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Sensing of SARS-CoV-2-infected cells by pDCs is more efficient compared with cell-free virions. ( A ) Quantification of IFN-I (IFN-α/β) in the supernatant of PBMCs that were co-cultured for 24 h with SARS-CoV-2 (LSPQ1)-infected <t>HeLa-hACE2,</t> exposed to cell-free SARS-CoV-2 at different MOIs, or treated with supernatant of infected cells (MOI 0.1) (sup) collected at 24 h post-infection. PBMCs used in the co-cultures and in “cell-free virus exposure” conditions were from the same donors with the same number of cells. In the latter conditions, the MOI was based on the number of PBMCs. UI, uninfected cells. Each dot represents independent experiments/donors. Mann-Whitney U test. ** P < 0.01. ( B ) (Upper panel) Frequency of SARS-CoV-2 infection in HeLa-hACE2 as determined by flow cytometry using αN Ab and a mouse IgG1 isotype control (ctrl). (Lower panel) A summary dot plot shows the results from six independent infections. ( C ) Quantification of IFN-I in the supernatant of SARS-CoV-2-infected HeLa-hACE2 (MOI 0.1, 24 h) that were co-cultured with pDC-depleted PBMCs or total PBMCs ( n = 3 donors). ( D ) The analysis was as described for panel C but with cell-free viruses instead of infected cells ( n = 2 donors). The MOI (0.1) used was based on the number of PBMCs, as done in . In all relevant panels, the co-cultures were done for 24 h. Shown is the mean ± SEM. The dotted line indicates the limit of detection (LOD) of the assay (60 U/mL). See also .

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1) Product Images from "Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin"

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

Journal: Journal of Virology

doi: 10.1128/jvi.01235-24

Figure Legend Snippet: Sensing of SARS-CoV-2-infected cells by pDCs is more efficient compared with cell-free virions. ( A ) Quantification of IFN-I (IFN-α/β) in the supernatant of PBMCs that were co-cultured for 24 h with SARS-CoV-2 (LSPQ1)-infected HeLa-hACE2, exposed to cell-free SARS-CoV-2 at different MOIs, or treated with supernatant of infected cells (MOI 0.1) (sup) collected at 24 h post-infection. PBMCs used in the co-cultures and in “cell-free virus exposure” conditions were from the same donors with the same number of cells. In the latter conditions, the MOI was based on the number of PBMCs. UI, uninfected cells. Each dot represents independent experiments/donors. Mann-Whitney U test. ** P < 0.01. ( B ) (Upper panel) Frequency of SARS-CoV-2 infection in HeLa-hACE2 as determined by flow cytometry using αN Ab and a mouse IgG1 isotype control (ctrl). (Lower panel) A summary dot plot shows the results from six independent infections. ( C ) Quantification of IFN-I in the supernatant of SARS-CoV-2-infected HeLa-hACE2 (MOI 0.1, 24 h) that were co-cultured with pDC-depleted PBMCs or total PBMCs ( n = 3 donors). ( D ) The analysis was as described for panel C but with cell-free viruses instead of infected cells ( n = 2 donors). The MOI (0.1) used was based on the number of PBMCs, as done in . In all relevant panels, the co-cultures were done for 24 h. Shown is the mean ± SEM. The dotted line indicates the limit of detection (LOD) of the assay (60 U/mL). See also .

Techniques Used: Infection, Cell Culture, Virus, MANN-WHITNEY, Flow Cytometry, Control

pDC sensing of SARS-CoV-2-infected cells requires physical contact. ( A ) (Left panel) Schematic representation of the experimental design (created with BioRender.com). HeLa-hACE2 cells were infected with SARS-CoV-2 at MOI of 0.1 for 24 h and then co-cultured with PBMCs in the presence or absence of a 0.4 µm transwell membrane insert. PBMCs were added to the upper chamber of the insert. IFN-I was measured from the lower chamber, at 18–24 h after the co-cultures. (Right panel) Quantification of IFN-I in the supernatant of PBMCs co-cultured with mock- or SARS-CoV-2-infected HeLa-hACE2 (Inf cells) in the presence (+TW) or absence (–TW) of a transwell insert. Inf cells, infected cells. Data shown are mean ± SEM from independent experiments with seven donors. Each dot depicts a donor. Mann-Whitney U test. ** P < 0.01. ( B ) (Upper panel) Design of an experiment aimed at determining whether cell-free virus can trigger residual IFN-I production in the absence of physical contact (created with BioRender.com). (Lower panel) IFN-I was measured in supernatants of PBMCs either co-cultured with infected HeLa-hACE2 (MOI 0.1) in the presence or absence of transwell inserts or exposed to cell-free virus (MOI 0.1, based on PBMC number) in the same conditions. The number of PBMCs used for the co-cultures or exposed to cell-free viruses was the same. Data shown are mean ± SEM from independent experiments with four donors. Mann-Whitney U test. * P < 0.05. In both panels: LOD, limit of detection of the IFN-I assay as described in legend. See also .

