are means 6 SEM of six independent experiments. For all experiments, the CpG-A–activated group was set to 1. The p values were calculated using one-sample t test (B–D). *p # 0.05, **p # 0.01, ***p # 0.001. decreased IFN-g production by T cells in comparison with su- apparatus to the plasma membrane, followed by shuttling to the pernatants of only CpG-A stimulated pDCs (Fig. 4D). Importantly, endosomes, where ligand attachment takes place (37). Thus, en- by guest on September 24, 2021 readdition of IFN-a largely prevented these alterations in cytokine hanced TLR9 accumulation at the plasma membrane might be a production (Fig. 4D). Taken together, S1PR4 signaling in pDCs marker of cells responding weaker to TLR9 ligands. However, translated into altered T cell differentiation, which was largely S1PR4 signaling did not induce TLR9 expression at the plasma dependent on reduced IFN-a production. membrane, which was observed upon CpG-A administration, indicating recycling of the activated receptor. Rather, S1PR4 acti- Suppression of IFN-a production by S1PR4 signaling is not a vation reduced TLR9 accumulation, thus being another marker consequence of altered CpG uptake, TLR9 trafficking, or pDC (besides IFN-a production) of delayed/reduced cell activation in maturation the presence of an S1PR4 agonist (Supplemental Fig. 1, black Next, we were interested in the mechanism by which S1P, via bar). Primary pDCs were reported to acquire a more mature S1PR4, blocks IFN-a production in pDCs. To address this ques- phenotype after isolation upon overnight culture in vitro, which tion, we first analyzed the binding and uptake of FITC-labeled affects IFN-a production (38). Thus, we wondered whether CpG-A by human primary pDCs. The first steps of endosomal S1PR4 signaling affects human pDC maturation. After over- TLR7/9 activation are the recognition and uptake of the ligand, night culture indeed two pDC subpopulations could be dis- followed by intracellular trafficking to early endosomes. Because tinguished based on their light scatter profile as reported before only one CpG uptake receptor (DEC-205) has been described (4), (Supplemental Fig. 2A) (38). Interestingly, CpG-A uptake we analyzed its surface expression on human primary pDCs. As was higher in the reported mature (forward light scatterlowside shown in Fig. 5A, human blood pDCs express DEC-205. How- scatterhigh) pDC subpopulation (Supplemental Fig. 2B). Adding ever, activation with CpG-A for different time points did not result CpG-A to freshly isolated pDCs also increased the forward light in receptor internalization (Fig. 5B). Recently, it was shown that scatterlowside scatterhigh pDC population compared with control CpG ODNs enter the cell rapidly, and colocalization of CpG cells. However, S1PR4 signaling slightly, but not significantly, ODNs with TLR9 within early endosomes takes place 5 min after decreased this population (Supplemental Fig. 2C, 2D), pointing to treatment (36). Time kinetics of CpG-A-FITC binding/uptake in an S1PR4-independent in vitro maturation and arguing against human primary pDCs by FACS showed that CpG-A-FITC binds to maturation as the underlying principle of S1PR4-dependent IFN-a pDCs during the first 5 min of activation (Fig. 5C). However, there suppression. was no difference between the control group (Fig. 5C, black line) TLR ligand uptake reduces ILT7 surface expression to enable and Cym50138 prestimulated cells (Fig. 5C, red line). Moreover, IFN-a production we also failed to detect differences in intracellular accumulation of FITC-labeled CpG-A by immunofluorescence analysis To avoid an overshooting IFN-I response, IFN-a production by (Fig. 5D). A next option was the availability of the receptor for pDCs is highly regulated. Various regulatory surface receptors on CpG-A. Recently, it was shown that TLR9 traffics from the Golgi pDCs signal via ITAM/ITIM pathways that involve members of The Journal of Immunology 7

FIGURE 5. Influence of S1PR4 signaling on CpG uptake. (A–D) Human pDCs were isolated from buffy coats and cultured in X-Vivo medium containing IL-3 (20 ng/ml). (A) Ex- pression of the CpG uptake receptor DEC-205 on human primary pDCs was analyzed by FACS. Data are representative of four independent experiments. (B) DEC-205 surface expression on primary pDCs was analyzed after different time points of CpG-A treatment. Untreated cells were set to 1. Data are means 6 SEM of five independent experiments. (C) Human primary pDCs were controls or prestimulated with Cym50138 for 30 min, followed by activation for different time points with FITC-labeled CpG-A and FACS analysis. Data are means 6 SEM of five independent experiments. (D) Immunofluores- cence staining of human primary pDCs. Cells were incubated for 5 min with FITC-labeled CpG-A (green) with or without Cym50138 prestimulation, followed by fixation, DAPI (blue) staining, and immunofluorescence imaging (original magnifi- cation 3640). Data are representative of three independent Downloaded from experiments. MFI, mean fluorescence intensity.

