Which Modulates T Cell Activation Adapter, Expressed by Regulatory T Cells, MS4A4B Is a GITR-Associated Membrane

MS4A4B Is a GITR-Associated Membrane Adapter, Expressed by Regulatory T Cells, Which Modulates T Cell Activation

This information is current as Duncan Howie, Kathleen F. Nolan, Stephen Daley, Emma of October 3, 2021. Butterfield, Elizabeth Adams, Hugo Garcia-Rueda, Claire Thompson, Nigel J. Saunders, Stephen P. Cobbold, Yukiko Tone, Masahide Tone and Herman Waldmann J Immunol 2009; 183:4197-4204; Prepublished online 14 September 2009; doi: 10.4049/jimmunol.0901070 Downloaded from http://www.jimmunol.org/content/183/7/4197

Supplementary http://www.jimmunol.org/content/suppl/2009/09/14/jimmunol.090107 http://www.jimmunol.org/ Material 0.DC1 References This article cites 36 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/183/7/4197.full#ref-list-1

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MS4A4B Is a GITR-Associated Membrane Adapter, Expressed by Regulatory T Cells, Which Modulates T Cell Activation1

Duncan Howie,2* Kathleen F. Nolan,* Stephen Daley,* Emma Butterﬁeld,* Elizabeth Adams,* Hugo Garcia-Rueda,* Claire Thompson,* Nigel J. Saunders,* Stephen P. Cobbold,* Yukiko Tone,† Masahide Tone,† and Herman Waldmann*

In the aftermath of thymic negative selection, natural and adaptive regulatory T cells (Tregs) must acknowledge peripheral, “danger-free” self-Ag to ensure their sustained activity. In this paper, we show that natural and adaptive Tregs or T cells transduced with cDNA for Foxp3, just like Th1 cells, express members of the MS4A family of transmembrane molecules. Naive T cells transduced with MS4A4B become able to respond to lower levels of Ag. Using two family members, MS4A4B and MS4A6B, as baits in a yeast split-ubiquitin Treg library screen, we demonstrate their interaction with each other and with GITR, Orai1, and

other surface receptors. Interaction of 4B with GITR augments GITR signaling and T cell IL-2 production in response to Downloaded from triggering with GITR ligand or anti-GITR Abs. This interaction provides a mechanism whereby MS4A family members, through lateral coassociation with costimulatory molecules, may amplify Ag signals. We propose that T cells preoccupied with immune defense use this MS4A family to enhance sensitivity to extrinsic Ag stimulation, ensuring its elimination, while Tregs use these adaptors to allow low level Ag signals to sustain regulatory function. The Journal of Immunology, 2009, 183: 4197–4204.

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mmune tolerance is an essential aspect of the immune system which TGF is experienced by T cells likely governs the outcome http://www.jimmunol.org/ and has evolved multiple mechanisms to ensure that host for the cell. I tissues are spared damage while pathogens are eradicated ef- Using serial analysis of gene expression (SAGE) and DNA mi- ficiently. Its major mechanisms are deletion of autoreactive cells croarrays, we investigated whether there were any genes shared by and the generation of regulatory T cells (Tregs)3. Tregs are func- Tregs induced by over-expression of Foxp3 and by TGF␤.We tionally characterized as T lymphocytes that have acquired the identified a family of four-pass membrane receptors, the MS4A ability to inhibit both T cell-mediated and innate immune re- family, with transcription up-regulated by both Foxp3 and TGF␤. sponses (1). Multiple routes by which Tregs can acquire this ca- Using a split-ubiquitin genetic screen, we showed interactions be- pacity have been described, including central education in the thy- tween MS4A4B and MS4A6B, the most Treg cell-specific MS4A ϩ ϩ ϩ mus, giving rise to the CD4 CD25 Foxp3 natural Tregs (2–4), members with multiple surface receptors, including the costimu- by guest on October 3, 2021 and peripheral induction through exposure to chronic, incomplete latory receptor GITR (TNFRSF18). One of these tetraspan family signals, along with the immunomodulatory cytokines TGF␤ or members has already been described in Th1 cells but their func- IL-10 (5–7). tional relevance to T cells has not been resolved. We show that TGF␤ has an essential role in immune homeostasis and is ca- transduction of MS4A4B into naive T cells can heighten their sen- pable, depending on the tissue-specific context, of inducing anti- sitivity to Ag. MS4A4B overexpression in EL4 cells results in inflammatory peripheral Foxp3-expressing Tregs, as demonstrated augmented signaling and IL-2 production in response to GITR in TGF␤1Ϫ/Ϫ and dominant negative TGF␤R mice, which develop triggering. We propose that Th1 cells (36) and Tregs use these a fatal early-onset autoimmune disease characterized by loss of adaptors to increase their ability to perceive Ag immunity and for Foxp3-positive Tregs. TGF␤ is, however, a pleiotropic cytokine ensuring adequate regulation of self-reactivity. and, in concert with IL-6, can steer T cells toward a proinflam- matory Th17 phenotype. Therefore, the physiological context in Materials and Methods Mice *Sir William Dunn School of Pathology, University of Oxford, Oxford, United King- dom; and †Department of Pathology and Laboratory Medicine, University of Penn- A1(M).RAG1null TCR-transgenic mice and CBA/Ca mice were bred and sylvania, Philadelphia, PA 19104 maintained in specific pathogen-free conditions at the Sir William Dunn Received for publication April 2, 2009. Accepted for publication July 21, 2009. School of Pathology. All procedures were conducted in accordance with the Home Office Animals (Scientific Procedures) Act of 1986. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Generation of SAGE libraries 1 This work was funded by a programme grant from the Medical Research Council ϩ and a grant from the EU FP6 RISET consortium. Four long SAGE libraries were generated from A1.RAGnull CD4 T cells. 2 Address correspondence and reprint requests to Dr. Duncan Howie, Sir William SAGE libraries were generated as previously described (33). Two li- Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, United King- braries were constructed from cells stimulated for 7 days with (DBYT) dom. E-mail address: [email protected] or without (DBY) exogenous human rTGF␤ added at 2 ng/ml. The other two libraries were identical to DBY and DBYT apart from isolation of 3 Abbreviations used in this paper: Treg, regulatory T cell; BMDC, bone marrow- derived dendritic cell; DBY, cells stimulated without exogenous human TGF␤; CD4 T cells from the bone marrow-derived dendritic cell (BMDCs) by DBYT, cells stimulated with exogenous human TGF␤; DC, dendritic cell; iTreg, Ficoll centrifugation after 7 days and reculture in IL-2 at 2000 U/ml for TGF␤-induced Tregs; nTreg, natural Treg; SAGE, serial analysis of gene expression. a further week. These libraries were named DBY.IL2 and DBYT.IL2. Data analysis was performed using the software package !SAGEClus Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 (Ref. 33; www.molbiol.ox.ac.uk/pathology/tig). www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901070 4198 MS4A EXPRESSION BY REGULATORY T CELLS

