Formation T Cell Motility and Immunological Synapse CD80

CD28−CD80 Interactions Control Regulatory T Cell Motility and Immunological Synapse Formation

This information is current as Timothy J. Thauland, Yoshinobu Koguchi, Michael L. of October 1, 2021. Dustin and David C. Parker J Immunol published online 29 October 2014 http://www.jimmunol.org/content/early/2014/10/29/jimmun ol.1401752 Downloaded from

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 29, 2014, doi:10.4049/jimmunol.1401752 The Journal of Immunology

CD28–CD80 Interactions Control Regulatory T Cell Motility and Immunological Synapse Formation

Timothy J. Thauland,* Yoshinobu Koguchi,*,1 Michael L. Dustin,†,‡ and David C. Parker*

Regulatory T cells (Tregs) are essential for tolerance to self and environmental Ags, acting in part by downmodulating costimulatory molecules on the surface of dendritic cells (DCs) and altering naive CD4 T cell–DC interactions. In this study, we show that Tregs form stable conjugates with DCs before, but not after, they decrease surface expression of the costimulatory molecule CD80 on the DCs. We use supported planar bilayers to show that Tregs dramatically slow down but maintain a highly polarized and motile phenotype after recognizing Ag in the absence of costimulation. These motile cells are characterized by distinct accumulations of LFA-1–ICAM-1 in the lamella and TCR-MHC in the uropod, consistent with a motile immunological synapse or “kinapse.” However, in the presence of high, but not low, concentrations of CD80, Tregs form stationary, symmetrical synapses. Using blocking Abs, we show that, whereas CTLA-4 is required for CD80 downmodulation, CD28–CD80 interactions are critical for

modulating Treg motility in the presence of Ag. Taken together, these results support the hypothesis that Tregs are tuned to alter Downloaded from their motility depending on costimulatory signals. The Journal of Immunology, 2014, 193: 000–000.

oxp3+ CD4 regulatory T cells (Tregs) are critical for shown that Tregs can downmodulate expression of CD80 and controlling immune responses and promoting tolerance to CD86 on dendritic cells (DCs) (6, 12–15), a process that involves F autoantigens and commensal gut microflora (1, 2). The transendocytosis (16) and depends on PKC-h (17). Down- transcription factor Foxp3 is necessary for Treg development and modulation of CD80/86 on DCs requires expression of CTLA-4 http://www.jimmunol.org/ function (3–5), and it controls the expression of hundreds of genes, by Tregs, and occurs in an Ag- and LFA-1–ICAM-1-dependent several of which, including Ctla4, are crucial for Treg development manner (14, 15). CD80 downmodulation is observed even when or function (2). Indeed, CTLA-4 is required for proper Treg func- nTregs and DCs are cocultured in the presence of LPS, IFN-g,or tioning, as specific ablation in Foxp3+ cells results in severe auto- other potent maturing stimuli, indicating that CTLA-4–mediated immunity (6). The costimulatory molecule CD28 is required for regulation of DCs by nTregs is a powerful mechanism for altering Treg development and survival (7, 8). In addition to thymus-derived DC function (15). In vivo imaging experiments have demonstrated or natural Tregs (nTregs), Foxp3+ Tregs can be induced from pe- that in the presence of Ag, Tregs prevent sustained naive T cell– ripheral CD4 T cells both in vitro and in vivo (9). DC interactions (18, 19) and induce dysfunction in CTLs in tumor Tregs use a variety of processes, involving both contact- tissue (20). Tregs were not observed directly interacting with by guest on October 1, 2021 dependent and cytokine-mediated mechanisms, to suppress im- naive T cells or CTLs in these experiments, and instead formed mune reactions in vitro and in vivo (10, 11). Several reports have Ag-dependent, unstable tethering interactions with DCs coincid- ing with marked downregulation of CD80 and CD86 (20). In experiments with TCR transgenic Tregs and naive CD4 T cells *Department of Molecular Microbiology and Immunology, Oregon Health & Science cocultured with peptide-pulsed splenic DCs, Tregs were highly University, Portland, OR 97239; †Department of Pathology, New York University School of Medicine, New York, NY 10016; and ‡Kennedy Institute of Rheumatology, motile, swarmed around Ag-loaded DCs, and outcompeted the Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, naive cells for space around the DCs (15). This swarming phe- University of Oxford, Oxford OX3 7FY, United Kingdom notype, possibly in conjunction with CTLA-4–mediated down- 1Current address: Earle A. Chiles Research Institute, Robert W. Franz Cancer modulation of CD80/86, could explain the in vivo imaging results Research Center, Providence Cancer Center, Portland, OR. described above. Owing to the importance of Tregs in modulating ORCIDs: 0000-0003-4983-6389 (M.L.D.); 0000-0003-4456-2788 (D.C.P.). the surface phenotype of DCs and the sparseness of studies ex- Received for publication July 10, 2014. Accepted for publication October 8, 2014. amining the immunological synapse (IS) formed by Tregs, the This work was supported by National Institutes of Health Grants R01 AI050823 (to factors governing Treg–DC interactions are of interest. D.C.P.), R01 AI092080 (to D.C.P.), and R01 AI043542 (to M.L.D.) and by a Well- come Trust Principal Research Fellowship (to M.L.D.). T.J.T. was supported by In this study, we tested the hypothesis that costimulatory mol- National Institute for Allergy and Infectious Diseases Grants T32 AI007472 and ecules on the surface of APCs are responsible for determining Treg T32 AI078903. motility and IS structure. Given that Tregs are capable of down- Address correspondence and reprint requests to Dr. David C. Parker, Department of modulating CD80 molecules on the surface of DCs, and that Molecular Microbiology and Immunology, L220, Oregon Health & Science Univer- sity, 3181 SW Sam Jackson Park Road, Portland, OR 97239 or Dr. Timothy J. CD28–CD80 interactions can modulate synapse structure (21), we Thauland at the current address: Division of Immunology, Allergy, and Rheumatol- examined the role that CD80 plays in Treg–DC IS formation. We ogy, Department of Pediatrics, Stanford University, 300 Pasteur Drive, Grant Build- show that in vitro–generated Tregs, similar to nTregs, are capable ing Room S374, Stanford, CA 94305. E-mail addresses: [email protected] (D.C.P.) or [email protected] (T.J.T.) of downmodulating CD80 on the surface of DCs, and that alter- The online version of this article contains supplemental material. ation of the DC surface phenotype affects subsequent Treg–DC Abbreviations used in this article: BMDC, bone marrow–derived DC; DC, dendritic interactions. Using supported planar bilayers as artificial APCs, cell; DIC, differential interference contrast; IS, immunological synapse; MCC, moth we demonstrate that Tregs slow down, but are still highly motile, cytochrome c peptide; MSD, mean square displacement; nTreg, natural regulatory when they recognize Ag in the absence of costimulation. How- T cell; pMHC, peptide-MHC; Treg, regulatory T cell. ever, the presence of high, but not low, levels of CD80 is sufficient Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 to induce the formation of symmetrical, nonmotile IS by Tregs.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401752 2 COSTIMULATION CONTROLS Treg MOTILITY

