CD99 Is a Novel Therapeutic Target for Control of T Cell–Mediated Central Nervous System Autoimmune Disease Ryan C

Home , CD99

Cutting Edge: CD99 Is a Novel Therapeutic Target for Control of T Cell−Mediated Central Nervous System Autoimmune Disease This information is current as of October 1, 2021. Ryan C. Winger, Christopher T. Harp, Ming-Yi Chiang, David P. Sullivan, Richard L. Watson, Evan W. Weber, Joseph R. Podojil, Stephen D. Miller and William A. Muller J Immunol 2016; 196:1443-1448; Prepublished online 15

January 2016; Downloaded from doi: 10.4049/jimmunol.1501634 http://www.jimmunol.org/content/196/4/1443

References This article cites 52 articles, 19 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/196/4/1443.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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. Th eJournal of Cutting Edge Immunology

Cutting Edge: CD99 Is a Novel Therapeutic Target for Control of T Cell–Mediated Central Nervous System Autoimmune Disease Ryan C. Winger,*,1 Christopher T. Harp,† Ming-Yi Chiang,† David P. Sullivan,* Richard L. Watson,* Evan W. Weber,* Joseph R. Podojil,† Stephen D. Miller,† and William A. Muller* Leukocyte trafficking into the CNS is a prominent fea- recruitment into the CNS is a prominent pathological feature ture driving the immunopathogenesis of multiple scle- driving multiple sclerosis (MS) and its animal model, exper- rosis and its animal model, experimental autoimmune imental autoimmune encephalomyelitis (EAE) (3, 4). encephalomyelitis. Blocking the recruitment of inflam- Before mediating tissue destruction in CNS autoimmunity, matory leukocytes into the CNS represents an exploit- lymphocytes interact with adhesion molecules on the BBB to Downloaded from able therapeutic target; however, the adhesion molecules reach their cognate Ag (5). In addition, the TEM of inflam- that specifically regulate the step of leukocyte diapede- matory APCs, including inflammatory myeloid-derived den- sis into the CNS remain poorly understood. We report dritic cells (iDCs), macrophages, and B cells, from the that CD99 is critical for lymphocyte transmigration periphery into the CNS is an early event, and their accumulation persists throughout the course of disease (6–8).

without affecting adhesion in a human blood–brain http://www.jimmunol.org/ barrier model. CD99 blockade in vivo ameliorated ex- Therefore, elucidating the mechanisms governing the TEM of lymphocytes and inflammatory APCs into the CNS is perimental autoimmune encephalomyelitis and decreased critical to understanding the pathogenesis of disease. The the accumulation of CNS inflammatory infiltrates, in- + + integrin VLA-4 mediates firm adhesion to endothelium in the cluding dendritic cells, B cells, and CD4 and CD8 periphery (9–13) and the CNS (14–16) and was demon- T cells. Anti-CD99 therapy was effective when admin- strated to play an integral role in inflammatory WBC accu- istered after the onset of disease symptoms and blocked mulation in the CNS (17–20). However, inflammatory relapse when administered therapeutically after disease WBCs may use alternative mechanisms that specifically gov-

symptoms had recurred. These findings underscore ern the step of diapedesis to infiltrate the CNS and mediate by guest on October 1, 2021 an important role for CD99 in the pathogenesis disease. of CNS autoimmunity and suggest that it may CD99 is a 32-kDa surface glycoprotein that is concentrated serve as a novel therapeutic target for controlling at cell borders of all endothelium and is expressed diffusely on neuroinflammation. The Journal of Immunology, the surface of WBCs (21, 22). Homophilic WBC–endothelial 2016, 196: 1443–1448. CD99 interactions are critical for TEM in vitro (21–23) and in inflammatory settings within the periphery in vivo (24– 26). Blocking WBC CD99 inhibits TEM to the same extent eukocyte diapedesis (transendothelial migration [TEM]) as blocking endothelial CD99 (21, 22, 24), making it an is a tightly regulated process that is vital to inflam- attractive therapy for inflammatory-mediated diseases. Re- L mation and immune responses. TEM is mediated by a cently, we reported that CD99 mediates monocyte TEM set of adhesive molecular interactions between circulating across human BBB models in vitro (23). However, the role of leukocytes (WBCs) and the vascular endothelium. During CD99 for other WBC subsets at the BBB may be different, immune surveillance, a limited number of WBCs enter the and its role in the pathogenesis of CNS disease is unknown. CNS by crossing the blood–brain barrier (BBB) to detect In the current study, we examined the requirement of CD99 potential damaging agents (1, 2); however, exacerbated WBC for lymphocyte TEM in the BBB model in vitro and inves-

