Cutting Edge: CD99 Is a Novel Therapeutic Target for Control of T Cell–Mediated Central Nervous System Autoimmune Disease Free

Leukocyte diapedesis (transendothelial migration [TEM]) is a tightly regulated process that is vital to inflammation and immune responses. TEM is mediated by a set of adhesive molecular interactions between circulating leukocytes (WBCs) and the vascular endothelium. During immune surveillance, a limited number of WBCs enter the CNS by crossing the blood–brain barrier (BBB) to detect potential damaging agents (1, 2); however, exacerbated WBC recruitment into the CNS is a prominent pathological feature driving multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) (3, 4).

Before mediating tissue destruction in CNS autoimmunity, lymphocytes interact with adhesion molecules on the BBB to reach their cognate Ag (5). In addition, the TEM of inflammatory APCs, including inflammatory myeloid-derived dendritic cells (iDCs), macrophages, and B cells, from the periphery into the CNS is an early event, and their accumulation persists throughout the course of disease (6–8). Therefore, elucidating the mechanisms governing the TEM of lymphocytes and inflammatory APCs into the CNS is critical to understanding the pathogenesis of disease. The integrin VLA-4 mediates firm adhesion to endothelium in the periphery (9–13) and the CNS (14–16) and was demonstrated to play an integral role in inflammatory WBC accumulation in the CNS (17–20). However, inflammatory WBCs may use alternative mechanisms that specifically govern the step of diapedesis to infiltrate the CNS and mediate disease.

CD99 is a 32-kDa surface glycoprotein that is concentrated at cell borders of all endothelium and is expressed diffusely on the surface of WBCs (21, 22). Homophilic WBC–endothelial 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 as blocking endothelial CD99 (21, 22, 24), making it an attractive therapy for inflammatory-mediated diseases. Recently, we reported that CD99 mediates monocyte TEM across human BBB models in vitro (23). However, the role of CD99 for other WBC subsets at the BBB may be different, and its role in the pathogenesis of CNS disease is unknown.

In the current study, we examined the requirement of CD99 for lymphocyte TEM in the BBB model in vitro and investigated the mechanism and efficacy of anti-CD99 therapy to regulate relapsing-remitting EAE (RR-EAE). RR-EAE in the SJL/J strain shares similar clinical and histopathological hallmarks that are germane to MS (27) and, thus, serves as a powerful tool for studying the immunopathogenesis of disease and potential therapeutic interventions (28). We show that CD99 is required for lymphocyte diapedesis, but not adhesion, in a human BBB model in vitro. More importantly, anti-CD99 therapy reduced the clinical severity of RR-EAE and diminished T cell, iDC, and B cell accumulation in the CNS. Thus, CD99 is a novel therapeutic target for the management of CNS inflammation and autoimmunity.

All procedures involving human subjects/materials were approved by the Institutional Review Board of the Northwestern University Feinberg School of Medicine.

Mouse anti-human mAbs for VE-cadherin (clone hec1) and CD99 (clone hec2), Armenian hamster anti-mouse PECAM (clone 2H8), and rat anti-mouse CD99 (clone 3F11) mAbs were generated by hybridoma methodologies, as previously described (21, 24, 29–31). Rat anti-mouse CD3 (clone 17A2) mAb was purchased from eBioscience.

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).

Lymphocytes were isolated from human PBMCs and mitogen activated (1 μg/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 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).

Quantitative endpoint TEM assays were performed as previously described (21, 23, 24, 32–34).

Female SJL/J mice (The Jackson Laboratory, Bar Harbor, ME) were housed under specific pathogen–free conditions in the Northwestern University Center for Comparative Medicine Animal Facility. The Northwestern University Animal Care and Use Committee approved all protocols.

Six- to eight-week-old female SJL/J mice were immunized s.c. over three sites on the flank with 100 μl an emulsion containing 50 μg proteolipid protein 139–151 and CFA, as previously described (7, 8). Disease was scored on scale of 0 to 5, as previously described (8, 35).

RR-EAE mice were randomized at the onset of clinical symptoms and treated i.p. with 100 μg rat–anti-CD99 mAb (clone 3F11) or control IgG (rat IgG; Jackson ImmunoResearch), beginning either at the onset of symptoms or at relapse and every other day thereafter.

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 then fixed in 4% paraformaldehyde overnight at 4°C. This method removes the meninges. SCs were blocked and permeabilized in a solution of 0.2% 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 against PECAM-1 and CD3 in blocking solution. SCs were incubated with 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 sagittal orientation, and parenchymal postcapillary venules (identified by PECAM and 20–40 μm in diameter) in the lumbar region associated with perivascular lymphocyte infiltrates (identified by CD3) were imaged using an UltraVIEW Vox imaging system equipped with a Yokogawa CSU-1 spinning disk. Images were acquired using Volocity software (Perkin Elmer), which renders the optical sections into three-dimensional images. Image processing and analysis were performed with ImageJ. Fig. 2 shows representative projections of the whole stack using the maximum-intensity method.

WBCs were isolated from digested SC and brain of individual PBS-perfused mice via Percoll gradient, as described previously (8, 38).

CNS cells were stained with the indicated Abs (eBioscience and BD), as described previously (8, 38, 39): CD45, CD39, CD11b, CD11c, Ly6c, CD19, plasmacytoid dendritic cell Ag (PDCA)-1, F4/80, CD3, CD4, CD8, and CD25. Intracellular staining for Foxp3 was performed per the manufacturer’s instructions with an eBioscience staining buffer kit. Aqua/VID LIVE/DEAD Cell Stain was purchased from Life Technologies.

