Journal: ACS nano

Article Title: Mucosal Immunization with a pH-Responsive Nanoparticle Vaccine Induces Protective CD8 + Lung-Resident Memory T Cells

doi: 10.1021/acsnano.9b00326

Figure Lengend Snippet: (A) Schematic of the experimental timeline. (B) Flow cytometry was used to identify antigen-specific (Tet+) CD8+ T cells in distinct lung compartments (airway, AW; interstitium, IST; marginated vascular, MV) and the spleen. BAL was collected to discriminate AW vs. IST cells, and i.v. staining with αCD45 antibody discriminated IST (CD45−) vs. MV (CD45+) cells. Samples were stained with PE-labeled SIINFEKL/MHC-I tetramer to identify antigen-specific CD8+ T cells. After gating out CD11b+, CD11c+, B220+, and CD4+ cells (“dump”), CD8α+CD45−Tet+ events in AW and IST, CD8α+CD45+Tet+ events in MV, and CD8α+Tet+ events in the spleen were quantified. Dot plots are representative of the gating strategy used in multiple experiments (see Figures S3A–S3C). (C) In conjunction with i.v. staining, microscopy was used to visualize fluorescent OVA-NP conjugates in the lower airways 24 h after immunization. Lungs were stained with αCD45 antibody, which labels vascular leukocytes, and tomato lectin, which binds to capillary endothelial cells and allows for visualization of lung structure. Purple: OVA-NP; blue: vascular leukocytes; green: lung vasculature. Scale bar = 100 μm. Immunization dose: 25 μg NP, 7.5 μg OVA.

Article Snippet: Recombinant vaccinia virus expressing influenza virus nucleoprotein, ovalbumin SIINFEKL peptide, and enhanced green fluorescent protein (VV.NP-S-EGFP) was obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH (NR-624; BEI Resources, Manassas, VA).

Techniques: Flow Cytometry, Staining, Labeling, Microscopy