Fig. 7. Blocking Gα_I-dependent chemotaxis in CX3CR1⁺ cells inhibits CCR2⁺Ly6C⁺ splenic monocyte clustering and impairs protection.

CX3CR1^CreERT2Rosa26^LSL-PTX mice transferred with OT-I cells were immunized with 10⁴ Lm-Ova. Six weeks later, mice received Tx or vehicle intraperitoneally daily for 5 days before 10⁶ Lm-Ova recall infection. (A) One hour before harvesting spleens and 16 hours after challenge, 5 μg of Ly6C-PE mAb was injected intravenously to the hosts. Cells were then stained for cell surface CD11b and Ly6C-PerCpCy5.5. After gating on Ly6C-PerCpCy5.5⁺ monocytes, the proportion of Ly6C-PE^hi and Ly6C-PE^low monocytes was quantified. Representative FACS dot plots are shown, and bar graphs pool two independent replicate experiments with n = 6. (B) Twenty-four hours after Lm infection (10⁶), spleens and livers were harvested and plated to enumerate Lm titers. A nonimmunized group of age-matched mice (“primary”) were also infected as control. Bar graphs show one of two independent experiments with n = 3 mice. P values are indicated. *P < 0.1, **P < 0.01, ***P < 0.001, and ****P < 0.0001.