Microarray analysis vealed using HRP-conjugated secondary Abs using ECL (SuperSignal; Pierce Biotechnology). Abs used were anti-␤-actin (Santa Cruz Biotech- Custom microarrays with immune system-directed bias were prepared nology), rabbit anti-MS4A4B (34), GITR (YGITR 765), Thy1.2 (30H12), using 70mer probes specific for 768 genes. Each probe was printed with p42/44 MAPK (3A7), and phospho p42/44 MAPK (Thr202/Tyr204) (both four replicates on nonadjacent areas onto amine slides (Genetix). De- from Cell Signaling Technology). tails of the microarray are available from ArrayExpress (Accession Number: A-MEXP-239; www.ebi.ac.uk/microarray-as/ae/). RNA integ- Flow cytometry rity was confirmed using a 2100 BioAnalyser (Agilent). Total RNA was reverse transcribed using Superscript III reverse transcriptase and la- Cells were stained with the following Abs: anti-GITR YGITR 765.4.2 beled as directed using the Cy3 and Cy5 3 DNA 900 dendrimer labeling (produced in house), fluorochrome-conjugated anti-CD25, anti-CD4, kit (Genisphere) using a two-step hybridization protocol with on-slide and anti-CD8 (BD Biosciences), PE- or allophycocyanin-conjugated anti- mixing on a Slide Booster (Advalytix). Arrays were scanned using a Foxp3 (eBioscience), and rabbit polyclonal anti-MS4A4B (34). Surface ScanArray Express HT scanner (PerkinElmer). Image analysis was per- staining for CD4 and CD25 was performed on ice for 30 min, followed by formed using BlueFuse v 2 (BlueGnome Ltd). washing in PBS and permeabilization with 0.5% saponin before staining for MS4A4B or rabbit Ig as a control for 30 min and detection with FITC-con- Quantitative real-time RT-PCR jugated goat anti-rabbit Ig. Blocking of MS4A4B staining with immunizing peptide (HQGTNVPGNVYKNHPGEIV) was performed in some cases for DNase I-treated RNA was reverse transcribed using StrataScript first strand 15 min before staining. Foxp3 was detected according to the protocol pro- synthesis kits (Stratagene) and random hexamer primers. For each exper- vided by eBioscience. For detection of IL-2 production by EL4 cells, cells imental sample, a minimum of two biological replicates was performed. cultured for 24 h with 10 ␮g/ml brefeldin A for the final 4 h were fixed in Individual gene assays were performed in triplicate in a 96-well format 2% formalin followed by permeabilization with 0.5% saponin. APC-con- using purpose-designed primers and fluorogenic probes (Eurogentec and jugated anti-IL2 (BD Biosciences) was used for IL-2 detection. supplementary Table I).4 Data was normalized to an endogenous control, such as hypoxanthine phosphoribosyltransferase or CD3␥-chain, and ex- Split-ubiquitin screening with Treg libraries pressed relative to a reference population using the comparative cycle Downloaded from Split-ubiquitin libraries were constructed by Dualsystems Biotech. The li- threshold method for relative quantification. braries DBYT.IL2/x-NubG and DBYT.IL2/NubG-x were directionally Retroviral transduction and FACS-sorting of A1.RAGnull T cells cloned into pPR3-C and pPR3-N, respectively. MS4A4B and MS4A6B cDNAs were cloned into the bait construct pBT3-STE. The bait constructs MS4A4B was cloned into the retrovirus vector MIGR1, which is bicis- were transformed into the yeast strain NMY51 (MATa his3⌬200 trp1–901