We suggest a model wherein highly motile Tregs are constantly Olympus 320/0.75 numerical aperture Plan Apo objective. This system scanning the surface of APCs, only stopping when the APC is included an Applied Precision chassis with a motorized XYZ stage, activated and displaying high levels of costimulatory activity. This WeatherStation environmental chamber, Olympus IX71 inverted fluorescent microscope, xenon lamp, and CoolSnap HQ2 camera. The DeltaVision behavior may allow Tregs specific for self and environmental Ags SoftWorx software package was used for image acquisition. to efficiently downmodulate costimulatory molecules on inappropriately activated cells while ignoring resting cells displaying Supported planar bilayer experiments those Ags. GPI-linked forms of Oregon Green 488 labeled I-Ek (200 molecules/mm2) and Cy5-labeled ICAM-1 (300 molecules/mm2) were incorporated into dioleoylphosphatidylcholine bilayers exactly as described (25, 26). For Materials and Methods some experiments, GPI-linked CD80 was incorporated into bilayers at 20, Animals 40, or 200 mm2 (27). These bilayers were supported on a coverslip in Heterozygous AD10 TCR transgenic mice on a B10.BR background, a Bioptechs flow cell and were loaded with 100 mM MCC or hemoglobin peptide (GKKVITAFNEGLK) in a PBS/citrate buffer (pH 4.5) for 24 h at specific for pigeon cytochrome c 88–104 and reactive against moth cyto- 7 chrome c (MCC) 88–103 (22), were provided by S. Hedrick (University of 37˚C (25). T cells (10 ) in 1 ml HBS buffer with 1% BSA were injected California at San Diego, La Jolla, CA) by way of P. Marrack (National onto bilayers at 37˚C. In some experiments, anti-CD28 and/or anti–CTLA- Jewish Medical Center, Denver, CO). B6.Cg-Foxp3tm2Tch/J (Foxp3-GFP) 4 Fab fragments were added to the T cells at 50 or 100 mg/ml 10 min prior + to injection onto the bilayers. Images were acquired every minute for 30– mice were obtained from The Jackson Laboratory. Male AD10 mice were 3 bred to homozygous female Foxp3-GFP mice and the AD10+ progeny 60 min on the DeltaVision system using an Olympus 60/1.42 numerical were used as a source of TCR transgenic GFP+ nTregs. All mice were aperture Plan Apo objective. housed in specific pathogen-free conditions and used in accordance with Imaris 6.3 (Bitplane) was used to track cells interacting with the bilayer. National Institutes of Health guidelines under an animal protocol approved To ensure that only cells productively interacting with the bilayer were by the Oregon Health & Science University Institutional Animal Care and analyzed, Cy5 fluorescence (ICAM-1 accumulation) was tracked auto- Downloaded from Use Committee. matically with the Spots tool in Surpass mode. In situations where the ICAM-1 signal was ambiguous, DIC images were examined to determine Abs whether the cell was flattened against the bilayer. DIC images were used to track cells interacting with bilayers loaded with irrelevant peptide, as there The Abs used for flow cytometry were as follows: anti-CD4 Alexa Fluor 488 was only sparse ICAM-1 accumulation under these conditions. (GK1.5; eBioscience), anti-CD4 PerCP (RM4-5; BioLegend), anti-CD152 PE (UC10-4B9; BioLegend), anti-Foxp3 Alexa Fluor 647 (150D; Bio- Scoring T cell–bilayer interactions