*Department of Pathology, Feinberg School of Medicine, Northwestern University, and R01 NS026543 (to S.D.M.), and National Cancer Institute Grant CA060553 to the Chicago, IL 60611; and †Department of Microbiology-Immunology, Feinberg School of Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core Facility. Medicine, Northwestern University, Chicago, IL 60611 Address correspondence and reprint requests to Dr. William A. Muller, Department of 1Current address: Department of Neurology, University of California San Francisco, San Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Francisco, CA. Avenue, Chicago, IL 60611. E-mail address: [email protected] ORCIDs: 0000-0001-9160-6646 (R.C.W.); 0000-0001-7000-8743 (C.T.H.); 0000- Abbreviations used in this article: BBB, blood–brain barrier; EAE, experimental auto- 0002-7462-3898 (D.P.S.); 0000-0002-5818-8515 (J.R.P.); 0000-0002-4802-2757 immune encephalomyelitis; iDC, inﬂammatory myeloid-derived dendritic cell; MS, (W.A.M.). multiple sclerosis; PDCA, plasmacytoid dendritic cell Ag; RR-EAE, relapsing- remitting EAE; SC, spinal cord; TEM, transendothelial migration. Received for publication July 29, 2015. Accepted for publication December 15, 2015. This work was supported by National Institutes of Health Grant T32 AI7476-17 and Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Heartland Afﬁliate American Heart Association Grant 15PRE22710025 (to R.C.W.), National Institutes of Health Grants R37 HL064774 and R01 HL046849 (to W.A.M.)

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501634 1444 CUTTING EDGE: ANTI-CD99 THERAPY AMELIORATES CNS INFLAMMATION AND EAE tigated the mechanism and efficacy of anti-CD99 therapy to TEM assay regulate relapsing-remitting EAE (RR-EAE). RR-EAE in the Quantitative endpoint TEM assays were performed as previously described SJL/J strain shares similar clinical and histopathological (21, 23, 24, 32–34). hallmarks that are germane to MS (27) and, thus, serves as a powerful tool for studying the immunopathogenesis of disease Mice and potential therapeutic interventions (28). We show that Female SJL/J mice (The Jackson Laboratory, Bar Harbor, ME) were housed CD99 is required for lymphocyte diapedesis, but not adhe- under specific pathogen–free conditions in the Northwestern University Center for Comparative Medicine Animal Facility. The Northwestern University sion, in a human BBB model in vitro. More importantly, Animal Care and Use Committee approved all protocols. anti-CD99 therapy reduced the clinical severity of RR-EAE and diminished T cell, iDC, and B cell accumulation in the Active induction and clinical evaluation of proteolipid protein 139– CNS. Thus, CD99 is a novel therapeutic target for the 151–mediated RR-EAE management of CNS inflammation and autoimmunity. Six- to eight-week-old female SJL/J mice were immunized s.c. over three sites on the flank with 100 ml an emulsion containing 50 mg proteolipid protein 139–151 and CFA, as previously described (7, 8). Disease was scored on scale Materials and Methods of 0 to 5, as previously described (8, 35). All procedures involving human subjects/materials were approved by the Institutional Review Board of the Northwestern University Feinberg School of In vivo Ab treatment Medicine. RR-EAE mice were randomized at the onset of clinical symptoms and treated i.p. with 100 mg rat–anti-CD99 mAb (clone 3F11) or control IgG (rat IgG;

Abs and reagents Downloaded from Jackson ImmunoResearch), beginning either at the onset of symptoms or at Mouse anti-human mAbs for VE-cadherin (clone hec1) and CD99 (clone relapse and every other day thereafter. hec2), Armenian hamster anti-mouse PECAM (clone 2H8), and rat anti- mouse CD99 (clone 3F11) mAbs were generated by hybridoma methodol- In situ whole-mount immunostaining ogies, as previously described (21, 24, 29–31). Rat anti-mouse CD3 (clone 17A2) mAb was purchased from eBioscience. Whole-mount immunostaining was performed, acquired, and analyzed as previously described (24, 36). Spinal cords (SCs) were harvested intact at disease peak and relapse by ﬂushing the vertebral column with saline (37) and Isolation and culture of human endothelial cells for the BBB model http://www.jimmunol.org/ The BBB model was cultured as described previously (23). Prior to TEM experiments, monolayers were activated by TNF (20 ng/ml) for 24 h and preincubated with apical CXCL12 (200 ng/ml; both from R&D Systems) for 20–30 min, as described previously (32–34).

Isolation of human leukocytes Lymphocytes were isolated from human PBMCs and mitogen activated (1 mg/ml PHA, Sigma Aldrich; 20 ng/ml IL-2, R&D Systems), as previously described (33, 34). CD4+ and CD8+ T cells were isolated from human by guest on October 1, 2021 PBMCs by viable cell sorting using mAbs against CD3, CD4, and CD8 by the Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core Facility (Feinberg School of Medicine, Northwestern University, Chicago, IL).

