Treatment and Prevention of Ischemic Diseases And/Or Ischemic Tissue Damages

Abstract

The invention relates to a CD84 inhibitor for use in treating or preventing of an ischemic disease and/or ischemic tissue damage, the use of a CD84 inhibitor for inhibiting CD84 mediated T cell migration in vitro and a method for screening for a CD84 inhibitor.

Claims

1. A CD84 inhibitor for use in treating or preventing of an ischemic disease and/or ischemic tissue damage.

2. The CD84 inhibitor for use according to claim 1, wherein the inhibitor is selected from the group consisting of an antibody, a blocking polypeptide, a small molecule, or an inhibitory nucleic acid, in particular wherein the inhibitor is an antibody.

3. The CD84 inhibitor for use according to claim 2, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, an scFv, a multimer of an scFv, a tandab, a diabody, triabody, a flexibody, or a fragment thereof.

4. The CD84 inhibitor for use according to any of claims 1 to 3, wherein the CD84 inhibitor is capable of inhibiting homotypic receptor interaction and/or dimerization of CD84 molecules.

5. The CD84 inhibitor for use according to any of claims 1 to 4, wherein the CD84 inhibitor inhibits the interaction of a CD84 molecule on T cells with another CD84 molecule derived from platelets.

6. The CD84 inhibitor for use according to any of claims 1 to 5, wherein the CD84 inhibitor binds to the domain of CD84 that mediates homotypic receptor interaction of CD84.

7. The CD84 inhibitor for use according to any of claims 1 to 6, wherein the ischemic disease and/or ischemic tissue damage is selected from the group consisting of trauma, ischemic cerebrovascular disorder, in particular cerebral infarction, ischemic renal disease, ischemic pulmonary disease, infection-related ischemic disease, ischemic disease of limbs, ischemic heart disease, myocardial infarction, atherosclerosis, peripheral vascular disorder, a pulmonary embolus, a venous thrombosis, a transient ischemic attack, unstable angina, cerebral vascular ischemia, stroke, an ischemic neurological disorder, ischemic kidney disease, vasculitis, transplantation, endarterectomy, aneurysm repair surgery, an inflammatory disorder, hypothermia or traumatic injury.

8. The CD84 inhibitor for use according to any of claims 1 to 7, wherein the method is for treating or preventing ischemia/reperfusion injury and/or infarct growth.

9. The CD84 inhibitor for use according to any of claims 1 to 8, wherein the CD84 inhibitor is for preventing the ischemic disease and wherein the CD84 inhibitor is administered prior to ischemia.

10. The CD84 inhibitor for use according to any of claims 1 to 8, wherein the method is for treating or preventing the ischemic disease, and wherein the CD84 inhibitor is administered during or after ischemia.

11. The CD84 inhibitor for use according to any of claims 1 to 10, wherein the CD84 inhibitor is capable of inhibiting CD84 mediated T cell migration.

12. The CD84 inhibitor for use according to any of claims 1 to 11, wherein the method further comprises administering another therapeutic agent.

13. The CD84 inhibitor for use according to any of claims 1 to 12, wherein the method further comprises suppression of T cell migration with said CD84 inhibitor, in particular wherein the ischemic disease and/or ischemic tissue damage is at least partially mediated by T cell migration.

14. Use of a CD84 inhibitor for inhibiting CD84 mediated T cell migration in vitro.

15. The use according to claim 14, wherein the CD84 inhibitor is as defined in any of claims 2 to 13.

16. A method for screening for a CD84 inhibitor, in particular for a CD84 inhibitor as defined in any of claims 2 to 13, the method comprising identifying whether a potential CD84 inhibitor inhibits T cell migration.

Description

DESCRIPTION OF THE FIGURES

[0126] FIG. 1a: Infarct volumes in mouse brains (cross-section shown) in Cd84.sup.−/− mice compared to wild-type (WT) littermates at 24 h after tMCAO as measured by triphenyltetrazolium chloride (TTC) staining. Representative TTC-stained 2 mm brain slices of WT (top row) and Cd84.sup.−/− (bottom row) mice at day 1 after tMCAO. The bar on the bottom right represents 1 cm. Chequered regions represent brain regions, which were not stained with TTC, which means that the tissue of this region was affected by the infarct. Non-chequered regions represent brain regions that were stained with TTC and, therefore, were not affected by the infarct.

