Journal: Biomolecules

Article Title: Antiviral Functions of Type I and Type III Interferons in the Olfactory Epithelium

doi: 10.3390/biom13121762

Figure Lengend Snippet: Upregulation of type I and III interferon transcript levels. Relative expression of Ifna2, Ifna4, Ifnb2, and Ifnl2/3 in the olfactory mucosa using qRT-PCR ( A – D ). Relative expression of Ifnar1 and Ifnlr1 in the olfactory mucosa at 24 h PI ( E ). Biological triplicates were included ( A – E ). Immunostaining of IFNAR1 (green in ( F )) and IFNLR1 (green in ( G )) with OMP (red in ( F , G )) in the OE. Bar = 15 μm.

Article Snippet: The antibodies used were: chicken anti-OMP (custom, 1:1000), goat anti-GFP (Rockland (Baltimore, MD, USA), 2.2 µg/mL), rabbit anti-IFNAR1 (Sino Biological (Beijing, China), 1:20), goat anti-IFNLR1 (Novus Biologicals (Centennial, CO, USA), 10 μg/mL), and rabbit anti-pSTAT1 (Abcam, 3.0 µg/mL).

Techniques: Expressing, Quantitative RT-PCR, Immunostaining

Journal: Biomolecules

Article Title: Antiviral Functions of Type I and Type III Interferons in the Olfactory Epithelium

doi: 10.3390/biom13121762

Figure Lengend Snippet: Interferon signaling is required for suppressing VSV replication in the olfactory mucosa. The expression levels of the viral genes, VSV-GFP, VSV-M, and VSV-N, in olfactory mucosae at 24 h PI were measured in Ifnar1 −/− ( A ), Ifnlr1 −/− ( B ), Ifnar1 −/− ; Ifnlr1 −/− ( C ), and Stat1 −/− ( D ) and compared to wildtype littermates. Student t -test, * p < 0.05.

Article Snippet: The antibodies used were: chicken anti-OMP (custom, 1:1000), goat anti-GFP (Rockland (Baltimore, MD, USA), 2.2 µg/mL), rabbit anti-IFNAR1 (Sino Biological (Beijing, China), 1:20), goat anti-IFNLR1 (Novus Biologicals (Centennial, CO, USA), 10 μg/mL), and rabbit anti-pSTAT1 (Abcam, 3.0 µg/mL).

Techniques: Expressing

Journal: EBioMedicine

Article Title: RIG-I activating immunostimulatory RNA boosts the efficacy of anticancer vaccines and synergizes with immune checkpoint blockade

doi: 10.1016/j.ebiom.2019.02.056

Figure Lengend Snippet: DC activation via RIG-I is mediated by the adapter protein MAVS and type I IFN signaling. Bone marrow-derived dendritic cells (BMDCs) from wild-type (WT) and indicated genetically deficient mice were transfected with 3pRNA or synthetic non-triphosphorylated RNA of the same sequence (synRNA) or were stimulated with the TLR ligands CpG or LPS. (a-c) Levels of IFN-α, IL-6 and IL-12p40 in culture supernatants were determined by ELISA. (b-e) CD86 and MHC class I expression on BMDCs from wild-type, MAVS (MAVS −/−) - and IFNaR1 (IFNaR1 −/− )-deficient mice were analyzed by flow cytometry. (f-h) BMDCs from ASC-deficient (ASC −/− ) animals were stimulated identically. (f) Levels of IL-1β, (g) expression of CD86 and (h) MHC-I were determined. All data give mean ± S.E.M. of at least triplicate samples representative of three independent experiments. An asterisk without brackets indicates comparison to the WT unstimulated control group (* P < 0.05, ** P < 0.01, *** P < 0.001, one-way analysis of variance (ANOVA) for multiple comparisons). MFI, mean fluorescence intensity.

Article Snippet: In some experiments, mice were pre-treated intraperitoneally (ip) with 400 μg anti-murine IFNaR1 antibody (clone MAR1-5A3, BioXCell, West Lebanon, NH) one day prior to the above immunization.