Figure Legend Snippet: pDC sensing of SARS-CoV-2-infected cells requires physical contact. ( A ) (Left panel) Schematic representation of the experimental design (created with BioRender.com). HeLa-hACE2 cells were infected with SARS-CoV-2 at MOI of 0.1 for 24 h and then co-cultured with PBMCs in the presence or absence of a 0.4 µm transwell membrane insert. PBMCs were added to the upper chamber of the insert. IFN-I was measured from the lower chamber, at 18–24 h after the co-cultures. (Right panel) Quantification of IFN-I in the supernatant of PBMCs co-cultured with mock- or SARS-CoV-2-infected HeLa-hACE2 (Inf cells) in the presence (+TW) or absence (–TW) of a transwell insert. Inf cells, infected cells. Data shown are mean ± SEM from independent experiments with seven donors. Each dot depicts a donor. Mann-Whitney U test. ** P < 0.01. ( B ) (Upper panel) Design of an experiment aimed at determining whether cell-free virus can trigger residual IFN-I production in the absence of physical contact (created with BioRender.com). (Lower panel) IFN-I was measured in supernatants of PBMCs either co-cultured with infected HeLa-hACE2 (MOI 0.1) in the presence or absence of transwell inserts or exposed to cell-free virus (MOI 0.1, based on PBMC number) in the same conditions. The number of PBMCs used for the co-cultures or exposed to cell-free viruses was the same. Data shown are mean ± SEM from independent experiments with four donors. Mann-Whitney U test. * P < 0.05. In both panels: LOD, limit of detection of the IFN-I assay as described in legend. See also .

Techniques Used: Infection, Cell Culture, Membrane, MANN-WHITNEY, Virus

CD54/CD11a adhesion complex is involved in the recognition and sensing of SARS-CoV-2 infected cells by pDCs in a potentially bidirectional manner ( A ) Design of a CD54 blocking experiment (created with BioRender.com). PBMCs, enriched pDCs, or SARS-CoV-2-infected cells were pretreated with various concentrations of αCD54 Ab for 30 min at room temperature before the 24 h co-cultures. Cells treated with an isotype Ab control served as a negative control. ( B ) IFN-I in the supernatant of a co-culture between infected HeLa-hACE2 and αCD54 Ab-pretreated PBMCs. The IFN-I level in the Ab-untreated condition was set at 100%, six donors. ( C ) IFN-I production in the supernatant of a 24 h co-culture whereby infected HeLa-hACE2 were pretreated with αCD54 Ab and then exposed to PBMCs. The IFN-I level in the untreated condition was set at 100%, six donors. ( D ) IFN-I release from the supernatant of SARS-CoV-2-infected Calu-3 co-cultured with anti-CD54 Ab-pretreated PBMCs. Data analysis was as described for panel B, four donors. ( E ) IFN-I from a co-culture supernatant of PBMCs with infected Calu-3 that were treated with αCD54 Ab. Data analysis was as described for Panel B, four donors. ( F ) Experimental design of a CD11a blocking experiment (created with BioRender.com). Either PBMCs were pretreated with increasing concentrations of αCD11a Ab or the αCD11a Ab was added to a co-culture of PBMCs and infected Calu-3. IFN-I was determined 18–24 h post-co-culture. ( G and H ) IFN-I production in the supernatant of infected-Calu-3 co-cultured with PBMCs. ( G ) PBMCs were pretreated with the indicated concentrations of αCD11a Ab or isotype Ab control, and IFN-I release from a co-culture with untreated PBMCs was set at 100%. ( H ) αCD11a Ab was added to the co-culture of PBMCs and infected Calu-3. IFN-I production from the Ab-untreated co-culture was set as 100%, six donors. In all relevant panels, data are shown as mean ± SEM. Statistical analysis: Mann-Whitney U test; * P < 0.05, ** P < 0.01, ns, not significant. See also .