FIGURE 2. Anti-CD99 ameliorates RR-EAE and reduces histopathological burden. (A) Comparison of mean clinical score between mice treated (arrows) FIGURE 1. CD99 is required for lymphocyte TEM in a human BBB model with anti-CD99 or control IgG at the onset of clinical symptoms and every in vitro. TEM assays were performed with total lymphocytes (A) or purified other day thereafter. Error bars indicate SEM. (B) Whole-mount immuno- CD4+ and CD8+ T cells (C) on inflamed BBB model monolayers in the staining of SC parenchymal postcapillary venules (PECAM-1; red) and CD3+ presence of anti-CD99 or control mAbs (nonblocking anti–VE-cadherin). (B T cell (green) infiltrates from mice treated with anti-CD99 (lower panels)or and D) The number of lymphocytes that adhered to the monolayer was scored control IgG (upper panels) at peak of disease. Scale bars, 26 mm. Data are for multiple high-power fields in each monolayer. Data represent the mean 6 representative of three independent experiments (A and B) with a minimum SD of hundreds of TEM events from at least six replicates from two (C and D) of five mice/group and from .20 immunostainings performed on post- and three (A and B) independent experiments. **p , 0.001, unpaired t test mortem material from three animals/group (B). **p , 0.001, Mann–Whitney and two-way ANOVA. ns, not significant. U test. The Journal of Immunology 1445

Table I. Clinical scores of chronic treated RR-EAE mice

Treatment Onset Peak Remission Relapse Control IgG 0.34 6 0.12 2.50 6 0.24 0.71 6 0.22 1.50 6 0.31 Anti-CD99 0.36 6 0.12a 1.39 6 0.21* 0.61 6 0.24a 0.46 6 0.14* The mean clinical scores of anti-CD99– or control IgG–treated mice from three independent experiments were quantitated. Data are mean 6 SEM. aNonsignificant. *p # 0.001 versus control IgG, Mann–Whitney U test. then fixed in 4% paraformaldehyde overnight at 4˚C. This method removes CD99 reduces clinical severity and histopathological burden of the meninges. SCs were blocked and permeabilized in a solution of 0.2% RR-EAE Triton X-100, 2.5% BSA, 2% FBS, and 2.5% host-species serum in PBS overnight at 4˚C. SCs were incubated overnight at 4˚C with primary mAb To date, the role of CD99 in EAE is unknown. We determined against PECAM-1 and CD3 in blocking solution. SCs were incubated with the role of CD99 for WBC recruitment to the CNS in vivo DyLight 550 goat anti-Armenian hamster and DyLight 488 goat anti-rat secondary Ab in PBS at room temperature for 4 h. SCs were mounted in using proteolipid protein 139–151–induced RR-EAE in sagittal orientation, and parenchymal postcapillary venules (identified by SJL/J mice by administering anti-CD99 or control IgG i.p. PECAM and 20–40 mm in diameter) in the lumbar region associated with once mice became symptomatic. Anti-CD99 mAb given i.p. perivascular lymphocyte infiltrates (identified by CD3) were imaged using an was able to reach the CNS because it labeled endothelial UltraVIEW Vox imaging system equipped with a Yokogawa CSU-1 spinning disk. Images were acquired using Volocity software (Perkin Elmer), which junctions of the BBB (data not shown). Mice receiving anti- Downloaded from renders the optical sections into three-dimensional images. Image processing CD99 mAb displayed significantly lower clinical scores than and analysis were performed with ImageJ. Fig. 2 shows representative pro- controls (p # 0.001) at peak disease and relapse (Fig. 2A, jections of the whole stack using the maximum-intensity method. Table I). The majority of anti-CD99–treated mice displayed mild disease and were even protected against the development Cell isolation of relapse (Fig. 2A, Table II). Whole-mount immunostaining +

WBCs were isolated from digested SC and brain of individual PBS-perfused revealed reduced histopathological burden of CD3 T cells in http://www.jimmunol.org/ mice via Percoll gradient, as described previously (8, 38). the SCs of anti-CD99–treated mice compared with controls (Fig. 2B). Qualitatively similar results were seen in SCs of FACS mice examined at the peak of relapse (data not shown). CNS cells were stained with the indicated Abs (eBioscience and BD), as described previously (8, 38, 39): CD45, CD39, CD11b, CD11c, Ly6c, Anti-CD99 treatment diminishes inflammatory leukocyte infiltration CD19, plasmacytoid dendritic cell Ag (PDCA)-1, F4/80, CD3, CD4, CD8, in the CNS of RR-EAE mice and CD25. Intracellular staining for Foxp3 was performed per the manu- facturer’s instructions with an eBioscience staining buffer kit. Aqua/VID The in vitro findings from Fig. 1 and whole-mount staining in LIVE/DEAD Cell Stain was purchased from Life Technologies.

