Leukocyte isolation and phenotyping of cells

An intravascular staining method was used to discriminate between cells within the vasculature and those within the parenchyma of tissues. Three minutes prior to sacrifice, mice were intravenously injected with either 3 μg of biotinylated- or fluorophore-conjugated CD8α (53-6.7) antibody or 2 μg of fluorophore-conjugated CD45 (30-F11) antibody⁴². Tissues were harvested and leukocytes isolated as previously described⁸. Studies of the salivary gland focused exclusively on the submandibular gland. Isolated leukocytes were surface-stained with monoclonal anti-mouse monoclonal antibodies at a 1:100 dilution unless otherwise noted: CD4 (GK1.5), CD5 (53-7.3), CD8α (53-6.7), CD8β (53-5.8), CD11c (N418), CD43 (1B11), CD44 (IM7), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD64 (X54-5/7.1), CD69 (H1.2F3), CD103 (2E7), Thy1.1 (OX-7) (1:250), B220 (RA3-6B2), F4/80 (BM8), Ly6C (HK1.4) (1:400), Ly6G (1A8), NKp46 (29A1.4), PD-1 (29F.1A12), CX3CR1 (SA011F11), CXCR3 (CXCR3-173) from BioLegend; CD11b (M1/70), CD19 (1D3), NK1.1 (PK136), Siglec-F (E50-2440), TCF-1 (S33-966) (1:50) from BD; CD3ε (145-2C11), CD127 (A7R34), KLRG1 (2F1), TCRβ (H57-597) from Tonbo Biosciences. Cell viability was determined using Ghost Dye Violet 510 or Ghost Dye Red 780 (Tonbo Biosciences) (1:300). To identify VSV nucleoprotein-specific CD8⁺ T cells, leukocytes were stained with H-2K^b/N (MHC class I tetramer) (1:200), conjugated to PE. To identify gp33-specific CD8⁺ T cells, leukocytes were stained with H-2D^b/N (MHC class I tetramer) (1:200), conjugated to APC. Staining for intracellular transcription factors and proteins was performed using a transcription factor staining buffer kit (Tonbo Biosciences) with monoclonal anti-mouse antibodies: T-bet (4B10) from BioLegend; Eomes (Dan11mag), Foxp3 (FJK-16s), Gata-3 (TWAJ), Ki67 (SolA15) (1:400), Rorγt (B2D) from ThermoFisher Scientific. The stained samples were acquired using LSRII or LSR Fortessa flow cytometers (BD) and analyzed with FlowJo software (BD). Neutrophils were distinguished by expression of CD11b and Ly6G. Eosinophils were identified by Siglec-F expression. Innate lymphoid, natural killer, B cell, and monocyte/macrophage populations were distinguished after excluding lineage-positive cells using combinations of CD3, CD5, CD19, B220, and Ly6G and then using recommended lineage defining-markers as described^43,44. Monocytes were further subdivided into classical and patrolling populations on the basis of Ly6C expression. Lung macrophages were subdivided into alveolar and interstitial populations on the basis of Siglec-F and CD11b expression.