Figure Legend Snippet: CD54/CD11a adhesion complex is involved in the recognition and sensing of SARS-CoV-2 infected cells by pDCs in a potentially bidirectional manner ( A ) Design of a CD54 blocking experiment (created with BioRender.com). PBMCs, enriched pDCs, or SARS-CoV-2-infected cells were pretreated with various concentrations of αCD54 Ab for 30 min at room temperature before the 24 h co-cultures. Cells treated with an isotype Ab control served as a negative control. ( B ) IFN-I in the supernatant of a co-culture between infected HeLa-hACE2 and αCD54 Ab-pretreated PBMCs. The IFN-I level in the Ab-untreated condition was set at 100%, six donors. ( C ) IFN-I production in the supernatant of a 24 h co-culture whereby infected HeLa-hACE2 were pretreated with αCD54 Ab and then exposed to PBMCs. The IFN-I level in the untreated condition was set at 100%, six donors. ( D ) IFN-I release from the supernatant of SARS-CoV-2-infected Calu-3 co-cultured with anti-CD54 Ab-pretreated PBMCs. Data analysis was as described for panel B, four donors. ( E ) IFN-I from a co-culture supernatant of PBMCs with infected Calu-3 that were treated with αCD54 Ab. Data analysis was as described for Panel B, four donors. ( F ) Experimental design of a CD11a blocking experiment (created with BioRender.com). Either PBMCs were pretreated with increasing concentrations of αCD11a Ab or the αCD11a Ab was added to a co-culture of PBMCs and infected Calu-3. IFN-I was determined 18–24 h post-co-culture. ( G and H ) IFN-I production in the supernatant of infected-Calu-3 co-cultured with PBMCs. ( G ) PBMCs were pretreated with the indicated concentrations of αCD11a Ab or isotype Ab control, and IFN-I release from a co-culture with untreated PBMCs was set at 100%. ( H ) αCD11a Ab was added to the co-culture of PBMCs and infected Calu-3. IFN-I production from the Ab-untreated co-culture was set as 100%, six donors. In all relevant panels, data are shown as mean ± SEM. Statistical analysis: Mann-Whitney U test; * P < 0.05, ** P < 0.01, ns, not significant. See also .

Techniques Used: Infection, Blocking Assay, Control, Negative Control, Co-Culture Assay, Cell Culture, MANN-WHITNEY

Formation of conjugates between pDCs and SARS-CoV-2-infected cells is dependent on receptor engagement with CD54. ( A ) Schematic representation of the experimental design (created with BioRender.com). Mock- and SARS-CoV-2-infected HeLa-hACE2 were pre-labeled with cell-membrane dye eFluo 670 before co-culturing with PBMCs. Cell conjugates were analyzed by flow cytometry and defined as CD3 - CD14 - CD19 - ILT7 + BDCA2 + eFluor670 + population. Analysis was done 18–24 h after the co-culture. ( B ) (Upper panel) Dot plots showing the gating strategy to identify cell conjugates between pDCs (gated from PBMCs) and mock- or SARS-CoV-2-infected HeLa-hACE2. Numbers shown in the dot plots are cell percentages. (Lower panel) Frequency of cell conjugates from different experiments done with five donors. ( C ) (Upper panel) Representative dot plots showing the frequency of conjugates formed between enriched pDCs (CM-Dil + ) and mock- or SARS-CoV-2-infected HeLa-hACE2 (eFluor 670 + ). (Lower panel) Overall data from different experiments done with 7 PBMC donors. ( D ) (Upper panel) Representative dot plots showing the effect of blocking CD54 receptor engagement on conjugate formation between pDCs and infected HeLa-hACE2. Numbers indicated represent the percentage of conjugates. (Lower panel) Frequencies of conjugates between pDCs and eFluor670-labelled mock- and SARS-CoV-2-infected HeLa-hACE2 in the presence of αCD54 Ab or isotype control (5 µg/mL). Analysis was done with four donors. In all relevant panels, each line depicts a donor. Statistical analysis Mann-Whitney U test. * P < 0.05. See also .

