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

Journal: British Journal of Cancer

Article Title: The lytic activity of VSV-GP treatment dominates the therapeutic effects in a syngeneic model of lung cancer

doi: 10.1038/s41416-019-0574-7

Figure Lengend Snippet: Interferon responsiveness limits VSV-GP activity on murine lung cancer cell line LLC1 in vitro. a Surface expression of IFNAR1 on LLC1 wt and IFNAR1 −/− was analysed by flow cytometry using mIFNAR1 specific antibody with only secondary antibody (grey) as control. b For microscopic analysis of VSV-GP-GFP infection in LLC1 wildtype and IFNAR1 −/− , cells were pre-incubated with 500 U/mL universal IFN-α overnight or left untreated and subsequently infected with VSV-GP-GFP at an MOI 0.1 for 24 h. c Survival of LLC1 wt and IFNAR1 −/− cells upon VSV-GP infection at increasing dose (MOI 0.1,1,10) and IFN-α pre-treatment (0–1000 U/mL overnight) was measured 72 h post infection using MTT assay. Data are shown as mean ± SEM ( n = 4)

Article Snippet: For quantification of cell surface IFNAR1, selected clones were incubated using the same antibodies and analysed using FACS Canto II (BD Biosciences).

Techniques: Activity Assay, In Vitro, Expressing, Flow Cytometry, Infection, Incubation, MTT Assay

Journal: British Journal of Cancer

Article Title: The lytic activity of VSV-GP treatment dominates the therapeutic effects in a syngeneic model of lung cancer

doi: 10.1038/s41416-019-0574-7

Figure Lengend Snippet: The in vivo efficacy of VSV-GP in the syngeneic LLC1 lung cancer model is dose dependent and correlates with tumour interferon sensitivity. Tumours were implanted in C57BL/6J mice by subcutaneously injecting 5 × 10 5 LLC1 wt ( a ) or 1 × 10 6 IFNAR1 -/- cells ( b ) into the right flank. Intratumoural or intravenous treatment with VSV-GP at a dose of 10 8 TCID 50 was initiated when tumours reached a size of 0.05–0.07 cm 3 . Established LLC1-IFNAR1 -/- tumours were treated intravenously with a single dose of 10 6 , 10 7 or 10 8 TCID 50 VSV-GP ( c ) or 10 8 TCID 50 replication-deficient VSV-ΔG-GP via intratumoural or systemic route ( d ). Individual tumour volume graphs and Kaplan–Meier survival curves are shown (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001)

Article Snippet: For quantification of cell surface IFNAR1, selected clones were incubated using the same antibodies and analysed using FACS Canto II (BD Biosciences).

Techniques: In Vivo

Journal: British Journal of Cancer

Article Title: The lytic activity of VSV-GP treatment dominates the therapeutic effects in a syngeneic model of lung cancer

doi: 10.1038/s41416-019-0574-7

Figure Lengend Snippet: VSV-GP treatment effect on LLC1-IFNAR1 −/− tumours is independent of adaptive immune activation. a To selectively address the contribution of cytotoxic T cells to VSV-GP treatment, C57BL/6J mice bearing LLC1-IFNAR1 −/− tumours were depleted for CD8 + T cells using a monoclonal antibody at days -2, 0, 2, 6 and 10 respective to single systemic virus (10 8 TCID 50 ) treatment. Individual tumour volume graphs and Kaplan–Meier survival curve are shown (* p < 0.05; ** p < 0.01; *** p < 0.001). b Mice from two separate experiments that showed LLC1-IFNAR1 −/− tumour long-term remission after VSV-GP treatment were re-challenged subcutaneously with 1 × 10 6 of either parental or IFNAR1 −/− LLC1 cells into the left flank and monitored for tumour outgrowth. c Reactivity of splenocytes isolated from mock, VSV-GP or VSV-GP + CD8a depletion—treated or completely naïve mice against three different survivin epitopes, VSV-NP peptide as well as LLC1-IFNAR1 −/− tumour cells are depicted as number of IFNγ spots per 2.5 × 10 6 cells. Numbers of spots of three animals per treatment group are presented as mean of three technical replicates, after subtraction of background signal (medium only). TNTC = spots too numerous to count. d Flow cytometry was used to quantify overall and VSV-GP specific CD8 + T cell response in whole blood and spleen on days 6 and 7 post virus treatment, respectively. After gating out non-viable cells, monocytes, myeloid cells and B cells percentages of CD8 + CD90 + T cells among CD45 + leukocytes are shown. e Frequencies of VSV-GP specific CD8 + T cells labelled by VSV-NP tetramer are shown from blood and spleen samples

Article Snippet: For quantification of cell surface IFNAR1, selected clones were incubated using the same antibodies and analysed using FACS Canto II (BD Biosciences).

