Fig 1: Presence of pDCs in Naïve Corneas and Alterations in Their Density upon Sterile Inflammation and Acute HSV-1 Keratitis(A and B) Flow cytometric analysis of pooled corneal single-cell suspensions of naïve mice (n = 15–20 corneas) and 3 days post-thermal cautery (n = 4–8 corneas). (A) Flow cytometry density plots illustrating PDCA-1+ CD45R/B220+ cells among CD45+ cells in naïve and inflamed corneas. Numbers represent the frequency of CD45+ PDCA-1+ CD45R/B220+ cells among total corneal single cells, and parentheses show the relative frequency of CD45+ PDCA-1+ CD45R/B220+ cells among CD45+ cells. (B) Flow cytometric histograms on the phenotype of CD45+ PDCA-1+ CD45R/B220+ cells in naïve and inflamed corneas. Depicted flow cytometry data are representative of three independent experiments.(C and D) Representative confocal micrograph of freshly excised unfixed corneas of DPE-GFP × RAG-1-/- mice with GFP-tagged pDCs in the center (C) and periphery (D) of naïve corneas. Images are representative of n = 3 mice.(E) Flow cytometric histograms showing phenotype of GFP-tagged cells in the cornea of DPE-GFP × RAG-1-/- mice on day 3 following thermal cautery. Depicted flow cytometry data are representative of three independent experiments.(F) TEM of a resident corneal pDC in a naïve WT C57BL/6 mouse (magnification 7,500x). White arrowhead, cell body; N, nucleus; white asterisk, cell processes extending from cell body.(G-I) MPM of freshly excised corneas. (G) Stub-like extensions from cell bodies (asterisks) depicted in naïve DPE-GFP × RAG-1-/- mice. (H) A corneal cDC in a naïve CD11c-GFP-DTR mouse. (I) MPM of freshly excised inflamed cornea 3-days post-thermal cautery in DPE-GFP × RAG-1-/- mice. Dendritiform processes (white arrows) and cell body (white arrowheads) are depicted. Another population of pDCs with round cell bodies without dendritiform processes (red arrowheads) is depicted as well.(J) Single-cell PCR on GFP-tagged pDCs from naïve corneas of DPE-GFP × RAG-1-/- mice in comparison with naïve WT C57BL/6 splenic PDCA-1+ CD45R/B220+ Siglec-H+ pDCs and F4/80+ macrophages for mRNA levels of TLR7 and 9. Data represent three independent experiments.(K and L). Flow cytometric analysis of single-cell suspension of corneas of sham-infected (n = 15–20 pooled corneas) and HSV-1-infected (n = 4–8 pooled corneas) mice. (K) Density plots showing PDCA-1+ CD45R/B220+ cells among CD45+ cells. Numbers represent the frequency of CD45+ PDCA-1+ CD45R/B220+ cells among total corneal single cells, and parenthesis demonstrate relative frequency of CD45+ PDCA-1+ CD45R/B220+ cells among CD45+ cells. (L) Flow cytometric histograms on the phenotype of CD45+ PDCA-1+ CD45R/B220+ cells. Depicted flow cytometry data are representative of three independent experiments.Scale bars: 50 µm (C, D, and I), 12 µm (G and H), and 5 µm (F). pDC, plasmacytoid dendritic cell; N, cell nucleus; G, Golgi apparatus; white asterisk, thick process; white arrowhead, elongated cell body; red arrowhead, round cell body; white arrow, thin dendritiform process. Bars denote SD. *p < 0.05, **p < 0.01, and ***p < 0.001.