Techniques: Activation Assay, Derivative Assay, Transfection, Sequencing, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Comparison, Control, Fluorescence

Journal: EBioMedicine

Article Title: RIG-I activating immunostimulatory RNA boosts the efficacy of anticancer vaccines and synergizes with immune checkpoint blockade

doi: 10.1016/j.ebiom.2019.02.056

Figure Lengend Snippet: Enhanced cross-presentation upon RIG-I activation mediates efficient cross-priming of cytotoxic T cells in vitro . BMDCs were stimulated as described for and were then cultured in the presence of OVA protein. (a) Cross-presentation of the processed peptide-epitope SIINFEKL in the context of MHC-I was analyzed by flow cytometry 18 h later. Data show H-2Kb-SIINFEKL expression on wild-type, IFNaR1- and MAVS-deficient cells. Data show mean fold change in comparison to untreated cells ± S.E.M. of quadruplicate samples pooled from two independent experiments. (b-d) Stimulated DCs were co-cultured with magnetically purified, CFSE-labeled CD8 + OT-I T cells in the presence of OVA protein. (b) CD8 T cell proliferation by CFSE dye dilution as well as IFN-γ levels in the supernatant from co-cultures with MAVS- (c) and IFNaR1-deficient DCs (d) were analyzed by flow cytometry or ELISA, respectively. (e-f) BMDCs from ASC-deficient animals were stimulated as described. (e) H-2Kb-SIINFEKL expression on DCs and (f) IFN-γ levels in the supernatant from co-cultures with CD8 + OT-I T cells were analyzed as described. All data give mean ± S.E.M. of at least triplicate samples representative of three independent experiments. (* P < 0.05, ** P < 0.01, *** P < 0.001, one-way analysis of variance (ANOVA) for multiple comparisons). ns, not significant.

Article Snippet: In some experiments, mice were pre-treated intraperitoneally (ip) with 400 μg anti-murine IFNaR1 antibody (clone MAR1-5A3, BioXCell, West Lebanon, NH) one day prior to the above immunization.

Techniques: Activation Assay, In Vitro, Cell Culture, Flow Cytometry, Expressing, Comparison, Purification, Labeling, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: RIG-I activating immunostimulatory RNA boosts the efficacy of anticancer vaccines and synergizes with immune checkpoint blockade

doi: 10.1016/j.ebiom.2019.02.056

Figure Lengend Snippet: The RIG-I / MAVS / IFN-I pathway induces robust cross-priming of cytotoxic T cells in vivo . WT and MAVS-deficient mice were injected sc with OVA +3pRNA twice. (a) Frequency of H-2K b -SIINFEKL Tetramer + cytotoxic T cells in draining lymph nodes (LN) and spleen. Each data point represents one individual of at least n = 10 mice and the mean per group is depicted as a bar. (b) Representative blots are gated on CD8 + T cells from ex vivo OVA restimulated LN cell cultures and give the percentage of proliferating cells. (c) IFN-γ levels from the above cultures. Data give the mean ± S.E.M. of n = 5 independent cell cultures per group each derived from individual mice. (d) In vivo cytotoxic activity was measured by target cell elimination of fluorescently labeled, SIINFEKL peptide-pulsed syngenic splenocytes. Histograms show the frequency of transferred target cells in the spleen of a representative recipient mouse. Numbers give the concentration [nm] of SIINFEKL-pulsing and thus the immunogenicity of the indicated target cell population. The graph shows mean specific lysis ± S.E.M. of n = 5 individual mice. (e-f) WT mice were vaccinated iv with OVA +3pRNA once and were additionally treated with anti-IFNaR1 blocking antibody one day prior to vaccination. (e) Frequency of H-2K b -SIINFEKL Tetramer + cytotoxic T cells in the spleen and (f) cytolytic activity were analyzed as described above (individual mice, n = 3 for the ‘no adjuvant’ and n = 4 for the ‘3pRNA’ group). All data are representative of at least two independent experiments. (* P < 0.05, ** P < 0.01, *** P < 0.001, one-way analysis of variance (ANOVA) for multiple comparisons). ND, not determined.

Article Snippet: In some experiments, mice were pre-treated intraperitoneally (ip) with 400 μg anti-murine IFNaR1 antibody (clone MAR1-5A3, BioXCell, West Lebanon, NH) one day prior to the above immunization.

Techniques: In Vivo, Injection, Ex Vivo, Derivative Assay, Activity Assay, Labeling, Concentration Assay, Immunopeptidomics, Lysis, Blocking Assay, Adjuvant