Legend), anti-CD11c FITC and PE (HL3; BD Biosciences), anti-CD25 http://www.jimmunol.org/ PerCP (PC61; BioLegend), and anti-CD80 FITC and PerCP-Cy5.5 (16- Cells were scored as nonmotile when they moved one cell diameter or less 10A1; BD Biosciences). during the entire imaging session. Cells were considered to have reformed an IS when they stopped forward progress, lost their uropod and ICAM-1 DC culture arc, and remained nonmotile for at least 10 min. Cells were considered to have broken symmetry when they were nonmotile for 10 min or more before Bone marrow–derived DCs (BMDCs) were cultured as described (23). gaining motility and moving more than one cell diameter. The vast majority Briefly, bone marrow cells were cultured for 9 d in bacteriological Petri of cells were symmetrical when first contacting the bilayer, but this state was dishes in complete RPMI 1640 media supplemented with GM-CSF su- transient (much less than 10 min) and cells that subsequently became motile pernatant (final concentration, 20 ng/ml). On day 9 of culture, immature were not considered to have broken symmetry. Cells were scored as having DCs were plated in LabTek II eight-well chambers (no. 1.5; Nunc) or six- a uropod anchor when the uropod of a motile cell was attached to the same well plates in fresh media with 20 ng/ml GM-CSF and 1 mg/ml LPS. place for at least 10 min, causing the cell to pivot. by guest on October 1, 2021 In vitro Treg polarization Results CD4+ cells and B cells were purified from AD10 and B10.BR spleen cell suspensions, respectively, using EasySep immunomagnetic negative se- Tregs transition from stable to motile contacts with DCs, lection (StemCell Technologies). B cells (5 3 106) and CD4+ cells (2.5 3 concomitant with downmodulation of costimulatory molecules 106) were cultured in 1 ml complete RPMI 1640 media in six-well plates with 2.5 mM MCC peptide, 20 ng/ml TGF-b, 100 U/ml IL-2, and 10 nM We set out to study the IS formed between Tregs and APCs. To all-trans retinoic acid. On days 2 and 3, 1 ml media supplemented with generate a population of Ag-specific cells for use in imaging 100 U/ml IL-2 was added to the cultures. To confirm that T cells were experiments, Tregs were induced in vitro by culturing purified CD4 polarized to an Treg phenotype, cells were fixed, permeabilized, and T cells from AD10 TCR transgenic mice with naive B cells in the stained for CD4, CTLA-4, and Foxp3. Fixation and permeabilization presence of MCC peptide, TGF-b, IL-2, and all-trans retinoic acid, reagents were from BioLegend. Cells were used on day 4. as previously described (28). After 4 d of culture under Treg CD80 downmodulation assay conditions, the cells were almost completely polarized to a Treg Day 9 BMDCs (1.6 3 105) were seeded onto 12-well plates and treated phenotype with high levels of Foxp3 and CTLA-4 (Fig. 1A). LPS as described above. One day later, the indicated numbers of day 4 Day 4 Tregs were loaded with CFSE, introduced to peptide-loaded Tregs were added per well in the presence or absence of 2.5 mm MCC. In BMDCs, and imaged over time. some experiments, Fab fragments of anti-CD28 (E18; gift from Thomas The initial interactions between Tregs and BMDCs were very Hunig)€ (24) or anti–CTLA-4 (UC10-4F10-11; Bio X Cell) were added at 100 mg/ml. The Fab fragments were generated with a Pierce Fab prepa- stable, with the vast majority of Tregs flattening against BMDCs ration kit (Thermo Scientific). As a control, Tregs were not added to some and maintaining a rounded, unpolarized shape for the duration of wells. Twenty-four hours later, the cells were harvested and stained for the experiment (Fig. 1B, Supplemental Video 1). However, after 30 min on ice for CD4, CD11c, and CD80. All Abs were used at 1:200. 24 h of coculture, the Tregs became highly motile and formed EDTA (1 mM) was included in the FACS buffer to discourage continued large, swarming clusters around the BMDCs (Fig. 1C). To further interactions. Samples were collected on a BD FACSCalibur with CellQuest software and analyzed with FlowJo (Tree Star). investigate this change in motility, we added fresh Tregs labeled with CFSE to the Treg-BMDC cocultures and observed their Imaging Treg–DC interactions behavior. Surprisingly, the freshly added Tregs also displayed Day 9 BMDCs (3 3 104) were seeded onto coverslips in eight-well a motile, swarming phenotype. As shown in Fig. 1D and chambers and treated with LPS as described above. One day later, 1.5 3 Supplemental Video 2, the freshly added Tregs adopted a polar- 5 10 CFSE-loaded, day 4 Tregs were added to the wells. Imaging com- ized shape and crawled along the surface of the BMDCs. The menced as soon as Tregs were added to the wells. Differential interference contrast (DIC) and fluorescent images were obtained every minute for 2 h. tendency of freshly added cells to form motile, rather than stable, All imaging was conducted at 37˚C with 5% CO2. Wide-field imaging was contacts indicates a change in the BMDCs rather than a change in performed with an Applied Precision DeltaVision system using an the Tregs following 24 h of culture together. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Tregs modify DC phenotype, concomitant with a change in the stability of Treg–DC interactions. (A) TCR transgenic CD4 T cells were cultured under Treg-polarizing conditions and expressed high levels of Foxp3 and CTLA-4 at day 4 poststimulation. Naive B10.BR CD4 T cells are shown for comparison. (B) Tregs were labeled with CFSE and introduced to BMDCs loaded with MCC peptide. Most Treg–DC interactions were stable. For further examples of stable Treg–DC contacts, see Supplemental Video 1. (C) Twenty-four hours after addition, the Tregs were highly motile and swarmed around BMDCs. (D) Fresh, CFSE-labeled Tregs were added to BMDCs 24 h after the addition of unlabeled Tregs. These freshly added Tregs had a polarized phenotype and crawled along the surface of BMDCs. The still images shown in (D) are from Supplemental Video 2. (E–H) Tregs were incubated with BMDCs in the presence or absence of MCC peptide, and the BMDCs were assayed at 24 h for surface levels of CD80. The T cell/DC ratios were 1:1 (E), 5:1 (F), or 10:1 (G and H). Fab fragments of anti–CTLA-4 (G) or anti-CD28 (H) were included in the cultures at 100 mg/ml. Results are representative of three (B–D) or six (E–H) independent experiments. Scale bars, 10 mm.

