data analysis software (version prism 8.0)  (GraphPad Software Inc)

Journal: Frontiers in Immunology

Article Title: Tissue-Resident-Memory CD8 + T Cells Bridge Innate Immune Responses in Neighboring Epithelial Cells to Control Human Genital Herpes

doi: 10.3389/fimmu.2021.735643

Figure Lengend Snippet: Widespread expression of IFI16 in genital skin during and after HSV-2 recurrence. (A) IFI16 expression in a representative HSV-2 ulcer lesion. Right box depicting actively infected epidermis with HSV-2 antigen expression. Left box showing uninfected epidermis distal to HSV lesion. Scale bars, 500 µm. (B) IFI16 expression in a representative serial skin biopsies sequentially obtained at 2-, 4-, and 8-week post-healing and in matched arm control skin. Scale bars, 100 µm. (C) Distribution of CD4 + and CD8 + T cells and IFI16 expression in an ulcerative HSV-2 lesion. IFI16 and HSV-2 antigen expression staining were performed on adjacent tissue sections. Inserts are higher magnification of the indicated boxed areas. (D) Quantitative PCR measurement of HSV-2 DNA and host genome in the right and left portion of the lesion tissue as designated in (C) .

Article Snippet: For experiments in the IFI16 knockout fibroblasts, data analysis performed using GraphPad Prism version 8.0, with yields analyzed by two-way ANOVA and Holm-Sidak’s multiple comparisons test (α = 0.05).

Techniques: Expressing, Infection, Control, Staining, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Tissue-Resident-Memory CD8 + T Cells Bridge Innate Immune Responses in Neighboring Epithelial Cells to Control Human Genital Herpes

doi: 10.3389/fimmu.2021.735643

Figure Lengend Snippet: Elevated level of IMA gene signature expression correlates with IFNG expression by DEJ CD8TRM cells in HSV-2 post-healing human skin. (A) Cell density (upper) and IFNG gene expression (lower) of DEJ CD8TRM in skin biopsies taken at the sites of 8-week post-healing and their matched contralateral controls (n = 8). DEJ CD8TRM cells were laser microdissected and coupled with quantitative RT-PCR. IFNG gene expression relative to ACTB. Individual (B) and collective levels (C) of IMA gene signature expression in the 8-week and control set of skin biopsies with or without IFNG detection. (D) Micrographic images depicting spatial expression of IFI16 , CD8 and IFNG mRNA transcripts in skin epidermis using RNA FISH dual probe sets of IFI16 + IFNG or IFI16 + CD8 in adjacent tissue sections of an 8-week skin biopsy, and dual probe set for IFI16 + CD8 on matched normal control skin. Scale bar, 100 µm.

Article Snippet: For experiments in the IFI16 knockout fibroblasts, data analysis performed using GraphPad Prism version 8.0, with yields analyzed by two-way ANOVA and Holm-Sidak’s multiple comparisons test (α = 0.05).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control

Journal: Frontiers in Immunology

Article Title: Tissue-Resident-Memory CD8 + T Cells Bridge Innate Immune Responses in Neighboring Epithelial Cells to Control Human Genital Herpes

doi: 10.3389/fimmu.2021.735643

Figure Lengend Snippet: Mechanism, specificity and effectiveness of IFN- γ -mediated antiviral action in primary human skin cells. (A) Evaluation of IFN- γ effect on HSV immediate early (IE) gene transcription via RNA FISH. In situ detection and visualization of IE gene expression using pooled probes specific to each of the five IE genes. Primary human keratinocytes 6 h.p.i. with K26 (MOI=1) either mock-treated or pretreated with IFN-γ (10 U/ml or 100 U/ml). Scale bar, 50 µm. (B) Quantitative assessment of IFN-γ-mediated reduction of IE gene expression via qRT-PCR. Primary human fibroblasts infected with wildtype strain KOS, MOI=1, 4 h.p.i. Mock treated compared to IFN- γ treated, 100 U/ml. (C) IFN-γ effect on HSV gene transcription (ICP27, ICP8, and glycoprotein B) in the presence or absence of interferon blockers, GIR208 and B18R. Primary human keratinocytes infected with KOS, MOI=1, 4 h.p.i. (D) Time-dependent inhibition by IFN- γ . Percentage of GFP-expressing cells in K26-infected primary human keratinocytes 16 h.p.i. (MOI=1) with IFN- γ treatment initiated at 2, 4, 8, 16, 24, or 48 hours prior to infection. (E, F) Dose-dependent inhibition by IFN- γ . GFP expression (E) and viral DNA replication (F) under increasing dosage of IFN-γ in K26-infected keratinocytes. MOI=1, 16 h.p.i. HSV genome DNA were quantified by qPCR and compared between 2 and 16 h.p.i. (G) Growth kinetics of strain KOS under low and high MOI in primary human fibroblasts with (100 U/ml) or without IFN-γ. (H) Responsiveness to IFN-γ in IFI16 knockout and Cas9 control human fibroblasts. Immunoblot of whole-cell lysate from IFI16 knockout and Cas9 control cells mock-treated or pretreated with IFN-γ (500U/mL) for 20 hours. One of three biological replicates is shown. (I) Viral yield 12 h.p.i. following wildtype HSV-2 strain 186 infection (MOI=0.1) of IFI16 knockout and Cas9 control fibroblasts mock-treated or pretreated with IFN-γ (500U/mL) for 20 hours. Two-way ANOVA with Holm-Sidak’s multiple comparisons test. Values are mean ± S.D. ( error bars ), from three independent experiments.

Article Snippet: For experiments in the IFI16 knockout fibroblasts, data analysis performed using GraphPad Prism version 8.0, with yields analyzed by two-way ANOVA and Holm-Sidak’s multiple comparisons test (α = 0.05).

Techniques: In Situ, Gene Expression, Quantitative RT-PCR, Infection, Inhibition, Expressing, Knock-Out, Control, Western Blot