[0127] FIG. 1b: Infarct volumes in mouse brains in Cd84.sup.−/− mice compared to wild-type (WT) littermates at 24 h after tMCAO (n=14-15 mice per group). The x-axis represents the two groups of samples (left: WT mice, right: Cd84.sup.−/− mice), while the y-axis represents the infarct volume in mm.sup.3. Each dot represents a data point, i.e. the data from one mouse. Further marked are the median value as well as the 25.sup.th and the 75.sup.th percentiles forming bottom and top line of the rectangles. Finally, the lowest and highest value for each sample type is indicated by a line as well. Statistical significances analyzed by Student t test, *p<0.05.

[0128] FIG. 1c: Neuroscore of WT and Cd84.sup.−/− mice at day 1 after tMCAO (n=14-15 mice per group). Statistical significances analyzed by Student t test, **p<0.01. The x-axis represents the two sample groups: WT mice (left) and Cd84.sup.−/− mice (right). The y-axis represents the Neuroscore. Each black dot represents the Neuroscore of a WT mouse, while each circle filled with white color represents the Neuroscore of a Cd84.sup.−/− mouse. Medians of each sample group are indicated by a black line. Statistical significances analyzed by Student t test, **p<0.01.

[0129] FIG. 2a: Accumulation of lymphocytes and monocytes in the brain after tMCAO. Quantification of brain-infiltrating CD4-positive T lymphocytes in the ipsilateral hemisphere on day 1 after tMCAO of WT (x-axis, left, shaded) and Cd84.sup.−/− mice (x-axis, right) (n=5 mice per group). Bar, 100 pm. The y-axis shows the number of ipsilesional CD4.sup.+ cells. Statistical significances analyzed by Student t test, *p<0.05. Further marked are the median value as well as the 25.sup.th and the 75.sup.th percentiles forming bottom and top line of the rectangles. Finally, the lowest and highest value for each sample type is indicated by a line as well.

[0130] FIG. 2b: Representative quantification of CD11b-positive cells in the ipsilateral hemisphere 24 h after tMCAO of WT (x-axis, left, shaded) and Cd84.sup.−/− mice (x-axis, right) (n=5 mice per group). Bar, 100 pm. The y-axis shows the number of ipsilesional CD11b.sup.+ cells. Further marked are the median value as well as the 25.sup.th and the 75.sup.th percentiles forming bottom and top line of the rectangles. Finally the lowest and highest value for each sample type is indicated by a line as well.

[0131] FIG. 2c: Representative quantification of the percentage of occluded vessels in the infarcted hemispheres of WT (x-axis, left, shaded) and Cd84.sup.−/− mice (x-axis, right) (n=5 mice per group). The y-axis shows the number of ipsilesional occluded vessels in percent (%). Statistical significances analyzed by Student t test, ***P<0.001. Further marked are the median value as well as the 25.sup.th and the 75.sup.th percentiles forming bottom and top line of the rectangles. Finally the lowest and highest value for each sample type is indicated by a line as well.

[0132] FIG. 3a: Infarct volumes 24 h after tMCAO of the indicated groups (x-axis) (n=8-12 mice per group). The y-axis represents infarct volume in mm.sup.3. Further marked are the median value as well as the 25.sup.th and the 75.sup.th percentiles forming bottom and top line of the rectangles. Finally, the lowest and highest value for each sample type is indicated by a line as well. Statistical significances analyzed by Student t test, *p<0.05.

[0133] FIG. 3b: Neuroscore at day 1 after tMCAO of the indicated groups (x-axis) (n=8-12 mice per group). The y-axis represents the Neuroscore. Further marked are the median value as well as the 25.sup.th and the 75.sup.th percentiles forming bottom and top line of the rectangles. Finally, the lowest and highest value for each sample type is indicated by a line as well. Statistical significances analyzed by Mann-Whitney U test. *p<0.05.

[0134] FIG. 4: Soluble CD84 promotes T cell migration. In the diagrams 4a to 4f below, the x-axis shows the tested groups, while the y-axis represents the distance in pm. Each dot represents the migrated distance over 30 min of one T cell (n=59-80 cells per group of 3-4 independent experiments). For each group the median is indicated by a black line. Statistical significances analyzed by 1-way ANOVA with Bonferroni post hoc test. ***p<0.001. In detail, FIGS. 4a to 4f represent the following:

[0135] FIG. 4a: Migrated distance of CD4-positive WT and Cd84.sup.−/− T cells treated with vehicle, Phorbol-12-myristate-13-acetate (PMA) or C-C chemokine cysteine motif chemokine ligand 20 (CCL20).

