CCX8037 does NOT reduce accumulation of OT-I CD8 T cells in the skin.

<p>Animals were injected with 3e6 OT-I CD8 T cells, and epicutaneously immunized 24 hours later on the ear skin. Ear skin was painted with either 100 µg Cholera Toxin (CT) only, or 100 µg CT + 100 µg OVA_257–264. Animals treated with OVA_257–264 were split into two groups and received subcut. injections of CCX8037 or vehicle every 12 hours for the course of the study. Mice were sacrificed for analysis 5 days post immunization. Mean and SEM shown for each data point, <i>p</i> values indicate Bonferroni multiple comparison post test. (A) Representative flow cytometry plot showing the accumulation of CD44^hi CD8 T cells, and gating of OT-I (CD45.1) cells in the ear skin. Plots are pre-gated on CD3ε⁺ CD8α⁺ cells. (B) Quantification of OT-I CD8 T cell accumulation in ear skin. Mice treated with CT only did not exhibit substantial OT-I CD8 T cell homing into the ear skin. Mice treated CT + OVA_257-264 and treated with vehicle had significant OT-I CD8 T cell homing, with 79.6% of all resident CD8 T cells being OT-I derived, while those treated with CCX8037 had 80.2% of all CD8 T cells being OT-I derived. N = 5 groups for vehicle, 6 for CCX8037 and 3 for CT only. Each group was comprised of 3–5 pooled mice. (C) CCX8037 did not affect the proliferation of OT-I CD8 T cells in CLN after Ag exposure. In animals exposed to CT only, OT-I CD8 T cells composed 1.4% of all CD44^hi CD8 T cells. In animals exposed to CT + OVA, there was no significant difference in the percentage of CD44^hi CD8 T cells that are OT-I between those treated with vehicle (26.0%) and CCX8037 (30.1%). N = 3 groups for CT only treated mice, N = 6 groups for Vehicle and CCX8037 treated mice, where each group is 3–5 pooled mice. (D) Generation of skin homing associated molecule E-selectin ligand by OT-I CD8 T cells in CLN was not affected by CCX8037. When mice were immunized with OVA antigen, the E-lig was not significantly affected by the presence of the CCX8037 (38.4% vehicle, 43.3% CCX8037). In the absence of OVA antigen, E-lig production by OT-I CD8 T cells was negligible (<1% E-lig⁺). N = 4 for CT only, 16 for Vehicle and CCX8037.</p

CCX8037 is a potent and selective antagonist of CCR9.

<p>CCL25-induced chemotaxis was measured on Molt-4 cells and murine thymocytes by using a DNA intercalating fluorescent dye (CyQUANT) to quantify responding cells and is labeled as the “migration signal”, as the relative fluorescence of the migrated population is proportional to the number of cells that migrated. Six to eight replicates were performed per data point. Calcium flux was measured on Molt-4 cells or IL-2-cultured lymphocytes loaded with Indo-1AM dye and exposed to IC₅₀ concentrations of the chemokines indicated. A) CCX8037 inhibits CCL25-induced Molt-4 chemotaxis in buffer (0.1% BSA in HBSS) with an IC₅₀ of 12 nM (n = 31). B) CCX8037 inhibits CCL25-induced Molt-4 chemotaxis in the presence of 100% human AB serum with an IC₅₀ of 32 nM (n = 9). C) CCX8037 inhibits CCL25-mediated mobilization of intracellular Ca2+ in Molt-4 cells with an IC₅₀ of 19 nM (n = 3). D) CCX8037 (blue trace; 10 uM) does not inhibit chemokine induced mobilization of intracellular Ca2+ to EC₅₀ concentrations of CCL4(CCR5), CCL15 (CCR1) on IL-2 activated lymphocytes. E) CCX8037 inhibits CCL25-induced murine thymocyte chemotaxis in buffer with an IC₅₀ of 2.5 nM (n = 2).</p

CCX8037 reduces accumulation of OT-I CD8 T cells in the intestinal epithelium without affecting gut homing tropism, imprinting and proliferation.

<p>Animals were injected with 3e6 OT-I CD8 T cells, and immunized 24 hours via oral gavage with either 10 µg Cholera Toxin (CT) only, or CT + 25 mg Ovalbumin (OVA). Animals given OVA were also injected subcut. every 12 hours for the course of the study with CCX8037 (30 mg/kg) or vehicle. Mice were sacrificed for analysis 5 days post immunization. Mean and SEM shown for each data point, <i>p</i> values indicate Bonferroni multiple comparison post test. (A) Representative flow cytometry plot showing the accumulation of CD44^hi CD8 T cells, and gating of OT-I (CD45.1) cells in the intestinal epithelium. Plots are pre-gated on CD3ε⁺/CD8α⁺ cells. (B) Quantification of OT-I CD8 T cell accumulation in intestinal epithelium. Mice fed CT only did not exhibit substantial OT-I CD8 T cell homing into the intestinal epithelium. Animals fed CT + OVA and treated with vehicle had significant OT-I CD8 T cell homing, with 27.9% of all resident CD8 T cells being OT-I derived. Animals fed CT + OVA and injected with CCX8037 exhibited significantly reduced intestinal epithelium accumulation of OT-I CD8 T cells, to 4.7%. N = 13 mice for CCX8037 and vehicle groups, and 6 for CT only. (C) CCX8037 did not affect the proliferation of OT-I CD8 T cells in MLN after Ag exposure. In animals exposed to CT only, OT-I CD8 T cells composed 1.8% of all CD44^hi CD8 T cells. In animals exposed to CT + OVA, there was no significant difference in the percentage of CD44^hi CD8 T cells that are OT-I between those treated with vehicle (29.7%) and CCX8037 (27.6%). N = 6 for CT only treated mice, N = 13 for Vehicle and CCX8037 treated mice. (D) Generation of gut homing molecules on OT-I CD8 T cells in MLN was not affected by CCX8037. Animals not fed OVA antigen had significantly lower β7⁺, CCR9⁺, or β7⁺CCR9⁺ expression. However, the expression of gut homing molecules was not significantly affected by CCX8037 treatment, compared to vehicle. OT-I CD8 T cells of vehicle and CCX8037 treated mice were 45.6% and 49.1% β7⁺ respectively, 44.7% and 44.1% CCR9⁺ respectively, and 36.3% and 36.1% β7⁺CCR9⁺ respectively. N = 13 mice for CCX8037 and vehicle groups, and 6 for CT only.</p