the Src kinase family as downstream signaling modules (6, 39). point after CpG-A treatment, which was prevented by S1PR4 ac- Some of these receptors undergo internalization after ligand tivation (Fig. 6G, 6H). These data may reflect ILT7 internalization. http://www.jimmunol.org/ binding (reviewed in Ref. 1). To elucidate the mechanism behind Our hypothesis gained further indirect support by the observation the decreased IFN-a production downstream of S1PR4 activation, that not all TLR ligands altered ILT7 surface expression on human we analyzed, among others, the surface expression of the inhibi- blood pDCs. Activation of whole PBMCs with another TLR9 li- tory receptor ILT7 on primary blood pDCs by FACS (Fig 6A). gand, CpG-B (CpG2006), provoked ILT7 internalization on pDCs, Unexpectedly, ILT7 surface expression rapidly (5 min) decreased whereas TLR7 activation with IMQ did not (Fig. 7A). Interestingly, after CpG-A stimulation (Fig. 6B, black line) and reappeared after a more physiological TLR7 ligand, the tick-borne encephalitis additional 5 min. In contrast, pDCs prestimulated with Cym50138 vaccine (FSME), promoted a rapid and prolonged ILT7 internal- and activated with CpG-A did not show any changes in ILT7 ization (Fig. 7B, 7C). ILT7 internalization by CpG-B or FSME by guest on September 24, 2021 surface expression during uptake of the TLR ligand (Fig. 6B, red was reversed by S1PR4 activation, whereas ILT7 levels on IMQ- line, and 6C, 6D). These findings were confirmed by immuno- treated cells were not altered by S1PR4 signaling (Fig. 7A–C). fluorescence microscopy (Fig. 6E). Although a decrease in ILT7 Importantly, for all used TLR ligands, ILT7 internalization was mRNA after pDC activation was observed before (5), long-term inversely correlated with IFN-a production. Both CpG-A and activation (16 h) of pDCs with CpG-A had no effect on ILT7 CpG-B, as well as the tick-borne encephalitis vaccine FSME, a surface expression (Supplemental Fig. 3A). Because the ligand for induced robust amounts of extracellular IFN- , whereas IMQ administration elicited low IFN-a secretion (Fig. 7D). IFN-a ILT7, BST2, is expressed on human blood pDCs, we investigated levels induced by CpG-B or FSME were decreased by Cym50138. if the expression of BST2 was affected in our experimental In contrast, pDCs prestimulated with Cym50138 and activated setup. BST2 is an IFN-I–induced gene. Hence, surface ex- with IMQ produced equal amounts of IFN-a compared with pression was upregulated after long-time CpG-A stimulation only IMQ-treated cells (Fig. 7E). These data suggest that un- (16 h) (Supplemental Fig. 3B). Although S1PR4 signaling (30 min) altered ILT7 expression after IMQ stimulation might be re- slightly increased BST2 surface expression, activation of S1PR4 sponsible for low IFN-a production compared with TLR ligands in combination with TLR9 activation failed to alter BST2 ex- that induce ILT7 internalization. In contrast, S1PR4 signaling pression (Supplemental Fig. 3B), indicating that alterations in only prevents IFN-a secretion triggered by ligands that induce BST2 expression did not contribute to S1PR4-dependent IFN-a ILT7 depletion from the cell surface. To support this hypothesis, suppression. We therefore hypothesized that the internalization of we blocked ILT7 downstream signaling by inhibiting of the ILT7 was required for CpG-A–dependent IFN-a production and tyrosine-protein kinase Lyn. As shown in Fig. 7F, Cym50138- that inhibition of this process was the primary mechanism by pretreated cells decreased CpG-A–induced IFN-a production which S1PR4 attenuated IFN-a production. A requirement