[0136] FIG. 4b: WT and Cd84.sup.−/− T cell migration in response to stimulation with WT platelet releasate (PLT-R) compared to vehicle.

[0137] FIG. 4c: WT T cell migration in response to stimulation with wildtype or Cd84.sup.−/− PLT-R compared to control.

[0138] FIG. 4d: Migrated distance of CD4-positive WT and Cd84.sup.−/− T cells treated with vehicle, recombinant GPVI-Fc or CD84-Fc protein.

[0139] FIG. 4e: WT T cell migration in response to stimulation with WT or Cd84.sup.−/− PLT-R in the presence of Control-Fc or recombinant CD84-Fc protein.

[0140] FIG. 4f: Migrated distance of CD4+ WT and Cd84.sup.−/− T cells treated with GPVI-Fc or CD84-Fc on primary murine brain endothelial cells (MBMEC) of WT or Cd84.sup.−/− mice.

[0141] FIG. 5: Soluble CD84 increases T cell migratory capacity. In the diagrams 5a to 5f below, the x-axis shows the tested groups, while the y-axis represents the speed in pm/min. Each data point represents the speed of migration analyzed over 30 min of one T cell (n=59-80 cells per group of 3-4 independent experiments). For each group the median is indicated by a black line. Statistical significances analyzed by 1-way ANOVA with Bonferroni post hoc test. ***p<0.001. In detail, FIGS. 5a to 5f represent the following:

[0142] FIG. 5a: Velocity of WT and Cd84.sup.−/− CD4+ T cells treated with vehicle, PMA or C-C chemokine cysteine motif chemokine ligand 20 (CCL20).

[0143] FIG. 5b: WT and Cd84.sup.−/− T cell migration speed in response to stimulation with WT platelet releasate (PLT-R) compared to vehicle.

[0144] FIG. 5c: Speed of WT T cell migration in response to stimulation with WT or Cd84.sup.−/− PLT-R compared to vehicle-treated control.

[0145] FIG. 5d: Velocity of WT and Cd84.sup.−/− CD4+ T cells treated with vehicle, Ctrl-Fc or CD84-Fc protein.

[0146] FIG. 5e: WT T cell migration velocity in response to stimulation with WT or Cd84.sup.−/− PLT-R in the presence of CD84-Fc or Ctrl-Fc.

[0147] FIG. 5f: Velocity of WT (black) and Cd84.sup.−/− (white) CD4+ T cells treated with CD84-Fc or Ctrl-Fc on primary murine brain endothelial cells (MBMEC) of WT or Cd84.sup.−/− mice.

EXAMPLES

[0148] Part 1—CD84 in Ischemic Disease and Tissue Damage

[0149] Materials and Methods

[0150] Cohort validation. To assess the transferability of the findings from mouse model into humans, the association of CD84 expression on platelets and stroke severity at day three after hospital admission was analyzed in a subsample of the Stroke-Induced Cardiac FAILure in Mice and Men (SICFAIL) study. SICFAIL is a cohort study, aiming to describe the natural course of cardiac function in 750 unselected acute IS patients (DRKS00011615). The subsample of the SICFAIL study population, analyzed in this study, included 100 patients with the diagnosis of acute IS according to World Health Organisation (WHO) definition (Hatano et al.) aged 18 years or older, who were hospitalized at the stroke unit of university hospital Wuerzburg between June 2016 and January 2017. Written informed consent was obtained prior study enrollment. The Ethics Committee of the Medical Faculty of the University of Wuerzburg (vote176/13) approved the study. All patients or their legal representatives provided written informed consent to participate.

[0151] All patients underwent standard treatment and monitoring at stroke unit. Information on stroke severity and acute treatment (intravenous thrombolysis, thrombectomy) was retrieved by review of patient records. Stroke severity was assessed by a neurologist according to National Health Institute Stroke Severity Scale NIHSS (Berger et al.). Additionally, all study participants underwent a personal standardized interview to collect information on demographic characteristics and comorbidities. Poor outcome was defined as NIHSS 5 on day three of hospitalization.