Fig. 2 suggest that the therapeutic efficacy of anti-CD99 is by guest on October 1, 2021 due to its ability to block the recruitment of lymphocytes to Statistical analysis the CNS. However, similar to MS, RR-EAE is characterized All statistical analyses were done using GraphPad Prism software (GraphPad, by extensive CNS perivascular infiltration of inflammatory San Diego, CA). APCs that perpetuate damage (3, 8, 43). In particular, iDCs are major players in CNS autoimmune pathology (8, 44), and Results and Discussion their numbers correlate with disease severity and progression CD99 blockade restricts lymphocyte TEM but not adhesion at the BBB (5, 8, 42, 43, 45–48). The molecular mechanisms governing the TEM of this pathogenic subset into the CNS are poorly Studies in EAE demonstrated that CNS-infiltrating lymphocytes understood. play a critical role in CNS autoimmune disease. We investi- The frequency of CNS-infiltrating WBC subsets known to gated the role of CD99 for lymphocyte TEM at the inflamed play a role in the pathogenesis of disease that express CD99 BBB model in vitro. BBB model monolayers were activated as was characterized by FACS on cells from dissociated brains described in Materials and Methods. TEM assays with poly- andSCsharvestedatdiseasepeak(Fig.3A,3B)fromfive clonal-activated lymphocytes were performed in the presence mice. We next enumerated the different CNS-infiltrating of blocking mAb against CD99 or nonblocking control inflammatory WBC populations in anti-CD99 or control mAb against VE-cadherin. Under control conditions, 65% mice by performing FACS at disease peak and relapse. There of adherent lymphocytes transmigrated, whereas anti-CD99 was a significant reduction in the absolute numbers of CNS ∼ . blocked TEM down to 19% ( 70% block in TEM) CD45hi-infiltrating B cells, iDCs, and CD4+ and CD8+ Tcells (Fig. 1A). Anti-CD99 blocked TEM of CD4+ and CD8+ T cells to a similar extent (Fig. 1C). Under all of the conditions examined, none of the mAb treatments significantly Table II. EAE severity of chronic treated RR-EAE mice affected lymphocyte adhesion to the monolayers (Fig. 1B, 1D), demonstrating that our observations are specifically due No Clinical Mild (Score 1–2) Severe (Score 3–5) Treatment Score (% [n]) (% [n]) (% [n]) to a block in TEM, as observed for monocytes (21, 23, 24) and neutrophils (22, 24, 36). This is in contrast to ICAM-1 Peak (40), VCAM-1 (17), ALCAM (41), and Ninjurin-1 (42), Control IgG 10 (4/40) 35 (14/40) 55 (22/40) Anti-CD99 25 (10/40) 62.5 (25/40) 12.5 (5/40) which were demonstrated to play a role in adhesion and Relapse subsequent diapedesis of inflammatory WBC subsets across Control IgG 11 (2/18) 83 (15/18) 6 (1/18) the BBB. Anti-CD99 50 (9/18) 50 (9/18) 0 (0/18) 1446 CUTTING EDGE: ANTI-CD99 THERAPY AMELIORATES CNS INFLAMMATION AND EAE

FIGURE 3. Inhibiting CD99 function prevents accumulation of inflammatory infiltrates in the CNS of RR-EAE mice. The percentages of CD45hi-infiltrating iDCs (CD11b+CD11c+ 2 Ly6chi), macrophages (CD11b+CD11c F4/80+), 2 plasmacytoid DCs (CD11b CD11c+PDCA-1+), and B cells (CD19+)(A) and Th cells (CD3+ CD4+), cytotoxic T cells (CD3+CD8+), and regulatory T cells (CD3+CD4+CD25+Foxp3+) (B) expressing CD99 from the brain and SC of individual mice at disease peak were quantitated Downloaded from by FACS. Line graphs are representative of five individual mice. Filled graphs represent fluorescence-minus-one control; open graphs represent CD99-expressing cells. The numbers of CD45hi-infiltrating CD11c+ iDCs, PDCA-1+ plasmacytoid DCs, F4/80+ macrophages, CD19+ B cells, CD4+ T cells, CD4+CD25+Foxp3+ reg- http://www.jimmunol.org/ ulatory T cells, and CD8+ T cells at disease peak (C and D) and relapse (E and F) were enumerated, using FACS, from brain and SCs of individual mice treated with anti-CD99 or control IgG. Data in (C)–(F) are mean absolute number of CD45hi CNS-infiltrating cells pooled from three independent experiments obtained from $12 mice/group. Error bars indicate SEM. **p ,

0.05, two-way ANOVA. (G and H) Peripheral by guest on October 1, 2021 blood leukocytes from anti-CD99– or control IgG–treated RR-EAE mice were collected via cardiac puncture at disease peak and relapse and counted manually (from Fig. 2A). Error bars indicate SEM. **p , 0.05. ns, not signiﬁcant by t test.