for compared with control pDCs. However, preadministration of an such a mechanism would be that altered ILT7 expression in the LPI for 30 min blocked the effect of S1PR4 signaling on IFN-a observed short time frame translated into differences in ILT7 secretion. Lyn is not exclusively activated downstream of ILT7, receptor engagement. We approached this question by asking but also other inhibitory pDC receptors such as CD303 (40). whether accumulation of pDC in cell clusters, indicative of en- Therefore, extensive future studies will be required to link hanced receptor/ligand interaction, was altered after short-term these molecules in the context of our setting. Finally, we CpG-A treatment with or without S1PR4 activation. Human approached signaling pathways downstream of S1PR4 preactivation PBMCs were therefore prestimulated with Cym50138 followed by (Supplemental Figure 4) that are involved in blocking ILT7 in- activation with CpG-A for 5 min, fixation, and flow cytometric ternalization following pDC activation. S1PR4 couples to Ga12/ analysis (Fig. 6F). Indeed, we observed a small but very consistent 13, which triggers activation of the RhoA/ROCK pathway that is, decrease in pDC accumulation in cell clusters at this early time among others, involved in vesicle trafficking and membrane dynamics. 8 S1P INHIBITS IFN-a FROM pDCs Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 FIGURE 6. CpG-A–induced ILT7 internalization is counteracted by S1PR4. (A) Gating strategy to determine ILT7 expression on pDCs in whole PBMCs (as shown) or after negative selection from buffy coats and culture in X-Vivo medium containing IL-3 (20 ng/ml). Light scatter gating focuses roughly on the region between lymphocyte and monocyte population where DCs reside. pDCs are gated based on CD123 expression, but lack of CD11c expression. Finally, ILT7-expressing pDCs among CD123+ cells are identified. (B) Time kinetics of ILT7 surface expression on human primary pDCs with or without Cym50138 prestimulation for 30 min followed by CpG-A (5 mg/ml) treatment, determined by FACS, are shown. CpG-A–untreated groups (control [ctrl] and Cym50138 treated) were set to 1. Data are means 6 SEM of five independent experiments. (C and D) Quantification of ILT7 surface expression by FACS 5 min after CpG-A (5 mg/ml) activation, with or without Cym50138 prestimulation for 30 min. (C) Representative FACS histograms are shown. (D) Quantification of ILT7 surface expression. The untreated group was set to 1. Data are means 6 SEM of six independent experiments. (E) Immunofluo- rescence staining of ILT7 in human primary pDCs. Cells were controls or incubated for 5 min with FITC-labeled CpG-A (green) with or without Cym50138 prestimulation for 30 min, followed by fixation, ILT7 (red) and DAPI (blue) staining, and immunofluorescence imaging. Data are representative of three independent experiments. (F–H) Human PBMCs were fixed 5 min after CpG-A (5 mg/ml) activation, with or without Cym50138 prestimulation for 30 min to determine pDC accumulation in cell clusters by flow cytometry. (F) Gating strategy is displayed. Doublets were determined by light scatter analysis. CD123-expressing CD11c2 cells were quantified. (G) Representative FACS plots of CD123+ cell accumulation in doublets are shown. (H) Quantification of CD123+ cell accumulation in doublets. The CpG-A–treated group was set to 1. Data are means 6 SEM of six independent experiments. The p values were calculated using one-sample t test (H), Student t test (D), or two-way ANOVA with Bonferroni correction (B). *p . 0.05, **p . 0.01. MFI, mean fluorescence intensity.