[0152] For CD84 analysis, blood was drawn the morning after study enrollment in tubes containing citrate phosphate dextrose adenine. Samples were diluted within 30 min after blood collection in PBS and platelets were stained using CD42b-FITC (SZ2; Immunotech SAS, Marseille, France) and CD84-PE antibodies (CD84.1.21; BioLegend, San Diego, USA). After an incubation period of 30 minutes, the samples were analyzed by flow cytometry instrument FACSCalibur (Becton Dickinson, Franklin Lakes, USA). CD84 expression was defined as mean fluorescence intensity (MFI) of phycoerythrin (PE) in platelets gated by CD42b positivity.

[0153] Mice. Animals used in this study were matched for age, sex and genetic background. Experiments were conducted in accordance with the regulations of the local authorities (Regierung von Unterfranken) and performed in accordance with the current ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://www.nc3rs.org.uk/arrive-guidelines).

[0154] Animal treatment. For T cell transfer experiments into Rag1.sup.−/− and Cd84.sup.−/− mice, splenic CD4.sup.+ T cells were isolated by negative selection (Miltenyi Biotech). Cells were injected intravenously (750,000 cells/mouse) 1 day before tMCAO (Kleinschnitz et al. 2010).

[0155] Focal ischemia model. Focal cerebral ischemia was induced in 10-to-14-week-old C57BL/6, Cd84.sup.−/−,.sup.21 Rag1.sup.−/− (Mombaerts et al.), Cd84.sup.fl/fl,PF4-Cre-neg. or Cd84.sup.fl/fl,PF4-Cre mice by tMCAO as previously described (Schuhmann et al. 2015). Inhalation anesthesia was induced by 2% isoflurane. The duration of the surgical procedure per animal was kept below 10 min. A silicon rubber-coated 6.0 nylon monofilament (6021PK10, Doccol, Redlands, Calif., USA) was advanced through the carotid artery up to the origin of the MCA causing an MCA infarction. After an occlusion time of 60 min, the filament was removed allowing reperfusion. Animals were sacrificed 23 h after reperfusion and brains were checked for intracerebral hemorrhages. Neurologic function was analyzed calculating a Neuroscore (score 0-10) based on the direct sum of the Grip test (score 0-5) and the inverted Bederson score (score 0-5) (Schuhmann et al. 2018, Moran et al., Bederson et al.).

[0156] Infarction size measurement. The extent of infarction was quantitatively assessed 24 h after reperfusion. Animals were sacrificed and brains were cut in three 2 mm thick coronal sections. The slices were stained for 20 min at 37° C. with 2% 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich; 2% weight/volume (w/v) solution) to visualize the infarctions (Junge et al.). Edema-corrected infarct volumes were calculated by planimetry (Image J software, National Institutes of Health) according to the following equation: V.sub.indirect (mm.sup.3)=V.sub.infarct×(1−(VI−VC)/VC). (VI−VC) represents the volume difference between the ischemic hemisphere (VI) and the control hemisphere (VC) and (VI−VC)/VC) expresses this difference as a percentage of the control hemisphere.

[0157] Histology. Histology and immunohistochemistry were performed according to standard procedures (Schuhmann et al. 2015). Cryoembedded coronal brain sections (2 mm) were cut into 10-μm-thick slices. Every tenth slice was used for evaluation. The following antibodies were used: monoclonal antibody anti-CD11b (MCA711, Serotec) and polyclonal antibody anti-CD4 (10056, Bio Legend). For quantification of occluded microvessels, brain slices were stained with hematoxylin and eosin. Afterwards the numbers of occluded and opened vessels per hemisphere were counted to determine the percentage of occlusions as previously described. All immunohistologic stainings were analyzed and acquired using a Nikon Eclipse 50i microscope.