at disease peak and relapse in CD99-treated mice (Fig. 3C–F). WBC counts were slightly higher in CD99-treated animals CD3+CD4+ Th1 and Th17 cells were reduced to an equal compared with control IgG (Fig. 3G, 3H). Taken together, extent in anti-CD99–treated mice (data not shown). There these data suggest that anti-CD99 therapy specifically inhibited were no significant differences in the absolute numbers WBC migration into the CNS rather than depleting WBCs or of microglia (CD45loCD11bloCD11c+), macrophages, or inhibiting emigration from bone marrow. plasmacytoid dendritic cells between anti-CD99– and control IgG–treated RR-EAE mice. In contrast to the CNS, we ob- Anti-CD99 blocks disease relapse in ongoing EAE and reduces served no significant differences in absolute cell numbers in leukocyte accumulation in the CNS spleen and lymph nodes between anti-CD99–treated mice In the clinic, most patients with MS receive treatment fol- and controls at any time point assessed (data not shown). In lowing disease exacerbations, rather than prophylactically. addition, at disease onset and remission there were no signifi- Therefore, we took a more clinically relevant approach by cant differences in cell numbers between anti-CD99 and control treating mice with established EAE only during the secondary in any of the tissues examined. Importantly, peripheral blood phase of disease at relapse onset (∼23 d postimmunization). The Journal of Immunology 1447

Anti–VLA-4 prevents the binding of leukocyte a4 integrin to its endothelial ligand VCAM-1, thereby inhibiting adhesion and subsequent TEM into the CNS. The S1P receptor modulator FTY720 (fingolimod) sequesters WBCs in the lungs and secondary lymphoid organs (49), thus impeding homing of WBCs to the CNS. Our data demonstrate that targeting CD99 interrupts leukocyte extravasation into the CNS. Anti-CD99 specifically disrupts the step of diapedesis, but not adhesion or trafficking, making it a unique therapeutic target. Unlike the steps preceding TEM in the inflammatory response, diapedesis is arguably the only committed, nonreversible step of inflammation (50). Once WBCs undergo TEM, they are committed to carrying out their effector function in the inflammatory response (51). Preventing WBC infiltration into the CNS reduces the extent of CNS inflammation and, thereby, ameliorates MS symptoms; however, this does not come without risk (49, 52). Anti–VLA-4 and fingolimod therapies were reported to im- pede the ability to protect against latent and chronic CNS Downloaded from viral infections by impairing immune surveillance (53–56). It is unknown whether anti-CD99 therapy would increase the risk for opportunistic infection; fortunately, recent advances in the field have made it possible to assess the likelihood with simple Ab tests (49). Taken together, our study reveals a novel role for CD99 http://www.jimmunol.org/ in the TEM of inflammatory WBCs across the BBB in vivo and further emphasizes the importance of WBC recruitment in the pathogenesis of CNS autoimmune disease. Furthermore, because anti-CD99 specifically blocks diapedesis, anti-CD99 or small molecule antagonists of CD99 signaling (24) might serve as exploitable therapeutic targets for suppressing ongoing neuroinflammation in CNS autoimmune disease. by guest on October 1, 2021 Acknowledgments We thank Clifford D. Carpenter for excellent technical assistance. FIGURE 4. Anti-CD99 ameliorates relapse in ongoing RR-EAE and reduces CNS inflammatory infiltrates. (A) Animals with ongoing RR-EAE were treated with anti-CD99 or control IgG during the secondary phase of disease Disclosures at the onset of relapse. **p , 0.001, Mann–Whitney U test. CNS tissues were The authors have no financial conflicts of interest. harvested at the end of the experiment to enumerate inflammatory APC (B) and T cell (C) infiltrates in the CNS of RR-EAE mice, as described in Fig. 3. Data in (A) are representative of three independent experiments (n = 8 mice/ References group for each experiment). Data in (B) and (C) are pooled from 12 mice/ 1. Hickey, W. F. 2001. Basic principles of immunological surveillance of the normal treatment group. Error bars indicate SEM. **p , 0.05, versus control IgG, central nervous system. Glia 36: 118–124. two-way ANOVA. ns, not significant. 2. Ousman, S. S., and P. Kubes. 2012. Immune surveillance in the central nervous system. Nat. Neurosci. 15: 1096–1101. 3. Frohman, E. M., M. K. Racke, and C. S. Raine. 2006. Multiple sclerosis–the plaque and its pathogenesis. N. Engl. J. Med. 354: 942–955. 4. Lucchinetti, C., W. Bruck,€ J. Parisi, B. Scheithauer, M. Rodriguez, and Anti-CD99 therapy ameliorated the development and severity H. Lassmann. 2000. Heterogeneity of multiple sclerosis lesions: implications for the of the secondary phase of disease (Fig. 4A). Similar to our pathogenesis of demyelination. Ann. Neurol. 47: 707–717. hi 5. Ifergan, I., H. Kebir, M. Bernard, K. Wosik, A. Dodelet-Devillers, R. Cayrol, observations in Fig. 3, CD45 CNS-infiltrating iDCs, B cells, N. Arbour, and A. Prat. 2008. The blood-brain barrier induces differentiation of and CD4+ and CD8+ T cells were significantly reduced in migrating monocytes into Th17-polarizing dendritic cells. Brain 131: 785–799. 6. Henderson, A. P., M. H. Barnett, J. D. Parratt, and J. W. Prineas. 2009. Multiple mice that received anti-CD99 therapy at the onset of relapse sclerosis: distribution of inflammatory cells in newly forming lesions. Ann. Neurol. compared with controls. Importantly, as observed in Fig. 3B 66: 739–753. + + + + 7. Miller, S. D., E. J. McMahon, B. Schreiner, and S. L. Bailey. 2007. Antigen pre- and 3D, there was no reduction in CD3 CD4 CD25 Foxp3 sentation in the CNS by myeloid dendritic cells drives progression of relapsing regulatory T cell accumulation in the CNS following treat- experimental autoimmune encephalomyelitis. Ann. N. Y. Acad. Sci. 1103: 179–191. ment with anti-CD99 (Fig. 4C). Selective inhibition of ef- 8. Bailey, S. L., B. Schreiner, E. J. McMahon, and S. D. Miller. 2007. CNS myeloid DCs presenting endogenous myelin peptides ‘preferentially’ polarize CD4+ T(H)- fector, but not regulatory, T cell TEM by anti-CD99 has 17 cells in relapsing EAE. Nat. Immunol. 8: 172–180. important implications for translation into the clinical setting. 9. Elices, M. J., L. Osborn, Y. Takada, C. Crouse, S. Luhowskyj, M. E. Hemler, and R. R. Lobb. 1990. VCAM-1 on activated endothelium interacts with the leukocyte Blocking WBC extravasation into the CNS is an exploitable integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 60: and effective therapeutic target for treating CNS-directed 577–584. 10. Siegelman, M. H., D. Stanescu, and P. Estess. 2000. The CD44-initiated pathway autoimmune disease. Anti–VLA-4 (natalizumab) is a highly of T-cell extravasation uses VLA-4 but not LFA-1 for firm adhesion. J. Clin. Invest. potent and efficacious therapy for the management of MS. 105: 683–691. 1448 CUTTING EDGE: ANTI-CD99 THERAPY AMELIORATES CNS INFLAMMATION AND EAE