nTregs have been shown to cluster around DCs after a 12-h the DCs in these experiments were often highly motile. To care- incubation, and this phenotype was associated with down- fully measure the interactions between Tregs and APCs in two modulation of CD80 and CD86 on the DC surface (15). To de- dimensions, we used supported planar bilayers containing GPI- termine whether changes in the surface phenotype of the DCs linked, fluorescently labeled ICAM-1 and MHC class II (I-Ek) could be responsible for the altered motility of Tregs after a 24-h loaded with MCC peptide (25). coculture, we conducted assays to measure downmodulation of Day 4 Tregs were injected onto the supported planar bilayers and CD80. Incubating Tregs with BMDCs resulted in Ag-specific examined for the distribution of peptide-MHC (pMHC)–TCR and downmodulation of CD80 at both 1:1 and 5:1 Treg/BMDC ratios ICAM-1–LFA-1 interactions at the Treg–APC interface. Although (Fig. 1E, 1F). In agreement with published work (15), Fab frag- the cells fluxed calcium immediately upon touching the bilayers ments of an anti–CTLA-4 Ab completely inhibited CD80 down- (data not shown), we did not observe symmetrical IS with well- modulation, even at a 10:1 Treg/BMDC ratio (Fig. 1G). Treatment defined supramolecular activation clusters, in agreement with a with Fab fragments of an anti-CD28 Ab known to efficiently block recent report (Fig. 2A) (29). Instead, the Tregs interacting with CD28–CD80 interactions (24) resulted in a decrease in the effi- the bilayer had the polarized morphology of motile cells, with ciency of CD80 downmodulation, but the effect was relatively a well-defined uropod. An arc of ICAM-1 was located in the mid- minor compared with anti–CTLA-4 treatment (Fig. 1H). cell region (Fig. 2B), as previously described for motile T cells (30). The strongest accumulation of pMHC-TCR was in the uro- CD80 modulates IS formation in Tregs pod of the crawling cells (Fig. 2B), consistent with a phenotype Although qualitatively informative, the Treg–DC interactions were seen for CD8 T cell blasts (31). As shown in Fig. 2C, the Tregs not conducive to precise measurements of Treg motility because broke radial symmetry and formed motile synapses almost im- 4 COSTIMULATION CONTROLS Treg MOTILITY