tronic and uses GFP as a reporter (a gift from Shohei Hori, RIKEN Re- leu2–3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-LacZ ade2:: search Centre for Allergy and Immunology, Yokohama City, Japan) using (lexAop)8-ADE2 GAL4) using standard procedures (35). For two-hybrid PCR. MIGR1 and MS4A4B-MIGR1 were packaged in Plat-E 293 cells (a screens, transformants were grown on selective medium lacking leucine, http://www.jimmunol.org/ gift from Dr. Siamon Gordon, Oxford University, Oxford, United King- tryptophan, histidine, and adenine, with addition of 20 mM 3-AT. Positive dom) by transfection using Fugene-6 reagent (Roche). Following 3 days of clones were sequenced by colony PCR using the primer set pPR3NFOR culture at 32°C, polybrene was added at 8 ␮g/ml. CD4 T cells from 5Ј-GTCGAAAATTCAAGACAAGG-3Ј and pPR3NREV 5Ј-AAGCGT A1.RAGnull mice were stimulated with peptide-pulsed dendritic cells GACATAACTAATTAC-3Ј for the NubGx library and pPRCFOR 5Ј- (DCs) for 3 days before transduction with MIGR1 or MS4A4B-MIGR1 TTTCTGCACAATATTTCAAGC-3Ј AND pPRCREV 5Ј-CTTGAC constructs. Plat-E supernatants containing the packaged MIGR1 or GAAAATCTGCATGG-3Ј for the xNubG library. Library plasmids were MS4A4B-MIGR1 virions were added to BMDC/T cell cultures and the isolated from positive clones and retransformed into NMY51 to test bait- culture plates were centrifuged at 3000 rpm for one hour at 37°C followed dependency. Only preys which activated the histidine and adenine reporters by 72 h of culture at 37°C. in the presence of MS4A4B or MS4A6B and not pCCW-Alg5 or pBT3STE

were considered true interactors. by guest on October 3, 2021 Production of stable MS4A4B-expressing EL4 cells Production of GITRL-Fc Fusion proteins The coding sequence of mouse MS4A4B was cloned into the BglII and BamH1 sites of the expression vector pMTF, which contains an EF1␣ Mouse GITRL (41 to 174 aa) was cloned into the expression plasmid promoter and neomycin resistance gene. EL4 cells were transfected by pMF-neo, which encodes EF1␣ promoter, neomycin resistance, murine electroporation with the pMTF-MS4A4B construct and cloned by limiting IgG2a signal sequence, and a human aglycosyl IgG1 Fc segment. The dilution. MS4A4B-expressing cells were selected by neomycin resistance resulting plasmid was transfected into CHO-S cells (Invitrogen) and stable using G418 at 1 mg per ml. Positive clones were confirmed using flow transfectants selected using G418. Fc-GITRL was purified using protein A cytometry for MS4A4B. agarose (Invitrogen). Proliferation assays Results ␤ CD4 T cells were stimulated with peptide-pulsed BMDCs or plate-bound Up-regulation of MS4A transcripts by TGF and Foxp3 anti-CD3 (145.2C11) in 96-well culture plates for 48 h. Tritiated thymidine transduction (Amersham Biosciences) at 0.018 MBq per well was added 16 hours before ␤ harvesting the cultures onto glass-fiber filters and scintillation counting TGF has an important role in the peripheral induction and main- using standard techniques. tenance of Foxp3 expression by Tregs (5, 8–10). SAGE library analysis and custom immune-biased DNA microarrays were used ELISA to gain insight into the molecular mechanisms underlying the IFN-␥ and IL-2 were measured by ELISA using paired Ab sets from BD TGF␤-induced conversion process. In the first approach, four new Biosciences. IFN-␥ was assayed using R46A2 and biotinylated XMG1.2 SAGE libraries were constructed from activated TCR-transgenic ␮ for detection. IL-2 was measured using 4 g/ml JES6–1A12 and biotin- CD4ϩ T cells from mice mono-specific for the male Ag H-Y in the ylated JES6–5H4 for detection. context of H2-Ek (A1.RAG1null), with or without added TGF␤ Western blotting according to Materials and Methods. A1.RAGnull mice contain no Cell lysis was conducted with ice-cold lysis buffer (Tris 20 mM pH 7.6, Foxp3-positive natural Tregs (nTregs), so any experimentally in- ϩ NaCl 140 mM, EDTA 2 mM, NaF 0.5 mM, sodium orthovanadate 1 mM, duced emergence of Foxp3-positive CD4 cells is due to de novo ␤-glycerophosphate 25 mM, sodium pyrophosphate 2 mM, benzamidine 2 induction. The suppressive properties of TGF␤-induced Tregs mM, DTT 0.5 mM) and complete EDTA-free protease inhibitors contain- (iTregs), Foxp3-transduced Tregs, and CD4ϩCD25ϩ nTregs are ing 0.5% Triton X-100 (Roche). Cell lysates were clarified by centrifugation at 14,000g for 15 min at 4°C. Lysates were subjected to SDS-PAGE, equivalent (data not shown). The data set of 48 libraries (described using ϳ10 ␮g protein per lane, and transferred onto polyvinylidene diflu- in detail in Ref. 11) was interrogated with a seed tag representing oride filters (Invitrogen). Filters were blocked for 1 h with 5% skim milk nTregs and iTregs; those tags which best fit the cell distribution of or 5% BSA and then probed with the indicated Abs. Bound Ab was re- the seed tag libraries indicated by bold typeface are listed in Fig. 1. Previously described Treg or anergy-associated transcripts were 4 The online version of this article contains supplemental material. abundantly expressed by these cells, CD27 (12), GITR (13), Tgtp The Journal of Immunology 4199