11. Berlin, C., R. F. Bargatze, J. J. Campbell, U. H. von Andrian, M. C. Szabo, 35. McCarthy, D. P., M. H. Richards, and S. D. Miller. 2012. Mouse models of S. R. Hasslen, R. D. Nelson, E. L. Berg, S. L. Erlandsen, and E. C. Butcher. 1995. multiple sclerosis: experimental autoimmune encephalomyelitis and Theiler’s virus- alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic induced demyelinating disease. Methods Mol. Biol. 900: 381–401. flow. Cell 80: 413–422. 36.Weber,E.W.,F.Han,M.Tauseef,L.Birnbaumer,D.Mehta,and 12. Chan, J. R., S. J. Hyduk, and M. I. Cybulsky. 2001. Chemoattractants induce a W. A. Muller. 2015. TRPC6 is the endothelial calcium channel that regulates rapid and transient upregulation of monocyte alpha4 integrin affinity for vascular leukocyte transendothelial migration during the inflammatory response. J. Exp. cell adhesion molecule 1 which mediates arrest: an early step in the process of Med. 212: 1883–1899. emigration. J. Exp. Med. 193: 1149–1158. 37. Terry, R. L., I. Ifergan, and S. D. Miller. 2016. Experimental Autoimmune 13. Huo, Y., A. Hafezi-Moghadam, and K. Ley. 2000. Role of vascular cell adhesion Encephalomyelitis in Mice. Methods Mol. Biol. 1304: 145–160. molecule-1 and fibronectin connecting segment-1 in monocyte rolling and adhesion 38. McMahon, E. J., S. L. Bailey, C. V. Castenada, H. Waldner, and S. D. Miller. on early atherosclerotic lesions. Circ. Res. 87: 153–159. 2005. Epitope spreading initiates in the CNS in two mouse models of multiple 14. Laschinger, M., and B. Engelhardt. 2000. Interaction of alpha4-integrin with sclerosis. Nat. Med. 11: 335–339. VCAM-1 is involved in adhesion of encephalitogenic T cell blasts to brain endo- 39. Bailey-Bucktrout, S. L., S. C. Caulkins, G. Goings, J. A. Fischer, A. Dzionek, and thelium but not in their transendothelial migration in vitro. J. Neuroimmunol. 102: S. D. Miller. 2008. Cutting edge: central nervous system plasmacytoid dendritic 32–43. cells regulate the severity of relapsing experimental autoimmune encephalomyelitis. 15. Jain, P., C. Coisne, G. Enzmann, R. Rottapel, and B. Engelhardt. 2010. J. Immunol. 180: 6457–6461. Alpha4beta1 integrin mediates the recruitment of immature dendritic cells 40. Bullard, D. C., X. Hu, T. R. Schoeb, R. G. Collins, A. L. Beaudet, and across the blood-brain barrier during experimental autoimmune encephalomyelitis. S. R. Barnum. 2007. Intercellular adhesion molecule-1 expression is required on J. Immunol. 184: 7196–7206. multiple cell types for the development of experimental autoimmune encephalo- 16. Vajkoczy, P., M. Laschinger, and B. Engelhardt. 2001. Alpha4-integrin-VCAM-1 myelitis. J. Immunol. 178: 851–857. binding mediates G protein-independent capture of encephalitogenic T cell blasts to 41. Cayrol, R., K. Wosik, J. L. Berard, A. Dodelet-Devillers, I. Ifergan, H. Kebir, CNS white matter microvessels. J. Clin. Invest. 108: 557–565. A. S. Haqqani, K. Kreymborg, S. Krug, R. Moumdjian, et al. 2008. Activated 17. Yednock, T. A., C. Cannon, L. C. Fritz, F. Sanchez-Madrid, L. Steinman, and leukocyte cell adhesion molecule promotes leukocyte trafficking into the central N. Karin. 1992. Prevention of experimental autoimmune encephalomyelitis by nervous system. Nat. Immunol. 9: 137–145. antibodies against alpha 4 beta 1 integrin. Nature 356: 63–66. 42. Ifergan, I., H. Kebir, S. Terouz, J. I. Alvarez, M. A. Lećuyer, S. Gendron, 18. Baron, J. L., J. A. Madri, N. H. Ruddle, G. Hashim, and C. A. Janeway, Jr. 1993. L. Bourbonnie`re, I. R. Dunay, A. Bouthillier, R. Moumdjian, et al. 2011. Role of Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into Ninjurin-1 in the migration of myeloid cells to central nervous system inflammatory Downloaded from brain parenchyma. J. Exp. Med. 177: 57–68. lesions. Ann. Neurol. 70: 751–763. 19. Theien, B. E., C. L. Vanderlugt, T. N. Eagar, C. Nickerson-Nutter, R. Nazareno, 43. Greter, M., F. L. Heppner, M. P. Lemos, B. M. Odermatt, N. Goebels, T. Laufer, V. K. Kuchroo, and S. D. Miller. 2001. Discordant effects of anti-VLA-4 treatment R. J. Noelle, and B. Becher. 2005. Dendritic cells permit immune invasion of the before and after onset of relapsing experimental autoimmune encephalomyelitis. CNS in an animal model of multiple sclerosis. Nat. Med. 11: 328–334. J. Clin. Invest. 107: 995–1006. 44. Chastain, E. M., D. S. Duncan, J. M. Rodgers, and S. D. Miller. 2011. The role of 20. Lehmann-Horn, K., S. A. Sagan, C. C. Bernard, R. A. Sobel, and S. S. Zamvil. antigen presenting cells in multiple sclerosis. Biochim. Biophys. Acta 1812: 265–274. 2015. B-cell very late antigen-4 deficiency reduces leukocyte recruitment and sus- 45. Clarkson, B. D., A. Walker, M. G. Harris, A. Rayasam, M. Sandor, and Z. Fabry.