FIGURE 2. Tregs form immunological kinapses on supported planar bilayers containing pMHC and ICAM-1. (A) Representative field of Tregs interacting with bilayers containing pMHC and ICAM-1. DIC (left) and fluorescence (right) images with pMHC (green) and ICAM-1 (red) are shown. (B) Representative image of a motile Treg with ICAM-1 accumulated in the midbody of the cell and pMHC accumulation in the uropod. (C) Representative time-lapse images of the initial Treg–bilayer contact showing pMHC (green) and ICAM-1 (red). Tregs displayed a polarized phenotype rapidly upon Downloaded from contact with the bilayer. Images are representative of seven independent experiments. Scale bars, 10 mm. mediately upon interacting with the bilayers. Motile IS, or In the absence of CD80, the vast majority of Tregs moved freely “kinapses,” have been previously reported in naive T cells in vitro across the bilayer, but the addition of CD80 caused some motile (32) and in vivo (33). cells to become anchored to the substrate via their uropods, Our examination of Treg–DC interactions suggested that the frustrating the forward motion of the cells and causing them to turn http://www.jimmunol.org/ level of costimulatory molecules on the APC surface could in circles (Fig. 4E). This behavior was seen at both concentrations modulate Treg behavior. Therefore, we examined IS and kinapse of CD80, but was especially prominent when high levels were formation in the presence of 0, 40, and 200 molecules/mm2 GPI- used (Fig. 4F). linked CD80 in the bilayers. In the presence of low levels of CD80 It has been shown that TCR transgenic mice contain a pop- (40 molecules/mm2), the Tregs predominantly formed motile ulation of nTregs that are most likely derived from cells expressing kinapses, similar to the phenotype observed in the absence of endogenous TCR a-chains in addition to transgenic a-and CD80 (Fig. 3A, 3B, Supplemental Videos 3, 4) . However, when b-chains (34). Indeed, we found that a fraction of naive CD4+ Tregs were introduced to bilayers containing 200 molecules/mm2 cells from AD10 mice expressed Foxp3 (Fig. 5A). To confirm by guest on October 1, 2021 CD80, their movement was drastically reduced, with some of the that our findings were generalizable to nTregs, we crossed Tregs forming stable IS (Fig. 3C, Supplemental Video 5). Analysis Foxp3-GFP mice to our AD10 TCR transgenic mice and intro- of the motility of Tregs on bilayers containing varying amounts of duced purified CD4 cells to bilayers (Fig. 5B, 5C). Unlike acti- CD80 showed that high levels of CD80 significantly decreased the vated Tregs, a significant fraction of the nTregs was sessile mean square displacement (MSD) of Tregs over time (Fig. 3D, regardless of CD80 concentration (Fig. 5E), possibly because 3E). As shown in Fig. 3F, the average speed of Tregs was sig- these cells are relatively metabolically inactive compared with nificantly reduced in the presence of high levels of CD80. Because in vitro–generated Tregs. Nevertheless, the presence on CD80 changes in the centroid of relatively nonmotile cells can inflate in the bilayers resulted in a significant decrease in motility speed measurements, we also measured the total displacement of (Fig. 5D–F). Interestingly, naive non-Tregs were much less mo- each cell and divided by track duration. This measurement yielded tile than Tregs in the absence of CD80. These results demonstrate values close to 0 for nonmotile cells and showed that Tregs are that nTregs behave similarly to in vitro–induced Tregs in the dramatically less motile when introduced to bilayers with high presence and absence of CD80. levels of CD80 (Fig. 3G). We also noted a decrease in the straightness of Treg tracks in the presence of high levels of CD80 Treg motility decreases dramatically in the presence of Ag (Fig. 3H). Tregs interacting with bilayers accumulated pMHC-TCR in the Although the fraction of cells that maintained symmetrical, contact zone (Fig. 2B) and fluxed intracellular calcium (data not nonmotile interactions with the planar bilayers for the entirety of shown), but they were almost always continuously motile over a 45- to 60-min experiment was 3-fold higher in the presence of 200 the entire imaging experiment (Fig. 4B). This led us to ask molecules/mm2 CD80, this result did not quite meet the standard whether there were any changes in Treg motility upon Ag rec- for statistical significance (Fig. 4A). However, significantly fewer ognition, or whether motility was only altered in the presence of cells were motile for the entire experiment in the presence of high high levels of CD80. Therefore, we introduced AD10+ Tregs to levels of CD80 (Fig. 4B). These results indicate that in addition to bilayers containing ICAM-1 and MHC loaded with either cognate slowing the