CD84 Links T Cell and Platelet Activity in Cerebral Thrombo-Inflammation in Acute Stroke

Abstract

Rationale: Ischemic stroke is a leading cause of morbidity and mortality worldwide. Recanalization of the occluded vessel is essential but not sufficient to guarantee brain salvage. Experimental and clinical data suggest that infarcts often develop further due to a thromboinflammatory process critically involving platelets and T cells, but the underlying mechanisms are unknown.

Objective: We aimed to determine the role of CD (cluster of differentiation)-84 in acute ischemic stroke after recanalization and to dissect the underlying molecular thromboinflammatory mechanisms.

Methods and results: Here, we show that mice lacking CD84-a homophilic immunoreceptor of the SLAM (signaling lymphocyte activation molecule) family-on either platelets or T cells displayed reduced cerebral CD4⁺ T-cell infiltration and thrombotic activity following experimental stroke resulting in reduced neurological damage. In vitro, platelet-derived soluble CD84 enhanced motility of wild-type but not of Cd84^-/- CD4⁺ T cells suggesting homophilic CD84 interactions to drive this process. Clinically, human arterial blood directly sampled from the ischemic cerebral circulation indicated local shedding of platelet CD84. Moreover, high platelet CD84 expression levels were associated with poor outcome in patients with stroke.

Conclusions: These results establish CD84 as a critical pathogenic effector and thus a potential pharmacological target in ischemic stroke.

Keywords: T lymphocytes; blood platelets; brain ischemia; infarction; stroke.

Figure 1.

Cd84^−/− mice are protected after transient middle cerebral artery occlusion (tMCAO). A, Representative triphenyltetrazolium chloride–stained 2-mm brain slices from 1 WT and 1 Cd84^−/− mouse 24 h after tMCAO. Bar=1 cm. B, Infarct volumes 24 h after tMCAO of WT (n=16) and Cd84^−/− mice (n=18; P=0.0198). C, WT (n=18) and Cd84^−/− (n=21) neuroscore at 24 h after tMCAO (P=0.0269). Statistical significances analyzed by Mann-Whitney U test. *P<0.05.

Figure 2.

Role of CD (cluster of differentiation) 84 in long-term functional outcome following stroke. Neurological outcome over time in WT (gray boxes) and Cd84^−/− mice (green boxes) as assessed by modified neurological severity score (mNSS; A), Corner test (B), and Bederson score (C) after 30-min transient middle cerebral artery occlusion (tMCAO; n=7–10 mice per group). Each symbol represents one individual. Statistical significances between WT and Cd84^−/− mice were determined using the Holm-Sidak correction on P obtained by Mann-Whitney U tests. *P<0.05, **P<0.01, and P<0.001; exact n-numbers and P are listed in Table V in the Data Supplement.

Figure 3.

CD (cluster of differentiation) 4⁺ T-cell recruitment to the postischemic brain is diminished in CD84-deficient mice. A, Representative immunocytologic stainings and quantification of brain-infiltrating CD4⁺ T lymphocytes in the ipsilateral hemisphere on day 1 after transient middle cerebral artery occlusion (tMCAO) of WT (wild type) and Cd84^−/− mice (n=5 mice per group; P=0.0328). Bar=100 µm. B, Representative CD11b immunoreactivity and quantification of CD11b⁺ cells in the ipsilateral hemisphere 24 h after tMCAO of WT and Cd84^−/− mice (n=5 mice per group; P=0.5476). Bar=100 µm. C, Representative images and quantification of Ly6B.2⁺ cells in the ipsilateral hemisphere 24 h after tMCAO of WT and Cd84^−/− mice (n=5 mice per group; P=0.5794). Bar=100 µm. D, Representative hematoxylin and eosin (H&E) staining and quantification of the percentage of occluded vessels in the infarcted hemispheres of WT and Cd84^−/− mice (n=5 mice per group; P=0.0079). White arrows indicate occluded vessels; blue arrows indicate patent vessels. Bar=100 µm. Statistical significances analyzed by Mann-Whitney U test. *P<0.05 and **P<0.01. DAPI indicates 4′,6-diamidino-2-phenylindole.

Figure 4.

CD (cluster of differentiation) 84 expression on CD4⁺ T cells and platelets is required to promote infarct growth after transient middle cerebral artery occlusion (tMCAO). Representative triphenyltetrazolium chloride–stained 2-mm brain slices of (A, top to bottom) and infarct volumes 24 h after tMCAO (B) of Rag1^−/− mice (n=9), Rag1^−/− mice with adoptive transfer of WT (n=10), or Cd84^−/− CD4⁺ T cells (n=11; P=0.0050) at day 1 before surgery, Cd84^−/− mice with adoptive transfer of WT (n=8) or Cd84^−/− CD4⁺ T cells (n=8) at day 1 before surgery, and mice specifically lacking the expression of CD84 on platelets (n=11) compared with control mice (n=8; P=0.0409), at day 1 after tMCAO. Bar=1 cm. C, Quantification of brain-infiltrating CD4⁺ T lymphocytes in the ipsilateral hemisphere on day 1 after tMCAO of Cd84^{fl/fl, Cre-neg} and Cd84^{fl/fl, Pf4-Cre} mice (n=6 mice per group; P=0.0368). Statistical significances analyzed by Mann-Whitney U test. *P<0.05. AT indicates adoptive transfer.

Figure 5.

Soluble CD (cluster of differentiation) 84 promotes CD4⁺ T-cell migration. A, Migrated distance of CD4⁺ WT and Cd84^−/− T cells treated with vehicle, phorbol-12-myristate-13-acetate (PMA), or CCL20 (C-C motif chemokine ligand 20). B, WT and Cd84^−/− CD4⁺ T-cell migration in response to stimulation with WT platelet-releasate (PLT-R) compared with vehicle (control). C, WT CD4⁺ T-cell migration in response to stimulation with WT or Cd84^−/− PLT-R compared with control. D, Migrated distance of CD4⁺ WT and Cd84^−/− T cells treated with vehicle, recombinant control-Fc, or CD84-Fc (recombinant soluble CD84 fused to the Fc part of human IgG1) protein. E, WT CD4⁺ T-cell migration in response to stimulation with WT or Cd84^−/− PLT-R in the presence of control-Fc or recombinant CD84-Fc protein. F, Migrated distance of CD4⁺ WT and Cd84^−/− T cells treated with control-Fc or CD84-Fc on primary murine brain microvascular endothelial cells (MBMECs) of WT or Cd84^−/− mice. Each dot represents the migrated distance over 30 min of one CD4⁺ T cell (n=59–80 cells per group of 3–4 independent experiments). Horizontal red lines correspond to the mean and SD. Statistical significances analyzed by 1-way ANOVA with Bonferroni post hoc test or Kruskal-Wallis test with Dunn multiple comparison test, the exact test n-numbers and P are listed in Table VI in the Data Supplement. ***P<0.001.