ceptibility to central nervous system autoimmunity. Ann. Neurol. 77: 902–908. 2015. CCR2-dependent dendritic cell accumulation in the central nervous system http://www.jimmunol.org/ 21. Schenkel, A. R., Z. Mamdouh, X. Chen, R. M. Liebman, and W. A. Muller. 2002. during early effector experimental autoimmune encephalomyelitis is essential for CD99 plays a major role in the migration of monocytes through endothelial effector T cell restimulation in situ and disease progression. J. Immunol. 194: 531– junctions. Nat. Immunol. 3: 143–150. 541. 22. Lou, O., P. Alcaide, F. W. Luscinskas, and W. A. Muller. 2007. CD99 is a key 46. Zozulya, A. L., S. Ortler, J. Lee, C. Weidenfeller, M. Sandor, H. Wiendl, and mediator of the transendothelial migration of neutrophils. J. Immunol. 178: 1136– Z. Fabry. 2009. Intracerebral dendritic cells critically modulate encephalitogenic 1143. versus regulatory immune responses in the CNS. J. Neurosci. 29: 140–152. 23. Winger, R. C., J. E. Koblinski, T. Kanda, R. M. Ransohoff, and W. A. Muller. 47. Seraﬁni, B., B. Rosicarelli, R. Magliozzi, E. Stigliano, E. Capello, G. L. Mancardi, 2014. Rapid remodeling of tight junctions during paracellular diapedesis in a hu- and F. Aloisi. 2006. Dendritic cells in multiple sclerosis lesions: maturation stage, man model of the blood-brain barrier. J. Immunol. 193: 2427–2437. myelin uptake, and interaction with proliferating T cells. J. Neuropathol. Exp. 24. Watson, R. L., J. Buck, L. R. Levin, R. C. Winger, J. Wang, H. Arase, and Neurol. 65: 124–141. W. A. Muller. 2015. Endothelial CD99 signals through soluble adenylyl cyclase and 48. Seraﬁni, B., S. Columba-Cabezas, F. Di Rosa, and F. Aloisi. 2000. Intracerebral

