Figure 1.

Identification of Ly6C/G⁺ cells in EVL. Spleens from L. donovani infected mice were removed at day 28 after infection and analyzed alongside spleens from naïve mice by flow cytometry and by IHC. A and B: Spleen cells from naïve (A) and infected (B) mice were stained with Ly6G and CD11b and the expression of Ly6C was determined on neutrophils (upper histograms) and monocytes/DC (lower histograms). C and D: Sections from naïve (C) and infected (D) mice were stained for Ly6C/G (Gr-1; red) and F4/80 (green) and counterstained with DAPI (blue). Original magnification, ×100. Insets show boxed areas at higher magnification: Gr-1^bright neutrophils (C) and a nest of F4/80⁻Gr-1^dull cells with two neutrophils in close proximity (D). Original magnification, ×630. E: Spleen cells from infected mice treated with 1A8 or isotype control antibody were stained with Gr-1 and Ly6C. Gr-1⁺Ly6C⁺ neutrophils are highlighted in oval gated regions. F: Spleen cells from infected mice treated with Gr-1 or isotype control antibody were stained with 1A8 and Ly6C. 1A8⁺Ly6C⁺ neutrophils are highlighted in oval gated regions. G–J: Absolute numbers of splenic neutrophils (G and H) and monocytes/DC (I and J) in spleens of naïve mice and infected mice were determined using Ly6G (1A8) and Ly6C for Gr-1-depleted mice (G and I) and using Ly6C and Gr-1 for 1A8-depleted mice (H and J). Data represent means ± SEM from 7 mice per group, pooled from two independent experiments for Gr-1 depletion and 4 mice per group from one experiment for 1A8 depletion. Similar results were seen in an additional experiment in which 1A8-depleted mice were analyzed using CD11b and Ly6C (data not shown). **P < 0.01, ***P < 0.001. ns, not significant. Peripheral blood neutrophils were depleted by 91 ± 4% after 1A8 and 98 ± 0.5% after Gr-1 depletion (data not shown). K: Spleen sections from infected mice administered either isotype control (ISO) or 1A8 were stained for Gr-1 (red) and DAPI (blue). Original magnification, ×200.