FIGURE 1. Seed tag analysis of Treg SAGE libraries. A, Forty-eight SAGE libraries encompassing Th1, Th2, Tr1, iTregs, CD25ϩCD45RBlow Tregs, tolerant and rejecting skin grafts, and activated or Downloaded from tolerogenic DCs (BMDCs and embryonic stem cell derived) were analyzed by seed-tag analysis. An idealised library distribution, or “seed-tag,” was constructed with distribution heavily weighted to nTregs and iTregs. Ranked genes that best ﬁt this proﬁle are shown. B, Expression of MS4A family http://www.jimmunol.org/ members in T cell and DC SAGE libraries. For de- tails of the libraries see Ref. 33. by guest on October 3, 2021

(14), and Nkd2 (15); however, the highest ranked transcript which down-regulated vs 11 up-regulated in response to TGF␤. There was expressed preferentially in the Treg libraries was MS4A4B. was also overlap in the identity of transcripts that were down- MS4A4B is a four-pass membrane protein of the MS4A family regulated by both Foxp3 and TGF␤. Five transcripts significantly (Fig. 1A). The MS4A family has at least 13 members on chromo- down-regulated following Foxp3 transduction were also decreased some 19 in the mouse (16). Within the 48 SAGE libraries, four by TGF␤ treatment (indicated in Tables I and II). The Th2-related additional MS4A members were identified with widely different transcripts IL-4 and IL-10 were significantly down-regulated in re- expression patterns (Fig. 1B). Notably, MS4A6B had a broadly sponse to both Foxp3 transduction and TGF␤ treatment. MS4A4B similar expression pattern to MS4A4B, albeit with lower tag num- and MS4A6C transcripts were significantly up-regulated following bers. MS4A6D was found to be expressed at the highest tag fre- expression of Foxp3. quency in libraries derived from DCs with a tolerizing bias i.e., We quantified the observed increase in MS4A4B expression in immature DCs differentiated from embryonic stem cells or murine response to Foxp3 using real-time quantitative RT-PCR. As shown bone marrow in the presence of TGF␤ or IL10 (Fig. 1B). in Fig. 2A, there was a 3-fold increase in MS4A4B transcripts in The SAGE data were validated using a custom DNA microarray peptide-activated A1.RAGnull T cells following the addition of approach. This data is available from Array Express under acces- TGF␤ to the culture medium. If the cells were subsequently ex- sion number E-MEXP-1157, (www.ebi.ac.uk/microarray-as/ae/). panded in high-dose IL-2 (2000U/ml), the MS4A4B transcripts cDNA from A1.RAGnull T cells transduced with Foxp3 or treated increased 6-fold compared with the equivalent cells expanded in with or without TGF␤ were analyzed. These results are presented the absence of TGF␤. A 12-fold increase in MS4A4B transcripts in Tables I and II. Fourteen transcripts were significantly down- was observed following retroviral transduction of A1.RAGnull T regulated vs nine up-regulated by Foxp3 transduction and 26 cells with Foxp3 in comparison with those transduced with empty 4200 MS4A EXPRESSION BY REGULATORY T CELLS

Table I. Gene transcripts up and down modulated in response to Table II. Gene transcripts up and down modulated in response to TGF␤ treatment of A1.RAGnull CD4 T cellsa Foxp3 transduction of A1.RAGnull CD4 T cellsa