Blocking of RhoA as well as ROCK with pharmacological inhibitors in mune cell migration, the role of the immune cell-specific S1PR4 is combination with Cym50138 indeed restored ILT7 internalization after ill defined (14). There is evidence that S1PR4 signaling modulates pDC activation with CpG-A (Fig. 7G). Unfortunately, RhoA/ROCK immune cell phenotypes, because a lack of S1PR4 in mouse cDCs inhibition prevented CpG-A–induced IFN-a secretion. Therefore, these reduces Th17 and increases Th2 immune responses (17). More- ILT7 expression data could not be correlated with IFN-a levels. over, S1PR4 activation on human monocyte-derived DCs induced Together, our data suggest a mechanism of S1PR4-dependent IL-27 production to increase Treg-dependent suppression of cy- IFN-a suppression (Fig. 8). S1PR4 signaling through the RhoA/ totoxicity against human tumor cells (19). The present study ROCK pathway preserves surface expression of the inhibitory re- suggests that S1PR4 signaling also regulates critical functions of ceptor ILT7. Increased ILT7 signaling consequently limits IFN-a human pDCs, by blocking IFN-I secretion. production. Upon in vitro activation, pDCs rapidly produce significant amounts of IFN-a, which initiates a feed-forward loop that further Discussion increases IFN-a production at later time points (41, 42). The S1PR4- The sphingolipid S1P signals via G-protein–coupled receptors to triggered decrease in IFN-a secretion was apparent already 4 h after affect inflammation. Whereas S1PR1 and 2 mainly regulate im- TLR9 activation, pointing to a regulatory impact on the initial steps The Journal of Immunology 9 Downloaded from

FIGURE 7. S1PR4-dependent regulation of ILT7 surface expression is linked to IFN-a regulation. (A) pDCs were untreated or prestimulated with http://www.jimmunol.org/ Cym50138 for 30 min and activated with the TLR9 ligand CpG-B (1 mmol) or the TLR7 ligand IMQ (2.5 mg/ml) for 5 min. Untreated cells were controls. ILT7 surface expression was measured by FACS. Data are means 6 SEM of five independent experiments for each TLR ligand. (B) ILT7 surface expression on pDCs was measured at different time points following activation with an FSME vaccine (10% v/v; signals through TLR7). Data are means 6 SEM of six independent experiments. (C) pDCs were untreated or prestimulated with Cym50138 for 30 min and activated with FSME (10% v/v) for 5 min. Untreated cells were controls. ILT7 surface expression was measured by FACS. Data are means 6 SEM of six independent experiments. (D) IFN-a levels in pDC supernatants after activation with different TLR ligands (IMQ, 2.5 mg/ml; CpG-A, 5 mg/ml; CpG-B, 1 mmol; and FSME, 10% v/v) were measured using CBA Flex sets. Data are means 6 SEM of six independent experiments. (E) pDCs were activated with the TLR9 ligand CpG-B (1 mmol) and the TLR7 ligands FSME (10% v/v) or IMQ (2.5 mg/ml), with or without Cym50138 (200 nmol) prestimulation for 30 min. IFN-a levels were measured by CBA. The TLR ligand–treated group was set to 1 each. Data are means 6 SEM of at least three independent experiments for each TLR ligand. (F) Human primary by guest on September 24, 2021 pDCs were prestimulated with LPI (10 mmol) to block ILT7 downstream signaling in combination with Cym50138 for 30 min and activated overnight (16 h) with CpG-A. IFN-a levels were measured using CBA. (G) Quantification of ILT7 surface expression on human pDCs by FACS analysis 5 min after CpG- A(5mg/ml) activation, with or without prestimulation with Cym50138 (200 nmol), the RhoA inhibitor Rhosin (10 mmol), or the ROCK inhibitor Y-27632 (10 mmol) for 30 min. Data are means 6 SEM of six independent experiments. Data are means 6 SEM of four independent experiments. The p values were calculated using one-sample t test (E), Student t test (A, C, F, and G), or one-way ANOVAwith Bonferroni correction (B). *p . 0.05, **p . 