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. 2012 Nov 29;7(11):e50498. doi: 10.1371/journal.pone.0050498

Figure 3. CCX8037 does NOT reduce accumulation of OT-I CD8 T cells in the skin.

Figure 3

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 OVA257–264. Animals treated with OVA257–264 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, 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 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 + OVA257-264 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 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 (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.