motility of Tregs, high levels of CD80 also induced peptide (MCC) or an irrelevant peptide (hemoglobin). As shown stable IS formation. As shown in Fig. 2C, Tregs that formed in Fig. 6A, in the absence of cognate Ag, Tregs moved rapidly kinapses broke radial symmetry almost immediately upon con- across the bilayers with only transient accumulations of ICAM-1, tacting the bilayers. However, we observed that a fraction of the and at no time were pMHC-TCR clusters apparent. Analysis of cells regained symmetry and formed a stable IS during the course the tracks made by Tregs in the absence of Ag showed that Tregs of the experiment (Fig. 4C). The percentage of Tregs displaying scanning the surface of an APC lacking cognate Ag move much this behavior was dramatically increased in the presence of high faster and straighter than did Tregs that have recognized Ag levels of CD80 (Fig. 4D). (Fig. 6B–F). Thus, Tregs are highly motile when scanning the The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 3. High concentrations of CD80 reduce the motility of Tregs. Tregs were introduced to bilayers containing pMHC, ICAM-1 and 0 (A), 40 (B), 2 or 200 (C) molecules/mm CD80. Time-coded tracks (blue to white from the beginning to the end of the experiment) are overlaid on ICAM-1 images. Scale by guest on October 1, 2021 bars, 10 mm. The tracks shown are representative of four (A), two (B), or six (C) independent experiments. Supplemental Videos 3, 4, and 5 are time-lapse videos of Tregs interacting with bilayers containing 0, 40, and 200 molecules/mm2 CD80, respectively. (D) MSD over time is plotted for three densities of CD80, and the MSD at 10 min is shown (E). The average speed (F), displacement/duration (G), and straightness (track displacement/length) (H) of Treg tracks on bilayers with 0, 40, or 200 molecules/mm2 CD80 are shown. Results (D–H) are from one representative experiment of three with n = 38, 52, and 41 for 0, 40, and 200 molecules/mm2 CD80, respectively. Error bars represent SEM. One-way ANOVAs followed by Tukey HSD tests were used to determine p values. A p value .0.05 was considered not significant. surface with ICAM and MHC without specific Ag, slow consid- Taken together, these data show that CD28–CD80 interactions control erably in the presence of Ag, and slow even more, often to the the motility of Tregs. point of stopping, in the presence of Ag and high levels of costimulation. Discussion In this study, we propose a model wherein Treg–DC interactions CD28–CD80 interactions control Treg motility are modulated by relative levels of costimulatory molecules. In the To dissect the mechanism by which CD80 modulates Treg motility, absence of Ag recognition, Tregs rapidly migrate over the surface we incubated cells with Fab Abs against CD28 or CTLA-4 and of APCs, scanning for their cognate Ag. Upon Ag recognition, introduced them to bilayers containing pMHC, ICAM-1, and high Tregs form either relatively slow-moving kinapses or nonmotile IS levels of CD80. As shown in Fig. 7A, blockade of CD28 resulted in depending on the levels of costimulatory molecules. In the pres- highly motile Tregs in the presence of CD80. In contrast, many of ence of high levels of costimulation, symmetrical ISs are favored. the Tregs treated with anti–CTLA-4 formed relatively nonmotile After levels of costimulatory molecules are reduced, kinapse contacts with the bilayer or had their uropods anchored (Fig. 7B). formation is favored and the Treg starts migrating again. Thus, by We analyzed the tracks of Tregs loaded onto bilayers without alternating between kinapses and stable ISs, Tregs could effi- CD80 or with CD80 and either anti-CD28, anti–CTLA-4, or no ciently contact and modulate levels of costimulatory molecules or Ab. Tregs treated with anti-CD28 were much more motile than otherwise suppress inappropriately activated DCs. cells in the no Ab control condition and were almost as motile as Our results are consistent with a recent study showing that cells loaded onto bilayers lacking CD80 (Fig. 7C–G). Treatment nTregs interacting with supported planar bilayers containing 90 with anti–CTLA-4 did not have an appreciable effect on motility molecules/mm2 CD80 form unstable ISs, but because CD80 was by most measures, as 10 min MSD displacement/duration and not tested at higher concentrations in that study, the profound speed were not different from the no Ab control (Fig. 7C–E). effect that CD80 has on Treg motility was not discovered (29). In Treatment with anti–CTLA-4 did result in somewhat straighter contrast to our study, human Tregs were observed to form tracks, although the significance of this finding is unclear (Fig. 7G). hyperstable ISs compared with T effector cells (35). This dis- 6 COSTIMULATION CONTROLS Treg MOTILITY Downloaded from

