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