Chemerin stimulates transendothelial migration of MSCs and requires MMP-2.

<p><i>A</i>, Representative fields from MSC transendothelial migration experiments showing migration of PKH67-labelled MSCs (left). CCX832 (1 µM) inhibited chemerin- (center) and CAM-CM stimulated MSC transendothelial migration but CCX826 (1 µM) had no effect (right). <i>B</i>, Chemerin, and IGF-II used as a positive control, promptly (30 min) stimulated proMMP2 abundance in media as detected by Western blot but had no effect on cellular proMMP2 abundance (left); chemerin significantly increased MMP-2 enzyme activity in MSC media detected by the selective substrate MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH₂ (right). <i>C</i>, Human recombinant MMP-2 (80 ng/ml) stimulated transendothelial migration and there was dose-dependent inhibition by an MMP-2 selective inhibitor (MMP-2 inhibitor I) (left). The MMP-2 inhibitor (60 µM) significantly inhibited chemerin-stimulated MSC transendothelial migration (centre). Horizontal arrows, p<0.05, t- test (n = 3).</p

Chemerin exhibits increased expression in CAMs and stimulates MSC migration.

<p><i>A</i>. Representative Western analysis of chemerin in media from ESCC CAMs and ATMs (left). Quantitative analysis by densitometry of chemerin abundance in media from ESCC CAMs and ATMs (n = 4 different pairs of myofibroblasts) (right). <i>B</i>. Concentration-dependent stimulation of MSC migration by chemerin in scratch wound migration assays (left) and Boyden chamber migration assays (right)(n = 3). <i>C</i>. Increased migration of MSCs in Boyden chambers in response to conditioned media (CM) from CAMs and their respective ATMs (left) (n = 4 different pairs of myofibroblasts). Stimulation of MSC migration by CAM-CM was inhibited by chemerin neutralizing antibody (Chem.Ab; 10 µg/ml) (center). MSC migration was decreased in response to CM from CAM1 and CAM4 cells transfected with chemerin siRNA#3 (right). Horizontal arrows, p<0.05, t- test (n = 3).</p

ChemR23 mediates chemerin stimulation of MSC migration via PKC and MAP kinases.

<p><i>A</i>, Representative images from MSCs stained for vimentin (positive control) and chemR23 revealing knock-down (KD) after ChemR23 siRNA treatment (left). Knockdown of ChemR23, but not GPR1, inhibited MSC migration in response to chemerin (100 ng/ml)(center) and CAM-CM (right). <i>B</i>, Concentration-dependent inhibition of MSC migration in response to chemerin by the ChemR23 antagonist CCX832 (left) but not the control compound CCX826 (1 µM) (center). MSC migration in response to CAM-CM was inhibited similarly by chemerin neutralising antibody, and CCX832, but not CCX826 (1 µM)(right). <i>C</i>, Representative Western blot shows increased phosphorylation of p42/44, p38 and JNK-II kinases in MSCs treated with chemerin (100 ng/ml)(left). In Boyden chamber assays, chemerin-stimulated MSC migration was inhibited by the JNK-II inhibitor, SP600125 (50 µM), the p42/44 inhibitor, UO126 (10 µM), p38 inhibitor SB202190 (3 µM), and the PKC inhibitor Ro320432 (2 µM) but not by PIK3 inhibitor LY294002 (50 µM) (right). Horizontal arrows, p<0.05, ANOVA (n = 3 in each case).</p

Increased MSC homing to xenografts seeded with CAMs and inhibition of homing by the chemR23 receptor antagonist, CCX832.

<p><i>A</i>, Visualisation of PKH67-labelled MSCs in representative fields from xenografts established with OE21 cancer cells alone or co-injected with CAMs followed by treatment with vehicle (top) or CCX832 (bottom) and iv injection of PKH67-labelled MSCs. <i>B</i>, In xenografts with OE21 cancer cells and CAMs there was increased MSC homing expressed as labelled cells per unit area of xenograft compared with xenografts of OE21 cancer cell alone; treatment with CCX832 inhibited homing (OE21/vehicle, n = 3; OE21/CCX832, n = 4; OE21 and CAMs/vehicle, n = 6; OE21 and CAMs/CCX832, n = 6). Horizontal arrows, p<0.05, ANOVA.</p