FIGURE 4. Treg–APC interactions have an altered phenotype in the presence of CD80. The percentage of Tregs that were either nonmotile (A)or continuously motile (B) during the entire length of the experiment is shown. (C) An example of a cell transitioning from a motile phenotype to a stable IS. http://www.jimmunol.org/ DIC (top) and fluorescence (bottom) images with pMHC (green) and ICAM-1 (red) are shown. Scale bar, 10 mm. (D) The percentage of cells that reformed IS over the length of an experiment. (E) An example of a cell attached to the bilayer via its uropod. The cell’s track is overlaid on DIC images. Scale bar, 10 mm. (F) The percentage of cells that were anchored by their uropods over the course of an experiment. Data in (A), (B), (D), and (F) show the mean of the fraction of cells displaying a given phenotype from two (40 molecules/mm2 CD80) or three (0 and 200 molecules/mm2 CD80) independent experiments with n = 115, 87, and 129 for 0, 40, and 200 molecules/mm2 CD80, respectively. Error bars represent SEM. One-way ANOVAs followed by Tukey HSD tests were used to determine p values. A p value .0.05 was considered not significant. crepancy could be due to a species difference, but is probably best crosslinking may simply augment TCR-induced signaling and/or by guest on October 1, 2021 explained by differences in the TCR stimulus provided to the cytoskeletal remodeling, or it could provide a unique signal. A Tregs. Zanin-Zhorov et al. (35) introduced T cells to supported similar phenomenon has recently been observed in thymocytes, in planar bilayers containing anti-CD3ε. It is likely that the presence which CD28-dependent actin remodeling was required for maxi- of such a strong and qualitatively different TCR stimulus obviates mal activation after TCR triggering (39). the need for CD80 to induce stopping of Tregs, masking the dif- An annular ring of CTLA-4 surrounding the central supramo- ferences in motility we noted when using saturating concen- lecular activation cluster characterizes the IS formed by induced trations of a physiological TCR ligand. However, note that CD252 Tregs interacting with bilayers containing CD80 (40). This dense non-Tregs displayed rapid symmetry breaking and motility in the accumulation of CTLA-4 may increase the efficiency of costim- same conditions, demonstrating that anti-CD3 does not generate ulatory molecule downmodulation. Thus, the reduced motility and a durable stop signal for all human T cells. increased IS formation we observed in the presence of high levels How does signaling through CD28 alter Treg motility? Cross- of costimulation may serve two purposes: 1) to increase the dwell linking CD28 results in Lck-mediated phosphorylation of the time of a Treg on a particular activated APC, and 2) to enhance the YMNM motif and recruitment of PI3K and Grb2 (36). The activity efficiency of downmodulation. The IS functions as a platform for of PI3K leads to activation of Akt, whereas Grb2 recruits the the polarized secretion of effector molecules toward the APC (41, guanine nucleotide exchange factor Vav1, resulting in activation 42). Therefore, it is possible that additional Treg functions, in- of the small GTPases Rac1 and Cdc42, which in turn activate cluding Ag-specific delivery of perforin (43, 44) or IL-10 (42), WAVE2 and WASp, respectively, ultimately leading to Arp2/3- may require the formation of a stable IS. mediated actin remodeling (37). Additionally, a proline-rich do- CTLA-4 ligation has been reported to increase motility of T cells main of CD28 is known to recruit several kinases downstream of both in vitro and in vivo (45–47). Because CTLA-4 has a much the TCR, including Lck and PKC-u (36). We hypothesize that higher affinity for CD80 than CD28 (48), it follows that high Tregs have a relatively higher threshold for activation than do levels of CD80 are needed to trigger the CD28-mediated stop conventional T cells, which allows them to monitor their envi- signal we see in our experiments. We suggest that the abundant ronment without being inappropriately activated, even in the face expression of CTLA-4 on Tregs tunes these cells to be highly of ubiquitously expressed, low-affinity cognate Ags. Under this motile unless they recognize Ag in the context of large amounts scenario, Tregs would require a robust signal through CD28 before of costimulation. Conventional CD4 T cells, unlike self-reactive transitioning from motile kinapses to stable synapses. In support of Tregs that are constantly seeing Ag, are able to efficiently scan a model where CD28 signaling through PI3K is critical for Treg APCs without this mechanism. In disagreement with our model, function, it has been shown that Tregs expressing an inactive form a recent report showed that activated CD4+CD25+ cells from of the PI3K catalytic subunit, p110d, are poor suppressors and fail CD282/2 mice did not migrate differently in lymph node slices to prevent inflammation in a colitis model (38). Strong CD28 from CD4+CD25+ cells from CD28+/+ mice (49). The apparent The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. CD80 reduces the motility of nTregs on supported planar bilayers. (A) Naive CD4 cells from B10.BR or AD10 mice were assayed for the expression of CD25 and Foxp3. (B and C) Naive AD10 mice CD4 cells were purified from the spleens of AD10 3 Foxp3-GFP mice and introduced to by guest on October 1, 2021 supported planar bilayers containing pMHC, ICAM-1, and 0 (B) or 200 (C) molecules/mm2 CD80. Time-coded tracks (blue to white from the beginning to the end of the experiment) are overlaid on ICAM-1 images. The movements of both nTregs (GFP+) and naive CD4 cells (GFP2) were followed over time, but only the nTreg tracks are shown. Scale bars, 10 mm. (D) MSD over time is plotted for nTregs and naive CD4 cells on bilayers lacking CD80 and on bilayers with 200 molecules/mm2 CD80. The MSD at 10 min and the fraction of cells with an MSD of .500 um2 are shown (E and F). The results are pooled from three experiments with n = 48, 40, 73, and 77 for nTreg/no CD80, nTreg/CD80 200 molecules/mm2, naive/no CD80, and naive/CD80 200 molecules/mm2, respectively. Error bars represent SEM (E) or 95% confidence interval (F). The p values were determined with a one-way ANOVA followed by a Tukey HSD test (E) or a Fisher exact test (F). disparity between these results and ours could be explained by the described for WASp-deficient Tregs, defective IS formation may fact that the absence of CD28 has a profound effect on the de- also be important. Tregs incapable of IS formation could also be velopment and homeostasis of Tregs (8). In the absence of CD28, deficient in CD80/86 downmodulation. Thus, constant surveillance the small number of surviving peripheral Tregs may have com- and modulation of DCs (via IS-dependent downmodulation of pensatory mechanisms for activation and motility. costimulatory molecules) may be required to prevent autoimmune Mice that have CD28 specifically ablated in Foxp3+ cells de- disease. Consistent with this idea, Tang et al. (19) have demon- velop severe multiorgan autoimmunity (50). We suggest that strated that nTregs from BDC2.5 TCR Tg mice (which have TCRs efficient downmodulation of costimulatory molecules on acti- specific for an Ag associated with pancreatic b cells) are motile vated APCs presenting self-Ag may be a CD28-dependent in the pancreatic lymph node when transferred into NOD mice. function that is missing in these mice. This hypothesis leads to However, when the same nTregs are transferred into NOD. the prediction that blocking CD28 will result in less efficient CD282/2 mice lacking Tregs, they are nonmotile (19). Given the downmodulation, and we did see a modest decrease in down- results presented in the present study, it is likely that the DCs in modulation in the presence of anti-CD28 (Fig. 1H). Alternatively, the Treg-deficient animals had relatively high levels of CD80/86 CD28-dependent arrest and symmetrical synapse formation may compared with animals containing endogenous Tregs, thereby be required for other Treg suppressive functions, as mentioned altering the behavior of the transferred Tregs. Higher levels of above. costimulatory molecules on DCs in the pancreatic lymph node of Mice deficient in the actin regulatory protein WASp develop these already diabetes-prone animals could partially explain why chronic colitis, and Tregs from these mice are poor suppressors NOD mice lacking Tregs develop diabetes more quickly than do wild- both in vitro and in vivo (51–53). WASp is also required for type mice (54). T cells to maintain IS symmetry (30). Although defective mi- To maintain tolerance and immune homeostasis, Tregs need to gration to lymphoid tissue likely plays a role in the phenotype monitor the activation state of all the DCs in the body. Our finding 8 COSTIMULATION CONTROLS Treg MOTILITY Downloaded from