TGF␤/Peptide FoxP3/vector

Increased by TGF␤ Ͼ 2SD alone Increased by FoxP3 Ͼ 2SD Lipocalin 2 10.09 NKG2-D 4.92 Transcription factor Sp5 9.88 Placental calcium-binding protein (18A2) 2.88 Lysozyme C 9.52 Lymphotoxin-␤ 2.74 Cathepsin S 8.28 Paired immunoglobin-like type 2 RB 2.58 Integrin ␣-E 8.13 GABA receptor-associated protein-like 1 2.37 Cell division protein kinase 2 7.60 Dynein intermediate chain 2 2.10 Lymphotactin 6.88 MS4A4B 1.99 CD14 5.58 MS4A6C 1.94 CCR-7 5.32 Thymosin ␤-4 1.82 Glycogen phosphorylase, liver form 5.00 Decreased by FoxP3 Ͼ 2SD Sodium channel 3 4.94 Prostaglandin E2 receptor 0.34 Decreased by TGF␤ Ͼ 2SD GATA-3 0.34 Wolframin 0.90 Phosphatidylserine synthase 2 0.34 Lymphocyte Ag 6 complex, locus C 0.89 Leukotriene B4 receptor 1 (LTB4-R 1)b 0.31 Glutaredoxin 0.87 Chemokine (C-X-C motif) receptor 6 0.30 P-selectin glycoprotein ligand 1 0.84 Vitamin D3 receptor (VDR) 0.29 DNA-binding protein inhibitor ID-2 0.82 IL-10b 0.28 Downloaded from 5-hydroxytryptamine 5B receptor 0.74 Regulator of G-protein signaling 1 (RGS1) 0.23 Integrin ␣-L 0.70 Early growth response protein 1 (EGR-1) 0.23 Protein tyrosine phosphatase 4a1 0.69 Ring ﬁnger protein 128 0.20 Cathepsin D 0.68 Extracellular matrix protein 1b 0.07 Tissue transglutaminase 0.68 IL-4 precursor (IL-4)b 0.07 Serine palmitoyltransferase 2 0.67 Interleukin-1 receptor, type II 0.02 V-CAM 1 0.66 Preproenkephalin 1 (Penk1), mRNAb 0.01

␤ http://www.jimmunol.org/ Integrin -2 0.64 a Cytochrome c oxidase polypeptide VIIb 0.59 Transcripts whose normalized intensity ratios of Foxp3:vector alone were b greater than two standard deviations from the mean were considered significant Leukotriene B4 receptor 1 0.58 changes and are included in the results. Osteopontin 0.58 b Modulated in common with TGF␤. IL-4b 0.57 Extracellular matrix protein 1b 0.54 IL-4R-␣ 0.54 Granzyme A 0.40 b splenocytes for CD4, CD25, Foxp3, and MS4A4B shows that IL-10 0.38 ϩ ϩ ϩ Preproenkephalin 1b 0.35 around 99% of CD4 CD25 Foxp3 nTregs stain positively for CCR-5 0.31 MS4A4B (Fig. 2F). Thus it would appear that MS4A4B expres- by guest on October 3, 2021 Ly-6A.2 0.26 sion is strongly influenced by Foxp3 expression and is induced in Spermidine synthase 0.24 cells expressing Foxp3. Dynein intermediate chain 2 0.19 At least thirteen MS4A genes are located in close vicinity on a Transcripts whose normalized intensity ratios of TGF␤:peptide alone were mouse chromosome 19. Their relative locations and transcriptional greater than two standard deviations from the mean were considered significant changes and are included in the results. orientation are shown in Fig. 2G. It has been proposed that the b Modulated in common with Foxp3. different MS4A genes arose during evolution by gene duplication (17). It is possible therefore that transcription of the different MS4A members is driven by similar promoter sequences. We used retrovirus. Using flow cytometry on permeabilized A1.RAGnull T real-time quantitative RT-PCR to quantify all 13 members of the cells, the protein was increased by one order of magnitude in re- MS4A family (Fig. 2G) and found that all of the MS4A members, sponse to TGF␤ (Fig. 2B). Western blotting with this Ab revealed with the exception of MS4A2, 3, 6D, and 8, were strongly up- a protein band of the expected 22kD size, which increased in regulated (5- to 10-fold increase) by TGF␤ or Foxp3 transduction. A1.RAGnull T cells in response to TGF␤. MS4A4B is expressed early in thymic development independently MS4A4B is expressed by nTregs and its expression can be of Foxp3 induced by Foxp3 expression We next tested whether Foxp3 was an absolute requirement for T Flow cytometry was performed to test whether up-regulation of cell MS4A4B expression during development by staining thymo- MS4A4B protein observed in response to TGF␤ was secondary to cytes and splenocytes from the CBA/Ca mouse strain and induction of Foxp3 in these cells. A1.RAGnull T cells, which had A1.RAGnull mice (the latter having no detectable Foxp3 expres- been retrovirally transduced with MIGR1-Foxp3 or empty MIGR1 sion at any stage of T cell development). MS4A4B was detectable (Fig. 2C) and FACS sorted for the top 20% brightest GFP express- at equivalent low levels on double-negative thymocytes in both ing cells, showed a substantial increase in MS4A4B expression strains of mice (supplemental Fig. 1A). MS4A4B expression tran- compared with Foxp3 negative cells. To determine whether a pos- siently dropped in wild-type mice at the double-positive stage but itive correlation exists between cellular Foxp3 protein expression not in A1.RAGnull double positives, implying that MS4A4B may and MS4A4B, a further FACS sort experiment was performed be required for positive selection in A1.RAG thymocytes but not where four fractions based on GFP brightness were sorted from in conventional mice. CD4 single-positive thymocytes in both A1.RAGnull T cells retrovirally transduced with MIGR-Foxp3 strains expressed bimodal expression of MS4A4B at equivalent (Fig. 2D). As shown in Fig. 2, D and E, there was correlation intensities (supplemental Fig. 1A). MS4A4B was expressed by between the level of expression of Foxp3 as shown by FACS and Foxp3-positive thymocytes with a single peak of staining (supple- the intensity of MS4A4B by Western blot. Staining of CBA/Ca mental Fig. 1B). On analysis of splenic CD4ϩ and CD8ϩ T cells, The Journal of Immunology 4201 Downloaded from http://www.jimmunol.org/