PKA to regulate leukocyte transendothelial migration. J. Exp. Med. 212: 1021– recruitment and maturation of dendritic cells in the onset and progression of ex- by guest on October 1, 2021 1041. perimental autoimmune encephalomyelitis. Am. J. Pathol. 157: 1991–2002. 25. Dufour, E. M., A. Deroche, Y. Bae, and W. A. Muller. 2008. CD99 is essential for 49. Steinman, L. 2014. Immunology of relapse and remission in multiple sclerosis. leukocyte diapedesis in vivo. Cell Commun. Adhes. 15: 351–363. Annu. Rev. Immunol. 32: 257–281. 26. Bixel, G., S. Kloep, S. Butz, B. Petri, B. Engelhardt, and D. Vestweber. 2004. 50. Muller, W. A. 2007. PECAM: Regulating the start of diapedesis. In Adhesion Mouse CD99 participates in T-cell recruitment into inflamed skin. Blood 104: Molecules: Function and Inhibition.K.Ley,ed.BirkhauserVerlag,Basel, 3205–3213. Switzerland, p. 201–220. doi:10.1007/978-3-7643-7975-9_8 27. Brown, A. M., and D. E. McFarlin. 1981. Relapsing experimental allergic en- 51. Sumagin, R., and I. H. Sarelius. 2010. Intercellular adhesion molecule-1 enrichment cephalomyelitis in the SJL/J mouse. Lab. Invest. 45: 278–284. near tricellular endothelial junctions is preferentially associated with leukocyte 28. Robinson, A. P., C. T. Harp, A. Noronha, and S. D. Miller. 2014. The ex- transmigration and signals for reorganization of these junctions to accommodate perimental autoimmune encephalomyelitis (EAE) model of MS: utility for leukocyte passage. J. Immunol. 184: 5242–5252. understanding disease pathophysiology and treatment. Handb. Clin. Neurol. 52. Hussain, R. Z., L. Hayardeny, P. C. Cravens, F. Yarovinsky, T. N. Eagar, 122: 173–189. B. Arellano, K. Deason, C. Castro-Rojas, and O. Stuve.€ 2014. Immune surveillance 29. Ali, J., F. Liao, E. Martens, and W. A. Muller. 1997. Vascular endothelial cadherin of the central nervous system in multiple sclerosis–relevance for therapy and ex- (VE-cadherin): cloning and role in endothelial cell-cell adhesion. Microcirculation 4: perimental models. J. Neuroimmunol. 276: 9–17. 267–277. 53. Langer-Gould, A., S. W. Atlas, A. J. Green, A. W. Bollen, and D. Pelletier. 2005. 30. Muller, W. A., C. M. Ratti, S. L. McDonnell, and Z. A. Cohn. 1989. A human Progressive multifocal leukoencephalopathy in a patient treated with natalizumab. endothelial cell-restricted, externally disposed plasmalemmal protein enriched in N. Engl. J. Med. 353: 375–381. intercellular junctions. J. Exp. Med. 170: 399–414. 54. Stuve,€ O., C. M. Marra, K. R. Jerome, L. Cook, P. D. Cravens, S. Cepok, 31. Schenkel, A. R., T. W. Chew, and W. A. Muller. 2004. Platelet endothelial cell E. M. Frohman, J. T. Phillips, G. Arendt, B. Hemmer, et al. 2006. Immune sur- adhesion molecule deficiency or blockade significantly reduces leukocyte emigration veillance in multiple sclerosis patients treated with natalizumab. Ann. Neurol. 59: in a majority of mouse strains. J. Immunol. 173: 6403–6408. 743–747. 32. Mamdouh, Z., A. Mikhailov, and W. A. Muller. 2009. Transcellular migration of 55. del Pilar Martin, M., P. D. Cravens, R. Winger, E. M. Frohman, M. K. Racke, leukocytes is mediated by the endothelial lateral border recycling compartment. T. N. Eagar, S. S. Zamvil, M. S. Weber, B. Hemmer, N. J. Karandikar, et al. 2008. J. Exp. Med. 206: 2795–2808. Decrease in the numbers of dendritic cells and CD4+ T cells in cerebral perivascular 33. Mamdouh, Z., G. E. Kreitzer, and W. A. Muller. 2008. Leukocyte transmigration spaces due to natalizumab. Arch. Neurol. 65: 1596–1603. requires kinesin-mediated microtubule-dependent membrane trafficking from the 56. Calic, Z., C. Cappelen-Smith, S. J. Hodgkinson, A. McDougall, R. Cuganesan, and lateral border recycling compartment. J. Exp. Med. 205: 951–966. B. J. Brew. 2015. Treatment of progressive multifocal leukoencephalopathy- 34. Carman, C. V., and T. A. Springer. 2004. A transmigratory cup in leukocyte di- immune reconstitution inflammatory syndrome with intravenous immunoglobulin apedesis both through individual vascular endothelial cells and between them. J. Cell in a patient with multiple sclerosis treated with fingolimod after discontinuation of Biol. 167: 377–388. natalizumab. J. Clin. Neurosci. 22: 598–600.