[0158] Migration assays. The migratory capacity of T cells was measured using ibidi μ-Slides VI0.4 coated with poly-D-lysine (10 μg/mL, Merck) and laminin (20 μg/mL, Merck) or ibidi Glass Bottom μ-Slides 8 well cultured with primary MBMEC as previously described (Bittner et al.). The migration assays were performed in DMEM high glucose (31053-028, Thermo Fischer) with B27 supplement (2%, 17504044, Thermo Fischer) in triplicates for each different condition (PMA (8 μg/μL, Merck), CCL20 (24.5 μg/μL, PeproTech), PLT-R (1:1 diluted with migration media), Fc-proteins (0.2 μg/ml). PLT-R was obtained by stimulating platelets (500000/μL) with 10 μg/mL of the GPVI agonist collagen-related peptide (CRP) for 15 min, subsequently the platelet supernatant was harvested (5 min with 800 g, followed by 5 min with 22000 g, 4° C. The GPVI-Fc fusion protein control-Fc (Gruner et al.), that did not affect T cell migration compared to vehicle (not shown) served as control for CD84-Fc, both Fc fusion proteins were purified in house from transfected HEK cells using standard techniques. For time-lapse video microscopy, MACS isolated CD4.sup.+ T cells (130-104-454, Miltenyi; 100 cells/μL) were added to the chamber and images were collected every 30 seconds for 30 minutes on a Leica DMI8 inverted microscope with 20×objective. Cells were tracked with LAS X Imaging software and analyzed with ImageJ 1.51 software.

[0159] Statistical analyses. All data from animal experiments are given as box plots including median with the 25.sup.th percentile, the 75.sup.th percentile, minimum and maximum deviation except for the Neuroscore and the migration assays, which are depicted as scatter plots including the mean and standard error of the mean. Human data are presented as median and interquartile range (25.sup.th percentile, and 75.sup.th percentile) or, in case of categorical data, frequencies and percentages, respectively. Data were tested for Gaussian distribution e.g. with the D'Agostino and Pearson omnibus normality test and then analyzed by Student's t-test, 1-way ANOVA or Mann-Whitney U-test as applicable. In case of frequencies, χ.sup.2-test or Fisher's exact test was used to compare groups. Bonferroni correction was applied when comparing more than two groups in the animal experiments. Scores addressing the functional outcome were compared using the Mann-Whitney U-test. Multivariable logistic regression analysis, adjusted for age and NIHSS at baseline, was used to identify the association of CD84 expression and poor outcome on day three of hospitalization (NIHSS 5). P<0.05 was considered statistically significant. For statistical analysis, the GraphPad Prism 5.0 software package (GraphPad Software) and SAS 9.4 (SAS Institute Inc., Cary, N.C., USA) were used.

Example 1—Involvement of CD84 in Thrombo-Inflammation

[0160] To test for a possible role of CD84 in thrombo-inflammation, Cd84.sup.−/− mice were subjected to one hour of transient middle cerebral ischemia (tMCAO) and 23 h of reperfusion. Strikingly, infarct volumes in the mutant mice were significantly reduced compared to wild-type (WT) littermates at 24 h after tMCAO as measured by triphenyltetrazolium chloride (TTC) staining (Med.: 89.3 (25%: 75.1; 75%: 111.6) vs. 79.2 (25%: 42.8; 75%: 90.4) mm.sup.3; p<0.05; FIG. 1a, FIG. 1b). The reduction in infarct volumes corresponded with improved neurological outcome in Cd84.sup.−/− mice as assessed by the Neuroscore (4.6±0.2 vs. 5.7±0.2, respectively; p<0.01; FIG. 1c).

[0161] The results showed that CD84-deficient mice are protected after tMCAO.

Example 2—Accumulation of Lymphocytes and Monocytes in the Brain After tMCAO

[0162] Next, the accumulation of lymphocytes and monocytes in the brain after tMCAO was assessed. Cd84.sup.−/− mice showed significantly reduced numbers of CD4.sup.+ T cells in the infarcted brain (˜50%) compared with WT mice (FIG. 2a), whereas the amount of CD11b.sup.+ cells (FIG. 2b) was comparable between the two groups. Furthermore, the percentages of occluded vessels in the ipsilateral hemisphere were reduced by ˜35% in Cd84.sup.−/− mice 24 h after stroke induction (FIG. 2c). These data demonstrated that CD84-deficiency is protective after tMCAO and that CD84 contributes to T cell recruitment into the infarcted brain territory.

[0163] The results indicated that T cell recruitment to the postischemic brain is diminished in CD84-deficient mice.

