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

Abstract

Leukocyte trafficking into the CNS is a prominent feature driving the immunopathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Blocking the recruitment of inflammatory leukocytes into the CNS represents an exploitable therapeutic target; however, the adhesion molecules that specifically regulate the step of leukocyte diapedesis into the CNS remain poorly understood. We report that CD99 is critical for lymphocyte transmigration without affecting adhesion in a human blood-brain barrier model. CD99 blockade in vivo ameliorated experimental autoimmune encephalomyelitis and decreased the accumulation of CNS inflammatory infiltrates, including dendritic cells, B cells, and CD4(+) and CD8(+) T cells. Anti-CD99 therapy was effective when administered after the onset of disease symptoms and blocked relapse when administered therapeutically after disease symptoms had recurred. These findings underscore an important role for CD99 in the pathogenesis of CNS autoimmunity and suggest that it may serve as a novel therapeutic target for controlling neuroinflammation.

Figure 1. CD99 is required for lymphocyte TEM at the human BBB model in vitro

TEM assays were performed with total lymphocytes (A) or purified CD4+ and CD8+ T-cells (C) on inflamed BBB model monolayers in the presence of anti-CD99 or control mAbs (non-blocking anti-VE-cadherin). (B,D) The number of lymphocytes that adhered to the monolayer were scored for multiple high power fields in each monolayer. Data represent the mean ± SD of hundreds of TEM events from at least six replicates from two (C,D) and three (A,B) independent experiments. **p< 0.001 (unpaired t-Test and two-way ANOVA).

Figure 2. Anti-CD99 ameliorates RR-EAE and reduces histopathological burden

(A) Comparison of mean clinical score between mice treated (arrows) with anti-CD99 (open circles) or control IgG (closed circles) at onset of clinical symptoms and every other day thereafter. (B) Whole mount immunostaining of spinal cord parenchymal postcapillary venules (PECAM-1; red) and CD3+ T-cell (green) infiltrates from anti-CD99 (lower panels) or control-IgG (upper panels) treated mice at peak of disease. Data shown are representative of 3 independent experiments (A,B) with a minimum of 5 mice per group and from >20 immunostainings performed on post-mortem material from 3 animals/group (B). Scale bar = 26μm. Error bars indicate SEM. **p< 0.001 (Mann-Whitney U test).

Figure 3. Inhibiting CD99 function prevents accumulation of inflammatory infiltrates in the CNS of RR-EAE mice

(A) The percentage of CD45^hi infiltrating iDCs (CD11b+CD11c+Ly6c^hi), macrophages (CD11b+CD11c−F4/80+), plasmacytoid DCs (CD11b−CD11c+PDCA-1+) and B-cells (CD19+), and (B) T-helper cells (CD3+CD4+), cytotoxic T-cells (CD3+CD8+) and T-regulatory cells (CD3+CD4+CD25+Foxp3+) expressing CD99 from the brain and spinal cord of individual mice at disease peak was quantitated by FACS. Histograms are representative of 5 individual mice. Filled bar indicates fluorescence minus one control and open line represents CD99 expressing cells. (C–F). The number of CD45^hi infiltrating CD11c+ iDCs, F4/80+ macrophages, PDCA-1+ plasmacytoid DCs and CD19+ B-cells, and CD4+ T cells, CD8+ T-cells and CD4+CD25+Foxp3+ T-regulatory cells at disease peak (C–D) and relapse (E–F) were enumerated from brain and spinal cords of individual anti-CD99 (open bars) or control IgG (closed bars) treated mice by FACS. Bolded antigens denote respective labeling on the bar graphs. Data shown in C–F are mean absolute number of CD45^hi CNS infiltrating cells pooled from 3 independent experiments obtained from at least 12 mice/group. Error bars indicate standard error of the mean. **p< 0.05 by two-way ANOVA. (G–H) Peripheral blood leukocytes were from anti-CD99 or control IgG treated RR-EAE mice were collected via cardiac puncture at disease peak and relapse and manually counted (from Fig 2A). Error bars indicate SEM. **p< 0.05; ns= not significant by t-Test.

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 (open circles) or control IgG (closed circles) during the secondary phase of disease at the onset of relapse. **p< 0.001 by Mann-Whitney U test. (B–C) CNS tissues were 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 3 independent experiments (n= 8 mice/group for each experiment; **p< 0.001 by Mann-Whitney U test). Data in B–C are pooled from 12-mice/treatment group. Error bars indicate SEM. **p< 0.05 compared to control IgG by two way ANOVA (B,C).