FIGURE 2. TGF␤ and Foxp3 induces up-regulation of MS4A genes. A, Real-time RT-PCR of MS4A4B transcripts from peptide-activated A1.RAGnull T cells after TGF␤ treatment or Foxp3 retroviral transduction. B, Left, FACS analysis of MS4A4B expression on A1.RAGnull T cells following TGF␤ treatment. Shaded histogram indicates MS4A4B Ab with the immunizing peptide. Right, Western blot performed on lysates of activated CD4ϩ cells with by guest on October 3, 2021 anti-MS4A4B polyclonal Ab or anti-␤ actin as loading control. C, FACS analysis of MS4A4B expression following retroviral expression of Foxp3 in A1.RAGnull T cells. D, MoFlow FACS sorting profiles of Foxp3 expressing (by GFP signal) A1.RAGnull T cells. E, Western blot of MS4A4B on the sorted cells represented in the FACS plots of D. F, CD4, CD25, Foxp3, and MS4A4B on splenocytes from CBA/Ca mice. R1 represents gated CD4ϩCD25ϩ cells. R2 represents CD4ϩCD25ϩFoxp3ϩ cells. G, Upper panel, Diagrammatic representation of the MS4A family genomic organization on mouse chromosome 19. Vertical lines represent non-MS4A family genes. Lower left, Real-time RT-PCR using SYBR green quantification of the MS4A family. Ratios are null plotted on log10 scale of fold differences in signal between Foxp3-transduced A1.RAG T cells and empty MIGR1 retroviral vector-transduced cells. Lower right, Ratios of fold changes in A1.RAGnull T cells cultured in the presence of TGF␤ (DBYT) vs the absence of TGF␤ (DBY). Results are representative of two separate experiments. Primers used are listed in supplementary Table II. RQ, relative quantification; -ve, negative. it was clear that peripheral CD4ϩ T cells exhibit a nearly two-log LexA-VP16 fusions (Fig. 3A)inS. cerevisiae strain NMY51 and range in expression of MS4A4B in both A1.RAG and CBA/Ca tested for their ability to activate reporter transcription of the histidine mice (supplemental Fig. 1C). Thus, MS4A4B is expressed early in and adenine reporters only in the presence of a strong ubiquitin reas- T cell development in a Foxp3-independent manner, although in sociation (supplemental Fig. 2, C and D, middle and right panels). the absence of Foxp3 there is a greater range of expression than Having established that MS4A4B and MS4A6B activates transcrip- in nTregs, which express MS4A4B strongly. tion of the yeast nutritional reporters with a membrane-located positive control construct, we screened the two iTreg libraries with MS4A4B interacts in cis with costimulatory receptors MS4A4B and MS4A6B as baits. Six integral membrane proteins, We next explored possible signaling mechanisms for MS4A4B in T GITR, ORAI1, MS4A6B, Ly6E, GPR177 and H2-DMa, were shown effectors and Tregs using a yeast split-ubiquitin screen. The advantage to interact with MS4A4B in a bait-dependent fashion (Fig. 3B). We of this approach over conventional yeast two-hybrid screens is that confirmed the MS4A4B-MS4A6B interaction with our MS4A6B protein interaction is measured at the plasma membrane, rather than as screen, obtaining two MS4A4B interacting clones in addition to 12 truncated fusions in the yeast nucleus (18). Two libraries were con- clones of GITR and two of MS4A6B, suggesting MS4A6B self-as- structed from A1.RAGnull T cells (see Materials and Methods). RNA sociation. We then confirmed the GITR/MS4A4B interaction using used for library construction was validated to test expression of GITRL-Fc to coimmunoprecipitate MS4A4B-GITR complexes from Foxp3, MS4A4B, MS4A6B, and MS4A6C (supplemental Fig. 2A). iTreg lysates (Fig. 3C). GITR has been shown to enhance TCR-driven Additionally, an in vitro suppression assay was performed using signaling via p42/44 MAPK (ERK) (19). We next tested whether, in DBYT cells to confirm that they displayed