Example 3—Relevance of T Cell Expressed CD84 for Infarct Progression

[0164] Next, the relevance of T cell expressed CD84 for infarct progression by performing adoptive T cell transfer experiments was assessed. For this, immunodeficient Rag1.sup.−/− mice, which lack B and T cells, or Cd84.sup.−/− mice received WT or Cd84.sup.−/− T cells 24 h before subjecting them to tMCAO. While Rag1.sup.−/− displayed small infarcts 24 h after tMCAO (Yilmaz et al., Kleinschnitz et al. 2010, Schuhmann et al. 2017), Rag1.sup.−/− mice had fully evolved infarctions after adoptive transfer (AT) of WT T cells (FIG. 3a). In sharp contrast, Rag1.sup.−/− mice reconstituted with Cd84.sup.−/− T cells had significantly smaller infarcts, as measured by TTC staining (Med.: 100.9 (25%: 74.8; 75%: 126.0) vs. 30.0 (25%: 23.3; 75%: 113.4) mm.sup.3; p<0.05; FIG. 3a) and better neurological outcome (FIG. 3b) at day 1 after stroke than those transplanted with WT T cells. However, infarct volumes and functional outcome did not differ when adoptively transferring WT or CD84-deficient T cells into Cd84.sup.−/− mice indicating that an interaction of CD84.sup.+ T cells with another CD84-expressing cell type is required to induce I/R injury.

[0165] The results showed that CD84 expression on T cells is required to promote infarct growth after tMCAO.

Example 4—Role of Platelets

[0166] To address a possible role of platelets in this setting, mice with a platelet/megakaryocyte-specific CD84-deficiency (Cd84.sup.fl/fl,PF4-Cre; knockout efficacy was confirmed using flow cytometry and Western Blot—not shown) were generated and subjected to tMCAO. These mice developed significantly (˜30%) smaller infarcts at day 1 after tMCAO when compared to littermate control mice (Cd84.sup.fl/fl; Med.: 92.3 (25%: 61.0; 75%: 105.9) vs. 63.4 (25%: 26.2; 75%: 79.4) mm.sup.3; p<0.05; FIG. 3b) suggesting that platelet CD84 is required for the development of T cell dependent cerebral I/R injury. This is in agreement with the previous observation that platelet depletion abolished detrimental T cell effects during I/R in stroke after adoptive transfer into immune-deficient Rag1.sup.−/− mice (Kleinschnitz et al. 2013).

[0167] The results showed that CD84 expression on T cells as well as platelet CD84 are required to promote infarct growth after tMCAO.

Example 5—Requirement of CD84 for T Cell Migration

[0168] To assess whether CD84 is required for T cell migration, the motility of WT and Cd84.sup.−/− T cells in a Laminin/PDL-coated two dimensional in vitro system was measured. Differences between the two genotypes in response to PMA or the C-C chemokine cysteine motif chemokine ligand 20 (CCL20) were not observed (FIG. 4a; 5a). As it was previously shown that the activatory platelet receptor GPVI plays a critical role in cerebral ischemia (Kleinschnitz et al. 2007, Cherpokova et al.), the effect of the releasate from GPVI-stimulated platelets on T cell motility in this assay was tested. Treatment of WT T cells with platelet-releasate (PLT-R) induced an increase ˜25% in the distance and speed of migrating T cells whereas this effect was absent in Cd84.sup.−/−T cells (FIG. 4b; 5b).

[0169] The releasate of Cd84.sup.−/− platelets failed to increase the migratory capacity of WT T cells, suggesting that platelet-derived sCD84 acts as a co-stimulatory factor on T cells (FIG. 4c; 5c). To test this directly, WT and Cd84.sup.−/− T cells were treated with recombinant soluble CD84 fused to the Fc part of human IgG1 (CD84-Fc) or a GPVI-Fc protein as control (Ctrl-Fc) and their migratory behavior was compared to that of unstimulated T cells (Control). Notably, WT but not Cd84.sup.−/−T cells responded to the presence of CD84-Fc with an increase in velocity and migrated distance compared to the control groups, indicating that the homophilic CD84 interactions are important for the T cell modulating effects of sCD84 (FIG. 4d; 5d). Moreover, Cd84.sup.−/− PLT-R supplemented with CD84-Fc restored T cell motility to a level comparable to that induced with WT PLT-R (FIG. 4e; 5e) demonstrating that platelet-derived sCD84 enhances T cell migration. Of note, the migration-promoting effect of sCD84 on WT T cells was also observed in a migration assay in the presence of primary brain derived microvascular endothelial cells, and results were not affected by their genotype (FIG. 4f; 5f).

[0170] The results indicated that soluble CD84 promotes T cell migration.

