Antigen presentation by neutrophils. (A) Immunofluorescence staining of a mLN 48 hours after TBI showing ring-shaped localization of Ly6G⁺ cells (red) around lymphocytes next to lymphatic vessels (Lyve-1, green; 4′,6-diamidino-2-phenylindole, blue; blood vessels (CD31), white; scale bar, 200 µm). (B) MLN 48 hours after allo-HSCT showing neutrophils (Ly6G⁺ = red) close to transplanted CD45.1⁺ T cells (green, scale bar = 150 µm). (C) Representative histogram of MHC-II expressing CD45⁺CD11b⁺Ly6G⁺ neutrophils in spleen, iLN, aLN, mLN, and ileum in C57BL/6 mice 48 hours after TBI. (D) Frequency of MHC-II⁺ (percentage of CD45⁺CD11b⁺Ly6G⁺) neutrophils in different organs in CD57Bl/6 mice 48 hours after TBI. (E-F) MHC-II expression and peptide presentation (YAe⁺) on CD11b⁺Ly6G⁺ neutrophils in B6D2F1 mice were assessed by flow cytometry in untreated mice, 24 hours after TBI and 48 hours after BM transplantation. (E) Representative FACS plots showing gating strategy. (F, left) Relative numbers of YAe⁺MHC-II⁺ cells of host neutrophils. (Right) Absolute numbers of host YAe⁺MHC-II⁺ neutrophils. (G-H) For neutrophil depletion, BALB/c mice were injected with either anti-Ly6G (0.5 mg intraperitoneally) or an isotype control (0.5 mg) on day −1 and transplanted after TBI on day 0 with 1 × 10⁷ purified and CellTrace Violet-labeled CD4⁺/CD8⁺ T cells from C57BL/6 mice. Mice were sacrificed on day 3 and mLN analyzed for donor-derived (H2kb⁺) T cells that had proliferated. (G) Representative flow cytometry plots of CellTrace Violet-dilution of H2kb⁺CD4⁺ T cells. (H) Pooled data from 2 independent experiments showing proliferated H2kb⁺CD4⁺ T cells after anti-Ly6G neutrophils depletion. (I) BALB/c mice were treated on day −1 by anti-Ly6G (0.5 mg) or isotype control antibody (0.5 mg) injection and underwent allo-HCT (FVB into BALB/c; 5 × 10⁶ BM cells + 0.5 × 10⁶ CD4⁺/CD8⁺ T cells). BM controls were treated on day 0 by isotype control antibody (0.5 mg) injection and underwent transplantation using BM alone (FVB 5 × 10⁶ cells).