suppressive activity on the GITR-expressing EL4 cell line, over- expression of MS4A4B Dby Ag-activated A1.RAGnull T cells (supplemental Fig. 2B). might costimulate ERK signaling. As shown in Fig. 3D, following MS4A4B and MS4A6B were expressed as C-terminal ubiquitin- activation of EL4 cells with anti-CD3 Abs, a 3- to 8-fold increase in 4202 MS4A EXPRESSION BY REGULATORY T CELLS Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021 FIGURE 3. MS4A4B and MS4A6B associate with GITR. A, Schematic illustrating topology of bait and prey constructs used in the split-ubiquitin FIGURE 4. MS4A4B lowers the activation threshold of CD4 T cells. A, B screen. , Identities of bait-dependent MS4A4B and MS4A6B binding ELISA for IL-2 and IFN-␥ in cell culture supernatants of MIGR1- or partners from the yeast split-ubiquitin screen. C, Confirmation of GITR- MIGR1-MS4A4B-transduced A1.RAGnull CD4 T cells in response to the MS4A4B association on iTreg. Immunoprecipitation of GITR was per- indicated stimuli. B, Tritiated thymidine uptake of Moflow sorted MS4A4B formed using GITRL-Fc or human Fc as a control. Rat anti-Thy1.2 was transduced A1.RAGnull CD4 T cells in response to titrated amounts of Dby also used as an irrelevant surface receptor control. Immunoprecipitates peptide presented by female CBA/Ca BMDC. Data represent mean val- D .p Ͻ 0.05 ,ء .were Western blotted with the indicated Abs. , Results of Western anal- ues Ϯ SEMs of three separate experiments ysis of ERK phosphorylation following 72 h stimulation with plate-bound anti-CD3 with or without anti-CD28. Intensity of phosphorylated ERK and total ERK bands were quantified using the Aida image analysis program hancement of the proliferative response was more apparent at (Raytest). lower concentrations of antigenic peptide (0.1 nM) than at higher amounts. These differences were not due to alterations in surface TCR or CD28 levels (data not shown). Thus, MS4A4B lowers the ERK phosphorylation was seen in EL4 cells expressing MS4A4B threshold for Ag-induced activation of primary T cells, resulting in relative to those untransfected. Thus, the presence of MS4A4B en- increased IL-2 synthesis and proliferation. hances proximal TCR signaling. MS4A4B enhances GITR function MS4A4B enhances IL-2 synthesis and activation of T cells We next asked whether the interaction of MS4A4B with GITR had The two canonical members of the MS4A family MS4A1 (CD20) functional consequences for GITR costimulation. To this end we and MS4A2 (Fc␧R1␤) serve to amplify signals transduced through stimulated MS4A4B-expressing EL4 cells (Fig. 5A) with anti-CD3 the BCR and high affinity IgE receptor Fc␧RI, respectively. As and GITRL-Fc or anti-GITR. Upon stimulation of EL4–4B cells MS4A4B was observed to augment TCR signaling in EL4 cells, with anti-CD3 and anti-GITR, p44 ERK phosphorylation was in- we pursued the idea that it may serve to lower TCR sensitivity to creased at all time-points in comparison to EL4 cells lacking Ag in primary T cells. We transduced A1.RAGnull T cells with MS4A4B. To determine whether augmented functional re- MS4A4B, Moflow sorted the 20% highest MS4A4B-expressing sponses to GITR triggering could be measured in MS4A4B- cells, then tested their activation to Ag or anti-CD3 stimulation. T expressing cells, we analyzed IL-2 production by flow cytom- cell IL-2 and IFN-␥ production and proliferation were moderately etry (Fig. 5C). Measurement of IL-2 production by these cells but significantly increased in cells transduced with MS4A4B com- revealed that expression of MS4A4B in the absence of GITR stim- pared with those transduced with empty retrovirus (Fig. 4). En- ulation was sufficient to increase IL-2 production in response to The Journal of Immunology 4203