[0171] Together, these data demonstrated that platelet CD84 exerts a (co-)stimulatory effect on T cells and is a determinant of infarct progression in a model of ischemic stroke.

Example 6—CD84 Expression in 100 Participants of the Stroke-Induced Cardiac FAILure in Mice and Men (SICFAIL) Study

[0172] Finally, CD84 expression in 100 participants of the Stroke-Induced Cardiac FAILure in Mice and Men (SICFAIL) study was analyzed, to assess a possible link between platelet CD84 expression and disease course in acute ischemic stroke (IS) patients. Median age was 67.0 years (IQR 54.5-76.0 years), 68% of the patients were male and median National Institute of Health Stroke Scale (NIHSS) at admission was 3 (IQR 2-5.5). 19 patients had poor outcome at day three of hospitalization defined as NIHSS≥5. Patients with poor outcome tended to be older, have a history of hypertension and higher NIHSS at admission. Although not statistically significant in univariate analysis (p=0.24), platelet CD84 MFI was identified to be independently associated with poor outcome after adjustment of age and baseline NIHSS (OR=1.27, 95% CI (1.04, 1.57)) (Table 1) in an explanatory multivariable logistic regression approach. These results demonstrate the ability to translate the findings on CD84 in mice into clinical research.

TABLE-US-00001 TABLE 1 Multivariable logistic regression to assess the association of platelet CD84 expression and poor outcome (NIHSS ≥5) at day three of hospitalization, adjusted for age and NIHSS at admission. Odds Ratio (95 % CI) p-value Age 1.07 (1.01, 1.13) 0.02 Baseline NIHSS 1.38 (1.16, 1.64) 0.0003 CD84 mean MFI 1.27 (1.04, 1.57) 0.02

[0173] Part 2—Generation, Selection and Screening of CD84 Activity Blocking Anti-CD84 Antibodies for the Prevention of Cerebral Thrombo-Inflammation

[0174] Material and Methods

[0175] Mice. Animals used in this study are matched for age, sex and genetic background. Experiments are conducted in accordance with the regulations of the local authorities (Regierung von Unterfranken) and performed in accordance with the current ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://www.nc3rs.org.uk/arrive-guidelines).

[0176] Focal ischemia model. Focal cerebral ischemia is induced in 10-to-14-week-old C57BL/6 or Cd84.sup.hCD84/hCD84 mice by tMCAO as previously described (Schuhmann et al. 2015). Inhalation anesthesia is induced by 2% isoflurane. The duration of the surgical procedure per animal is kept below 10 min. A silicon rubber-coated 6.0 nylon monofilament (6021PK10, Doccol, Redlands, Calif., USA) is advanced through the carotid artery up to the origin of the MCA causing an MCA infarction. After an occlusion time of 60 min, the filament is removed allowing reperfusion. Animals are sacrificed 23 h after reperfusion and brains are checked for intracerebral hemorrhages. Neurologic function is analyzed calculating a Neuroscore (score 0-10) based on the direct sum of the Grip test (score 0-5) and the inverted Bederson score (score 0-5) (Schuhmann et al. 2018, Moran et al., Bederson et al.)

[0177] Infarction size measurement. The extent of infarction is quantitatively assessed 24 h after reperfusion. Animals were sacrificed and brains are cut in three 2 mm thick coronal sections. The slices are stained for 20 min at 37° C. with 2% 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich; 2% (w/v) solution) to visualize the infarctions (Junge et al.). Edema-corrected infarct volumes are calculated by planimetry (Image J software, National Institutes of Health) according to the following equation: V.sub.indirect (mm.sup.3)=V.sub.infarct×(1−(VI−VC)/VC). (VI−VC) represents the volume difference between the ischemic hemisphere (VI) and the control hemisphere (VC) and (VI−VC)/VC) expresses this difference as a percentage of the control hemisphere.

