Figure 1. CCX8037 is a potent and selective antagonist of CCR9. 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 CCL25mediated 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).
Extravasation of OT-I CD8 T Cells to Inflamed Skin is Unaffected by CCR9 Antagonist
The prior experiments did not rule out the possibility that CCX8037 caused a general defect in trafficking of CD8 T cells to all peripheral tissues. To see if this was the case, we tested the ability of CCX8037 to inhibit Ag-specific accumulation of OT-I cells within the skin [1]. OT-I CD8 T cells were adoptively transferred into WT CD45-congenic recipients exactly as performed in the gut-homing studies above. Animals were immunized topically on the ear skin with either CT only or with CT + OVA. As above, animals that received both antigen and adjuvant were split into two groups for injection of CCX8037 or vehicle every 12 hours. Mice were sacrificed 5 days post immunization and cell suspensions were prepared directly from the inflamed ear skin and from the cervical lymph nodes (cLN), which are the lymphoid organs that directly drain the site of inflammation. In striking contrast to the findings in gut, CCX8037 did not lead to any appreciable reduction in the proportion of CD8 T cells within skin that were derived from the OT-I donor (Figs. 3A and B). Ag-induced proliferation of OT-I cells within the cLN was not affected by CCX8037, nor was their imprinting with the skinhoming molecule E-lig (Fig. 3C and 3D). Unlike gut-selective homing of CD8 T cells, skin specific homing does not depend upon CCR9 function. Having shown that CCX8037 compromises CD8 T cell homing to the gut, but not to the skin in vivo, we find that a small molecule drug can be administered systemically to selectively inhibit CCR9-mediated trafficking of CD8 T cells to the small intestine.
Concluding Remarks
In this study, we have demonstrated in vivo that a small molecule inhibitor of CCR9 can prevent T cell accumulation within inflamed intestine without affecting inflamed skin. We believe this to be the first clear-cut in vivo example of a small molecule able to modulate tissue-selective T cell mediated immunity, a strategy that may well be used to modulate autoimmune syndromes in the intestine, as well as autoimmune syndromes in other tissues.
Materials and Methods Ethics Statement
All experimental procedures involving animals were approved by the Children’s Hospital Boston (CHB) IACUC (protocol number 10-04-1645R), which oversees the facility in which these procedures were performed.
Mice
C57BL/6 n mice (CD45.2+) were purchased from Charles River Labs (Wilmington, MA). OT-I Tg mice were bred onto the CD45.1 C57BL/6 n background ((B6.SJL-Ptprca Pep3b/BoyJ; The Jackson Laboratory). Animals were housed under SPF conditions.
Cell Culture, Chemotaxis and Mountain Peak Assays
Molt-4 cells were obtained from the ATCC (Manassas, VA) and cultured in RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Invitrogen, Carlsbad, CA) in a humidified 5% CO2 incubator at 37uC.
Figure 2. CCX8037 reduces accumulation of OT-I CD8 T cells in the intestinal epithelium without affecting gut homing tropism, imprinting and proliferation. Animals were injected with 3e6 OT-I CD8 T cells, and immunized 24 hours via oral gavage with either 10 mg 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, p values indicate Bonferroni multiple comparison post test. (A) Representative flow cytometry plot showing the accumulation of CD44hi CD8 T cells, and gating of OT-I (CD45.1) cells in the intestinal epithelium. Plots are pre-gated on CD3e+/CD8a+ 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 CD44hi CD8 T cells. In animals exposed to CT + OVA, there was no significant difference in the percentage of CD44hi 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 b7+, CCR9+, or b7+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% b7+ respectively, 44.7% and 44.1% CCR9+ respectively, and 36.3% and 36.1% b7+CCR9+ respectively. N = 13 mice for CCX8037 and vehicle groups, and 6 for CT only. addition of CyQUANT (Invitrogen) to the cells and measuring the resulting fluorescence using a Spectraflour Plus plate reader (Tecan, Grodig, Austria). Mouse thymocytes for determining the murine potency of CCX8037 were isolated from 3? week old C57BL/6 mice (The Jackson Laboratory, Sacramento CA). Mountain peak assays were performed using IL-2 cultured lymphocytes as previously described [11].