Journal: EBioMedicine

Article Title: RIG-I activating immunostimulatory RNA boosts the efficacy of anticancer vaccines and synergizes with immune checkpoint blockade

doi: 10.1016/j.ebiom.2019.02.056

Figure Lengend Snippet: RIG-I activation synergizes with anti-CTLA-4 blockade to enhance the efficacy of anticancer vaccines. (a) ‘Prophylactic’ vaccination scheme: WT and MAVS −/− mice were vaccinated with OVA and 3pRNA iv. Anti-CTLA-4 antibody was administered ip B16.OVA tumor cells were injected iv on day 7. (b) Serum levels of IFN-I after a single 3pRNA injection. (c) Frequency of H-2K b -SIINFEKL Tetramer + cytotoxic T cells in peripheral blood of WT and MAVS −/− mice on day 7 after vaccination. (d) Number of macroscopically visible pseudo-metastases in the lung at day 19 after tumor induction. Data are pooled from two independent experiments and are presented as percentage of the ‘No adjuvant’ control group. (e) Number of pseudo-metastases in WT animals that were additionally injected with anti-IFNaR1 or isotype control antibody one day prior to vaccination. All data are representative of at least two independent experiments. (f) ‘Therapeutic’ vaccination scheme: WT mice were injected sc with B16.OVA cells. Intravenous vaccination with OVA and 3pRNA was performed on day 6 after tumor inoculation; anti-CTLA-4 was administered ip on day 6, 9 and 12. Tumor growth in mice was analyzed. Data show mean tumor growth ± S.E.M. from n = 5 individual mice. (g) Tumor-bearing mice were vaccinated with OVA +3pRNA as described for f. Some mice were additionally treated with CD4 + T-cell, CD8 + T cell or NK-cell depleting antibodies. Data give mean tumor growth ± S.E.M. of n = 6 individual mice per group. All data are representative of at least two independent experiments. (* P < 0.05, ** P < 0.01, *** P < 0.001, ordinary one-way ANOVA for multiple comparisons or two-tailed unpaired t -test.)

Article Snippet: In some experiments, mice were pre-treated intraperitoneally (ip) with 400 μg anti-murine IFNaR1 antibody (clone MAR1-5A3, BioXCell, West Lebanon, NH) one day prior to the above immunization.

Techniques: Activation Assay, Vaccines, Injection, Adjuvant, Control, Two Tailed Test

Journal: Nature

Article Title: Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis

doi: 10.1038/s41586-024-07671-y

Figure Lengend Snippet: a , Immunohistology of human BM biopsies from healthy controls and patients with secondary ITP with non-Hodgkin lymphoma (without BM involvement). MKs (CD41 + , >15 μm; green), pDCs (CD123 + ; magenta), nuclei (DAPI; blue). Scale bar, 50 µm. b , c , Quantification of the number of pDCs, MKs per high power field (HPF) size of 0.9 mm × 0.7 mm and platelets ( b ) and the fraction of MKs with pDC contact ( c ) from the experiment in a . n = 5 patients. Data are mean ± s.d. Statistical analysis was performed using unpaired t -tests with Welch’s correction; NS, P = 0.158 (pDCs), ** P = 0.0011 (MKs), ** P = 0.0014 (platelets), **** P = 0.000002 (MKs/pDCs). d , Infection may alter the role of pDCs as homeostatic sensors. e , f , Immunohistology of human BM biopsies from healthy control patients (the same patients as shown in a and b ) and from autopsies of patients with COVID-19 (see also Extended Data Fig. ). Quantification of the number of pDCs and the fraction of MKs in contact with pDCs ( e ) and the number of MKs ( f ) is shown. n = 5 (control) and n = 12 (COVID-19) individuals. Data are mean ± s.d. Statistical analysis was performed using unpaired t -tests with Welch’s correction; **** P = 0.00007 (pDCs), **** P = 0.0000000007 (MKs/pDCs), ** P = 0.0018 (MKs). g , Increased activation of pDCs in the BM of patients with COVID-19. Quantification of activation marker CD69 (left) and IFNα expression (right) (Immunohistology; see also Extended Data Fig. ). n = 3 patients. Data are mean ± s.d. Statistical analysis was performed using unpaired t -tests with Welch’s correction; * P = 0.0304, ** P = 0.0069. h , BM from FVB;K18-hACE2 mice infected with SARS CoV-2 (10 5 median tissue culture infectious dose (TCID 50 ) SARS-CoV-2 per mouse in 25 μl intranasally (i.n.)) were analysed in the presence ( n = 3) or absence ( n = 3) of IFNAR1 blocking antibody and compared to untreated control mice (PBS, n = 2) (immunohistology). Data are mean ± s.d. Statistical analysis was performed using unpaired t -tests with Welch’s correction; ** P = 0.0015 (pDCs), ** P = 0.0041 (percentage of MK–pDC-contacts), ** P = 0.0011 (MKPs), ** P = 0.0014 (MKs).

Article Snippet: In selected experiments, K18-hACE2 mice were injected intraperitoneally with 2 mg per mouse of anti-IFNAR1 blocking antibody (BioXcell, BE0241, MAR1-5A3) 1 day before infection.

Techniques: Infection, Control, Activation Assay, Marker, Expressing, Blocking Assay