Neutrophils massively infiltrate ADLNs early after vaccination and shelter BCG. (A) At 24 hours after vaccination with BCG-egfp, ADLN EGFP⁺ gated cells were phenotyped as CD11b⁺, Ly-6G⁺, F4/80^-, and MHCII^-, indicating they were neutrophils. (B) CD11b⁺ ADLN cells were magnetically enriched and EGFP⁺ cells were sorted by fluorescence-activated cell sorting (FACS). The majority of the CD11b⁺/EGFP⁺ cells were positive for Ly-6G (red), and microscopic analysis revealed their characteristic polylobed nucleus stained with DAPI (blue). Several bacilli (green) were often detected inside the same neutrophil. Cells adhering to slides were labeled with Alexa fluor 594 (red) and DAPI (blue) and observed under a Zeiss Axioskop fluorescence microscope with an Achroplan 100 ×/1.25 NA oil iris objective. Images were acquired with a Leica 300F camera and processed with Leica Qwin software. (C) Neutrophil recruitment to the ADLNs was analyzed at 4, 12, 24, and 72 hours after BCG-egfp injection, by immunostaining cryosections with anti-Ly-6G. From 4 to 24 hours, neutrophils (red) massively infiltrated the subcapsular space and sinuses of the ADLNs and mainly colocalized with BCG (green). Bacilli were detected inside neutrophils under the ADLN capsule as observed by confocal microscopy and indicated by arrows. At 72 hours, neutrophils disappeared from the ADLNs. (i-ii, iv-vi) Images were acquired as in Figure 3B. (iii) Image was acquired as in Figure 4Diii.