0.01. MFI, mean fluorescence intensity. following TLR ligand uptake/recognition rather than on feed-forward showing increased maturation marker expression, which are signaling. This notion is supported by experiments with a reversed generated upon in vitro culture of primary human pDCs isolated experimental setup (i.e., TLR9 activation followed by S1PR4 agonist from blood and further expanded following TLR9 ligation (38) stimulation), in which S1PR4 signaling failed to diminish IFN-a (Supplemental Fig. 2). An S1PR4-dependent alteration of this secretion (Supplemental Fig. 4). The first step leading to endosomal pDC subset was not apparent (Supplemental Fig. 2C). However, TLR activation is, for a variety of agonists, receptor-dependent up- we noticed an impact of S1PR4 signaling on ILT7 expression, take. For instance, viruses and bacteria typically enter the cell by which was proposed as a maturation marker of human pDC receptor-mediated endocytosis or phagocytic pathways, followed by in vitro (38). We observed rapid ILT7 internalization after CpG-A their degradation and the release of immunogenic nucleic acids. The administration, which was prevented by S1PR4 signaling and uptake of synthetic TLR ligands such as CpG is also receptor me- coupled to IFN-a production. By using different TLR ligands, we diated, although the underlying mechanisms are largely unknown. confirmed the impact of S1PR4 on ILT7 trafficking and con- DEC-205, an Ag delivery receptor expressed by murine CD8+ DCs comitant IFN-a regulation. Strikingly, only stimulation with TLR and different human leukocytes including pDCs (43–45), was re- ligands that are taken up by receptor-mediated endocytosis (FSME cently identified as an uptake receptor for CpG ODNs (4). However, vaccine or CpG ODNs) triggered ILT7 internalization and were DEC-205 was irrelevant at least for CpG-A uptake in human primary responsive to S1PR4-dependent IFN-a suppression. In contrast, pDCs, because the receptor failed to internalize upon CpG-A ad- IMQ, which enters the cell via diffusion through the plasma ministration in our experimental setup. Moreover, uptake of FITC- membrane, failed to alter ILT7 surface expression, and IMQ- labeled CpG by the whole pDC population was not affected by dependent IFN-a production was not regulated by S1PR4. These S1PR4 signaling, indicating a regulatory impact downstream or in- data allow proposing a model of ILT7-dependent IFN-I regulation. dependent of ligand uptake/recognition. ILT7, when expressed at the cell surface, binds its ligand BST2 We observed that CpG was taken up at higher levels by a sub- (e.g., on bystander pDCs), thereby limiting IFN-a production. population of primary human pDCs, which was, to our knowledge, Full-blown pDC activation requires ILT7 internalization, which not reported before. This subpopulation corresponds to pDCs is coupled to endocytosis of TLR7/9 ligands via an unknown 10 S1P INHIBITS IFN-a FROM pDCs

of different ISGs, which might be due to the relatively high remaining IFN-a levels (mean .1000 pg/ml) and the high affinity of the IFN-a/b receptor 1/2 heterodimer for IFN-a. However, reduced IFN-a production was coupled to T cell polarization. The induction of adequate T cell responses requires the ability of APCs to process Ags, followed by precise presentation through the MHCs, the expression of costimulatory molecules, and, finally, a suitable milieu displayed by specific (inflammatory) cytokines (e.g., IFN-a and IL-12) (52). Human pDCs as APCs were shown to capture and cross-present viral Ags via MHC I to naive T cells, resulting in CD8+ T cell responses (53). Furthermore, human blood pDCs express several costimulatory and T cell–activating molecules, which are required for optimal T cell activation. S1PR4 activation failed to alter the expression of MHCs and costimulatory molecules or others regulators of T cell activation/ proliferation such as GrB (29). Taken together, our data do not reveal an impact of S1PR4 on APC functions of human blood pDCs, corroborating a previous observation that S1PR4 did not affect Ag presentation by human cDCs (19). T cells require, be- Downloaded from sides Ag presentation and costimulation, a third signal to develop strong effector functions (52). IFN-a was shown to maintain proliferation of CD8+ T cells (54, 55). However, activation of S1PR4 signaling in pDCs and thus reducing IFN-a failed to alter FIGURE 8. Proposed mechanism of ILT7 regulation during pDC activation T cell proliferation. Beside its role in lymphocyte activation,

1 http://www.jimmunol.org/ and the impact of S1PR4 signaling. ( ) IMQ enters the cells via diffusion, IFN-a modulates the outcome of T cell responses. It has been reaches endosomes, and triggers IFN-a production via TLR7. This results in described that IFN-a directly affects human naive CD4+ Tcellsto moderate cytokine levels, due to ILT7 surface expression, concomitant ligand (e.g., BST2) engagement, and a negative signaling input via Lyn kinase. (2) TLR favor their differentiation into IFN-g–producing Th1 cells (33). ligands (TLR-L), such as CpGs or viral particles, are taken up by receptor- Furthermore, IFN-a suppresses the expansion of regulatory mediated endocytosis, thereby inducing ILT7 internalization, resulting in massive T cells, while increasing IFN-g–producing effector T cells (56). IFN-a production. (3) S1PR4 activation stabilizes ILT7 at the cell surface. Triggering S1PR4 on Ag-pulsed and TLR-activated pDCs, fol- ILT7 binds its ligand on bystander cells, thereby reducing IFN-a levels. lowed by cocultivation with autologous T cells, shifted cytokine secretion from IFN-g to IL-10, corroborating these previous re-