Techniques: Activation Assay, Isolation, Flow Cytometry

Journal: British Journal of Cancer

Article Title: The lytic activity of VSV-GP treatment dominates the therapeutic effects in a syngeneic model of lung cancer

doi: 10.1038/s41416-019-0574-7

Figure Lengend Snippet: Interferon insensitivity and CD8 + T cell deficiency correlate with enhanced and prolonged intratumoural virus replication. Unilateral LLC1 wt or IFNAR1 −/− mouse lung tumours were grown in syngeneic C57BL/6 J mice ( a – c ) or athymic NMRI-nu/nu mice ( d – f ) and treated intratumourally with a single dose of 10 8 TCID 50 VSV-GP-Luciferase. Tumours were monitored every second day post treatment using the in vivo bioluminescence imaging (BLI) system IVIS. Representative BLI pictures of treated mice ( a , d ) and quantification of the average radiance in the tumour area ( b , e ) are shown (mean ± SD). Tumour growth after VSV-GP-Luciferase treatment is depicted with aligned time axis as mean ± SD in C57BL/6 J ( c ) and NMRI-nu/nu mice ( f ). Colour scale displays luminescence as photons/second/cm 2 /steradian (p/s/cm 2 /sr)

Article Snippet: For quantification of cell surface IFNAR1, selected clones were incubated using the same antibodies and analysed using FACS Canto II (BD Biosciences).

Techniques: Luciferase, In Vivo, Imaging

Journal: British Journal of Cancer

Article Title: The lytic activity of VSV-GP treatment dominates the therapeutic effects in a syngeneic model of lung cancer

doi: 10.1038/s41416-019-0574-7

Figure Lengend Snippet: Lytic activity of VSV-GP on LLC1-IFNAR1 −/− tumours associated with T cell infiltration and immune activation. Established LLC1-IFNAR1 −/− tumours were treated with a single intravenous dose of 10 8 TCID 50 of VSV-GP and resected after 3 or 7 days, respectively. Immunostaining against VSV-N, activated caspase 3 (aCas3), CD8a, CD4 and PD-L1 was performed on fixed microsections. a The panel depicts representative pictures of a group size of six animals each. Scale bar 3 mm. The density of IHC positive cells in sections was analysed 3 days ( b ) and 7 days ( c ) post treatment and revealed significant immune activation at the latter time point. Data are shown as mean ± SEM ( n = 6). d VSV-specific CD8 + T cells in spleen and tumour of the same animal were measured 7 days post treatment in flow cytometry using VSV-NP-MHC multimer. Frequencies of VSV-specific CD8 + T cell among CD90 + CD8 + T cells are depicted in a graph (left panel) and as dot plots (right panel)

Article Snippet: For quantification of cell surface IFNAR1, selected clones were incubated using the same antibodies and analysed using FACS Canto II (BD Biosciences).

Techniques: Activity Assay, Activation Assay, Immunostaining, Flow Cytometry

Journal: British Journal of Cancer

Article Title: The lytic activity of VSV-GP treatment dominates the therapeutic effects in a syngeneic model of lung cancer

doi: 10.1038/s41416-019-0574-7

Figure Lengend Snippet: Proinflammatory cytokine release and activation of innate and adaptive immune responses after VSV-GP treatment. Established LLC1-IFNAR1 −/− tumours were treated with a single intravenous dose of 10 8 TCID 50 of VSV-GP and resected after 3 or 7 days, respectively. For quantification of cytokine levels, tumour lysates were assayed using Luminex multiplex technology to detect proinflammatory ( a ) and immune-suppressive cytokines ( b ). Data presented as mean ± SEM ( n = 3) (* p < 0.05; ** p < 0.01). RNA from tumour homogenates was used for transcriptome analysis via NanoString technology. A hierarchical cluster analysis (Euclidean distance; average linkage) for sample data were performed. The z-score-normalised gene expression intensity is presented with each column representing one individual tumour. Four tumours per group were used for analysis. Data shown are representative from two independent experiments. c Heatmap presents T cell signature genes from treated vs untreated tumours at 3 and 7 days post treatment. Genes were selected based on the cell type–annotation from the NanoString nCounter PanCancer Immune Profiling Panel gene list. Genes are ordered according to the cell type annotation. d Heatmaps visualising the differential expression of gene panels representative for innate and adaptive immune response, respectively

Article Snippet: For quantification of cell surface IFNAR1, selected clones were incubated using the same antibodies and analysed using FACS Canto II (BD Biosciences).

Techniques: Activation Assay, Luminex, Multiplex Assay, Expressing