FIGURE 6. Tregs migrate rapidly in the absence of cognate Ag. Tregs were introduced to bilayers containing ICAM-1 and I-Ek loaded with either MCC or irrelevant (hemoglobin) peptides. (A) Representative time-coded tracks (blue to white from the beginning to the end of the experiment) of Tregs migrating on bilayers containing MHC loaded with hemoglobin peptide overlaid on an ICAM-1 image are shown. Scale bar, 10 mm. (B) MSD over time is plotted for Tregs interacting with bilayers loaded with MCC or hemoglobin peptide, and the MSD at 10 min is shown (C). The straightness (D), speed (E), and average displacement/duration (F) of Treg tracks on bilayers with MCC or hemoglobin peptide are shown. Results (B–F) are from one representative

experiment of two with n = 62 and 84 for MCC and hemoglobin peptide, respectively. Error bars represent SEM. The p values were determined with a two- http://www.jimmunol.org/ tailed Student t tests. Hb, hemoglobin peptide. that Ag can slow Treg migration may help explain how Tregs suppressive signals. The functional consequences of regulation of accumulate where their Ags are displayed. High CD80/CD86 levels Treg motility and synapse formation by CD28 will be a fertile ﬁeld then alter Treg behavior further to enable Ag-speciﬁc delivery of of investigation in future experiments. by guest on October 1, 2021

FIGURE 7. CD28–CD80 interactions are responsible for modulating Treg motility. Tregs were introduced to bilayers containing pMHC, ICAM-1, and 200 molecules/mm2 CD80 in the presence of anti–CTLA-4 (A) or anti-CD28 (B) Fab Abs. Time-coded tracks (blue to white from the beginning to the end of the experiment) are overlaid on ICAM-1 images. Scale bars, 10 mm. The tracks shown are representative of two (A) or three (B) independent experiments. (C) MSD over time is plotted for Tregs on bilayers lacking CD80 and on bilayers with 200 molecules/mm2 CD80 with or without anti–CTLA- 4 and anti-CD28 Fab Abs, and the MSD at 10 min is shown (D). The average speed (E), average displacement/duration (F), and straightness (track displacement/length) (G) of Treg tracks on bilayers with or without CD80 and Fab Abs are shown. Results (C–G) are from one representative experiment of three with n = 135, 92, 113, and 109 for no CD80, CD80, CD80 plus anti-CD28, and CD80 plus anti–CTLA-4, respectively. Error bars represent SEM. One- way ANOVAs followed by Tukey HSD tests were used to determine p values. A p value .0.05 was considered not signiﬁcant. The Journal of Immunology 9

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