ports. In this regard, S1PR4 signaling enables pDCs to adopt a by guest on September 24, 2021 mechanism, to prevent ILT7 interaction with its ligand and regulatory phenotype inducing tolerogenic T cell responses. downstream inhibitory signaling (Fig. 8). Interestingly, ILT7 in- Our findings illustrate one possible mechanism by which the ternalization was prolonged when using an immobilized virus tumor microenvironment, via enhanced S1P production, avoids instead of synthetic TLR ligands, thus strengthening the physio- tumor eradication by high levels of IFN-I (57, 58). In concordance logical relevance of this mechanism during pDC activation. with this hypothesis, tumor-associated pDCs show a partially ac- S1PR4 signaling was previously coupled to receptor trafficking. tivated phenotype and produce very low amounts of IFN-a com- We observed that production of inflammatory cytokines by tumor- associated macrophages required shuttling of the nerve growth pared with pDCs isolated from patient’s blood (59). Furthermore, factor receptor TrKA to the plasma membrane, which was initiated high numbers of infiltrating pDCs in tumors were correlated with by S1P and S1PR4 (18). Membrane trafficking of cell-surface poor patient prognosis in ovarian cancer (60). Targeting the S1P– receptors involves alterations of actin dynamics. Accordingly, S1PR axis is a promising tool to overcome several diseases, including S1PR4 predominantly couples to G12/13 that activates Rho autoimmune disorders and cancer. There are many studies dealing and regulates cell shape and stress fiber formation (46). Indeed, with the modulation of S1PR1 in different autoimmune diseases (e.g., inhibiting Rho and downstream ROCK activity prevented the multiple sclerosis and rheumatoid arthritis), in which the therapeutic impact of S1PR4 on ILT7 trafficking. Future studies will provide benefit is attributed to inhibition of immune cell trafficking (reviewed further mechanistic insight as to how S1PR4 signals locally pre- in Ref. 61). Interestingly, S1PR1 signaling did not affect pDC traf- vent ILT7 internalization and whether it involves the activation of ficking into diseased areas in a murine MS model, which proved known immunomodulatory mediator systems that are known to beneficial for disease outcome (62). This was attributed to a low affect IFN-a production by pDC. Recently, it was shown that other S1PR1 expression level in murine pDC, which selectively express S1PR4 (63). Whether the immunosuppressive effect of pDCs accu- G-protein–coupled receptor ligands, such as PGE2, can reduce IFN-a production by pDCs (47–49). PGE2 in immune cells signals mostly mulating in the brain of experimental animals in the multiple sclerosis via EP2 or EP4, which couple to Gs to elevate cAMP. Interestingly, model is at least partially transmitted through S1PR4 remains to be G12/13 are also capable of increasing cAMP via adenylate cyclase determined. However, the current study and previous studies (17–19) 7 that is highly expressed in human primary pDCs (50). It will be reveal a role for S1PR4 in modulating immune cell activation that is interesting to determine whether cAMP signaling contributes to relevant in an inflammatory context. S1PR4 might be a promising inhibiting IFN-a production downstream of S1PR4. therapeutic target to modulate pathogenic IFN-a production in IFN- The defense against viral infection is a hallmark of pDC function I–driven diseases or to relieve a block of IFN-I production in tumors. (51). Hence, production of IFN-I in response to viral components via TLR7/9 is indispensable to ensure upregulation of antiviral Acknowledgments IFN-stimulated genes. In our setup, decreased IFN-a levels after We thank Praveen Mathoor and Margarethe Mijatovic for excellent S1PR4 activation were not translated into decreased mRNA levels technical assistance. The Journal of Immunology 11

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