All statistical analyses were done using GraphPad Prism software (GraphPad, San Diego, CA).

Studies in EAE demonstrated that CNS-infiltrating lymphocytes play a critical role in CNS autoimmune disease. We investigated the role of CD99 for lymphocyte TEM at the inflamed BBB model in vitro. BBB model monolayers were activated as described in 2Materials and Methods. TEM assays with polyclonal-activated lymphocytes were performed in the presence of blocking mAb against CD99 or nonblocking control mAb against VE-cadherin. Under control conditions, 65% of adherent lymphocytes transmigrated, whereas anti-CD99 blocked TEM down to ∼19% (>70% block in TEM) (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 affected lymphocyte adhesion to the monolayers (Fig. 1B, 1D), demonstrating that our observations are specifically due to a block in TEM, as observed for monocytes (21, 23, 24) and neutrophils (22, 24, 36). This is in contrast to ICAM-1 (40), VCAM-1 (17), ALCAM (41), and Ninjurin-1 (42), which were demonstrated to play a role in adhesion and subsequent diapedesis of inflammatory WBC subsets across the BBB.

To date, the role of CD99 in EAE is unknown. We determined the role of CD99 for WBC recruitment to the CNS in vivo using proteolipid protein 139–151–induced RR-EAE in SJL/J mice by administering anti-CD99 or control IgG i.p. once mice became symptomatic. Anti-CD99 mAb given i.p. was able to reach the CNS because it labeled endothelial junctions of the BBB (data not shown). Mice receiving anti-CD99 mAb displayed significantly lower clinical scores than controls (p ≤ 0.001) at peak disease and relapse (Fig. 2A, Table I). The majority of anti-CD99–treated mice displayed mild disease and were even protected against the development of relapse (Fig. 2A, Table II). Whole-mount immunostaining revealed reduced histopathological burden of CD3⁺ T cells in the SCs of anti-CD99–treated mice compared with controls (Fig. 2B). Qualitatively similar results were seen in SCs of mice examined at the peak of relapse (data not shown).

The in vitro findings from Fig. 1 and whole-mount staining in Fig. 2 suggest that the therapeutic efficacy of anti-CD99 is due to its ability to block the recruitment of lymphocytes to the CNS. However, similar to MS, RR-EAE is characterized by extensive CNS perivascular infiltration of inflammatory APCs that perpetuate damage (3, 8, 43). In particular, iDCs are major players in CNS autoimmune pathology (8, 44), and their numbers correlate with disease severity and progression (5, 8, 42, 43, 45–48). The molecular mechanisms governing the TEM of this pathogenic subset into the CNS are poorly understood.

The frequency of CNS-infiltrating WBC subsets known to play a role in the pathogenesis of disease that express CD99 was characterized by FACS on cells from dissociated brains and SCs harvested at disease peak (Fig. 3A, 3B) from five mice. We next enumerated the different CNS-infiltrating inflammatory WBC populations in anti-CD99 or control mice by performing FACS at disease peak and relapse. There was a significant reduction in the absolute numbers of CNS CD45^hi-infiltrating B cells, iDCs, and CD4⁺ and CD8⁺ T cells at disease peak and relapse in CD99-treated mice (Fig. 3C–F). CD3⁺CD4⁺ Th1 and Th17 cells were reduced to an equal extent in anti-CD99–treated mice (data not shown). There were no significant differences in the absolute numbers of microglia (CD45^loCD11b^loCD11c⁺), macrophages, or plasmacytoid dendritic cells between anti-CD99– and control IgG–treated RR-EAE mice. In contrast to the CNS, we observed no significant differences in absolute cell numbers in spleen and lymph nodes between anti-CD99–treated mice and controls at any time point assessed (data not shown). In addition, at disease onset and remission there were no significant differences in cell numbers between anti-CD99 and control in any of the tissues examined. Importantly, peripheral blood WBC counts were slightly higher in CD99-treated animals compared with control IgG (Fig. 3G, 3H). Taken together, these data suggest that anti-CD99 therapy specifically inhibited WBC migration into the CNS rather than depleting WBCs or inhibiting emigration from bone marrow.

In the clinic, most patients with MS receive treatment following disease exacerbations, rather than prophylactically. Therefore, we took a more clinically relevant approach by treating mice with established EAE only during the secondary phase of disease at relapse onset (∼23 d postimmunization). Anti-CD99 therapy ameliorated the development and severity of the secondary phase of disease (Fig. 4A). Similar to our observations in Fig. 3, CD45^hi CNS-infiltrating iDCs, B cells, and CD4⁺ and CD8⁺ T cells were significantly reduced in mice that received anti-CD99 therapy at the onset of relapse compared with controls. Importantly, as observed in Fig. 3B and 3D, there was no reduction in CD3⁺CD4⁺CD25⁺Foxp3⁺ regulatory T cell accumulation in the CNS following treatment with anti-CD99 (Fig. 4C). Selective inhibition of effector, but not regulatory, T cell TEM by anti-CD99 has important implications for translation into the clinical setting.

Blocking WBC extravasation into the CNS is an exploitable and effective therapeutic target for treating CNS-directed autoimmune disease. Anti–VLA-4 (natalizumab) is a highly potent and efficacious therapy for the management of MS. Anti–VLA-4 prevents the binding of leukocyte α4 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 impede the ability to protect against latent and chronic CNS 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 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.

We thank Clifford D. Carpenter for excellent technical assistance.

The authors have no financial conflicts of interest.