A In this study we found multiple significantly up- and down- regulated genes in Foxp3- and iTreg populations. These observa- tions are in accordance with recent reports that Foxp3 acts as both a transcription repressor and activator of multiple genes, both di- rectly and indirectly (21). MS4A4B was up-regulated most strongly and specifically by Treg populations in response to both TGF␤ and transduction with Foxp3. A correlation was shown be- B tween Foxp3 expression and MS4A4B using retrovirally expressed Foxp3. CD4ϩCD25ϩFoxp3ϩ nTregs were uniformly shown to be strongly positive for MS4A4B expression. MS4A4B is a member of a family of 13 highly homologous MS4A genes on mouse chromosome 19, which is syntenic with human chromosome 11 (17). Control of expression of the MS4A members is highly cell type specific (22, 23). Likewise, MS4A4B, MS4A4C, MS4A6B, MS4A6C, and MS4A6D have differential C distributions in our SAGE libraries. Their transcription is coordi- nately up-regulated by TGF␤ or expression of Foxp3. The arche- typal MS4A members CD20 (MS4A1), Fc␧R1␤ (MS4A2), and HTm4 (MS4A3) are components of larger oligomeric complexes (22, 24–26) in which they transmit positive and negative signals Downloaded from (27–30). The possibility that MS4A4B amplifies positive TCR signals via clustering other surface costimulatory receptors is sup- ported by our data. This might occur in Tregs or cells destined to become such through interacting with multiple other cell surface receptors in a way analogous to that described for MS4A1–3 or as part of a structure similar to the tetraspanin web (reviewed in Ref. http://www.jimmunol.org/ 31). The results of our split-ubiquitin screens show clearly that lateral associations within the MS4A family can occur, such as FIGURE 5. Enhanced GITR costimulation in EL4 cells expressing MS4A4B7MS4A6B and MS4A6B7MS4A6B. In addition, MS4A4B. A, FACS plots of EL4 and EL4 cells stably expressing MS4A4B MS4A4B and MS4A6B have other interactors, both shared and (EL4–4B). The left panel shows MS4A4B staining on EL4 cells (gray unique, showing the potential for MS4A4B/6B-mediated recruit- histogram) and EL4–4B cells (black histogram). The middle panel shows ment of each other’s cis-ligands, such as GITR, into supramolecu- GITR staining on EL4 cells and the right panel shows EL4–4B. Isotype lar signaling clusters on Tregs. control staining is shown in gray. B, Western blot for phosphorylated-ERK Interaction of GITR with MS4A4B and MS4A6B is of consid- by guest on October 3, 2021 and nonphosphorylated ERK. EL4 and EL4–4B cells were stimulated with erable interest as GITR is highly expressed on CD4ϩCD25ϩ anti-CD3 (KT3) and anti-GITR (YGITR-765) cross-linked with anti-rat ϩ Foxp3 nTregs and was originally described in the context of mAb for the indicated times. All Abs were used at 3 ␮g per ml. Western blotting was performed according to Materials and Methods. C, Intracel- Tregs as a negative regulator of Treg activity (13). Subsequently, lular IL-2 staining on EL4 and EL4–4B cells stimulated for 24 h with a positive role in T cell activation has been demonstrated for GITR anti-CD3 with either human IgG1-Fc, GITRL-Fc, or anti-GITR added at 3 on non-Tregs when triggered by agonist anti-GITR mAbs or the ␮g/ml. Results representative of three separate experiments. natural ligand GITRL (32). Our data show that cells expressing MS4A4B have increased sensitivity to GITRL stimulation, resulting in increased IL-2 production. This may be a consequence of anti-CD3 stimulation by 4- to 5-fold. Remarkably, whereas EL4 GITR clustering in response to its cis-interaction with MS4A4B, cells lacking MS4A4B responded with little increase of IL-2 resulting in signal amplification. It is conceivable that different production to GITRL or anti-GITR stimulation, 10% of those signals may be generated by different GITR binding partners, such cells expressing MS4A4B consistently produced IL-2 in reas MS4A4B/6B. sponse to either GITRL or anti-GITR. Thus, MS4A4B appears What might be the value of this to the immune system and why to enhance the capacity of GITR to promote IL-2 production would such adapters be found in both Th1 cells and Tregs? We and T cell activation. propose that expression of MS4A family members may serve to reduce the threshold for TCR-mediated signaling directed toward Discussion danger-associated extrinsic Ags. In this way, the system would Strongly autoreactive T cells are eliminated in the thymus in the stay responsive until the last traces of Ag had gone. In contrast, course of T cell development. To ensure self-tolerance, a further expression of such members in Tregs would enable them to remain fraction of the positively selected T cells are directed toward Treg reactive to danger-free self Ags, even if, as a result of negative cell function and exported to the periphery as CD4ϩCD25ϩ selection, their TCR were of lower affinity. nTregs (2–4). Emerging evidence in recent years also points to In summary, Foxp3 and TGF␤ signaling are linked to up-regu- further routes of peripheral Treg induction (5, 6, 9, 20). The lation of the MS4A gene family. All Foxp3-positive cell popula- common theme arising from these peripheral Treg induction tions in our experiments expressed elevated levels of MS4A4B. modes is one of repeated stimulation of naive T cells under Moreover, MS4A4B and MS4A6B form complexes with each suboptimal activation conditions where danger signals are ab- other as well as GITR, in addition to multiple cell surface recep- sent. It is unknown what molecular mechanisms instruct some tors. MS4A4B interaction with GITR results in augmented GITR Ag-naive cells to preferentially expand to danger-free Ag with signals and T cell IL-2 production. These associations may explain the outcome of a Treg phenotype whereas other naive cells how MS4A4B over-expression was shown to increase activation remain quiescent to such stimuli. of T cells. 4204 MS4A EXPRESSION BY REGULATORY T CELLS

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