[0178] Migration assays. The migratory capacity of T cells is measured using ibidi μ-Slides VI0.4 coated with poly-D-lysine (10 μg/mL, Merck) and laminin (20 μg/mL, Merck) or ibidi Glass Bottom μ-Slides 8 well cultured with primary MBMEC as previously described (Bittner et al.). The migration assays are performed in DMEM high glucose (31053-028, Thermo Fischer) with B27 supplement (2%, 17504044, Thermo Fischer) in triplicates for each different condition (anti-CD84 antibodies (10 μg/μL), Fc-proteins (0.2 μg/ml)). A GPVI-Fc fusion protein control-Fc (Gruner et al.), that did not affect T cell migration compared to vehicle (not shown) or a Fc-only protein serves as control for CD84-Fc, all Fc fusion proteins are purified in house from transfected HEK cells using standard techniques. For time-lapse video microscopy, MACS isolated CD4.sup.+ T cells (130-104-454, Miltenyi; 100 cells/μL) are added to the chamber and images are collected every 30 seconds for 30 minutes on a Leica DM18 inverted microscope with 20×objective. Cells are tracked with LAS X Imaging software and analyzed with ImageJ 1.51 software.

[0179] Statistical analyses. Data are tested for Gaussian distribution e.g. with the D'Agostino and Pearson omnibus normality test and then analyzed by Student's t-test, 1-way ANOVA or Mann-Whitney U-test as applicable. Bonferroni correction is applied when comparing more than two groups in the animal experiments. Scores addressing the functional outcome are compared using the Mann-Whitney U-test. P<0.05 is considered statistically significant. For statistical analysis, the GraphPad Prism 5.0 software package (GraphPad Software) and SAS 9.4 (SAS Institute Inc., Cary, N.C., USA) are used.

Example 7—Generation of Anti-CD84 Antibodies

[0180] Cd84.sup.−/− mice, 6-8 weeks of age, are immunized with a CD84 immuno-precipitate from mouse or human platelet lysate. Each antigen is resolved in Freund's adjuvant and injected subcutaneously. The immunizations are performed repeatedly, before splenic B cells of an immunized mouse are harvested and fused with myeloma cells. The successfully fused Hybridoma cells are positively selected using a specific selection medium. The supernatants of antibody-producing hybridoma cells are screened for the presence of antibodies that recognize murine or human CD84 by enzyme-linked immunosorbent assays (ELISA) and/or flow cytometry.

Example 8—Studying the Effect of Anti-CD84 Antibodies on T Cell Motility; Selection of Blocking Anti-CD84 Antibodies as CD84 Inhibitors

[0181] To assess whether anti-CD84 antibodies affect the motility enhancing effect of soluble CD84 (sCD84) the motility of T cells is assessed in a Laminin/PDL-coated two dimensional in vitro system. Human T cells or T cells from wildtype (WT), or Cd84.sup.hCD84/hCD84 mice (mice that express the extracellular domain of human CD84 instead of murine CD84) are treated with recombinant soluble CD84 fused to the Fc part of human IgG1 (CD84-Fc) or control-Fc protein with or without anti-CD84 antibodies. The migratory behavior of these T cells is analyzed and compared with that of unstimulated T cells (control).

[0182] CD84-Fc should trigger an increase in velocity and migrated distance compared to the control groups, and this mobility promoting effect of CD84-Fc should be reduced by blocking anti-CD84 antibodies. Non-blocking anti-CD84 antibodies that do not reduce mobility-promoting effect of CD84-Fc are discarded and blocking anti-CD84 antibodies that significantly reduce mobility promoting effect of CD84-Fc are selected as inhibitors of CD84 activity for further experiments.

Example 9—Studying the Effect of Blocking Anti-CD84 Antibodies on Experimental Stroke

[0183] To confirm the effect of the anti-CD84 antibodies in thrombo-inflammation, wild-type (WT) or Cd84.sup.hCD84/hCD84 mice (mice that express the extracellular domain of human CD84 instead of murine CD84) receive anti-CD84 IgG or Fab or F(ab′).sub.2 fragments thereof (10-100 μg/mouse) before, during or after being subjected to one hour of transient middle cerebral ischemia (tMCAO) and 23 h of reperfusion. 24 h after tMCAO, the neurological outcome in anti-CD84 treated mice will be compared with that of control animals (mice of the same genotype that received control IgG (Fab or F(ab′).sub.2 fragments thereof) or vehicle instead of anti-CD84 antibodies (Fab or F(ab′).sub.2 fragments) by assessing the Neuroscore. In addition, infarct volumes of the mice are measured by triphenyltetrazolium chloride (TTC) staining. The expectation is that anti-CD84 antibodies or Fab or F(ab′).sub.2 fragments thereof that block the interaction between soluble CD84 and CD84 on T cells result in smaller infarct sizes and a better neurological outcome.

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