Figure Legend Snippet: Formation of conjugates between pDCs and SARS-CoV-2-infected cells is dependent on receptor engagement with CD54. ( A ) Schematic representation of the experimental design (created with BioRender.com). Mock- and SARS-CoV-2-infected HeLa-hACE2 were pre-labeled with cell-membrane dye eFluo 670 before co-culturing with PBMCs. Cell conjugates were analyzed by flow cytometry and defined as CD3 - CD14 - CD19 - ILT7 + BDCA2 + eFluor670 + population. Analysis was done 18–24 h after the co-culture. ( B ) (Upper panel) Dot plots showing the gating strategy to identify cell conjugates between pDCs (gated from PBMCs) and mock- or SARS-CoV-2-infected HeLa-hACE2. Numbers shown in the dot plots are cell percentages. (Lower panel) Frequency of cell conjugates from different experiments done with five donors. ( C ) (Upper panel) Representative dot plots showing the frequency of conjugates formed between enriched pDCs (CM-Dil + ) and mock- or SARS-CoV-2-infected HeLa-hACE2 (eFluor 670 + ). (Lower panel) Overall data from different experiments done with 7 PBMC donors. ( D ) (Upper panel) Representative dot plots showing the effect of blocking CD54 receptor engagement on conjugate formation between pDCs and infected HeLa-hACE2. Numbers indicated represent the percentage of conjugates. (Lower panel) Frequencies of conjugates between pDCs and eFluor670-labelled mock- and SARS-CoV-2-infected HeLa-hACE2 in the presence of αCD54 Ab or isotype control (5 µg/mL). Analysis was done with four donors. In all relevant panels, each line depicts a donor. Statistical analysis Mann-Whitney U test. * P < 0.05. See also .

Techniques Used: Infection, Labeling, Membrane, Flow Cytometry, Co-Culture Assay, Blocking Assay, Control, MANN-WHITNEY

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Journal: Journal of Virology

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

doi: 10.1128/jvi.01235-24

Figure Lengend Snippet: Sensing of SARS-CoV-2-infected cells by pDCs is more efficient compared with cell-free virions. ( A ) Quantification of IFN-I (IFN-α/β) in the supernatant of PBMCs that were co-cultured for 24 h with SARS-CoV-2 (LSPQ1)-infected HeLa-hACE2, exposed to cell-free SARS-CoV-2 at different MOIs, or treated with supernatant of infected cells (MOI 0.1) (sup) collected at 24 h post-infection. PBMCs used in the co-cultures and in “cell-free virus exposure” conditions were from the same donors with the same number of cells. In the latter conditions, the MOI was based on the number of PBMCs. UI, uninfected cells. Each dot represents independent experiments/donors. Mann-Whitney U test. ** P < 0.01. ( B ) (Upper panel) Frequency of SARS-CoV-2 infection in HeLa-hACE2 as determined by flow cytometry using αN Ab and a mouse IgG1 isotype control (ctrl). (Lower panel) A summary dot plot shows the results from six independent infections. ( C ) Quantification of IFN-I in the supernatant of SARS-CoV-2-infected HeLa-hACE2 (MOI 0.1, 24 h) that were co-cultured with pDC-depleted PBMCs or total PBMCs ( n = 3 donors). ( D ) The analysis was as described for panel C but with cell-free viruses instead of infected cells ( n = 2 donors). The MOI (0.1) used was based on the number of PBMCs, as done in . In all relevant panels, the co-cultures were done for 24 h. Shown is the mean ± SEM. The dotted line indicates the limit of detection (LOD) of the assay (60 U/mL). See also .

Article Snippet: HeLa cell line stably expressing the full-length human ACE2 receptor (HeLa-hACE2) was generated by transducing HeLa cells with lentiviral vector particles expressing the human ACE2 and blasticidin-resistance genes (the lentiviral vector pWPI-IRES-Bla-Ak-ACE2-TMPRSS2 was a kind gift from Dr. Sonja Best, Addgene plasmid #154983).

Techniques: Infection, Cell Culture, Virus, MANN-WHITNEY, Flow Cytometry, Control

Journal: Journal of Virology

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

doi: 10.1128/jvi.01235-24

Figure Lengend Snippet: pDC sensing of SARS-CoV-2-infected cells requires physical contact. ( A ) (Left panel) Schematic representation of the experimental design (created with BioRender.com). HeLa-hACE2 cells were infected with SARS-CoV-2 at MOI of 0.1 for 24 h and then co-cultured with PBMCs in the presence or absence of a 0.4 µm transwell membrane insert. PBMCs were added to the upper chamber of the insert. IFN-I was measured from the lower chamber, at 18–24 h after the co-cultures. (Right panel) Quantification of IFN-I in the supernatant of PBMCs co-cultured with mock- or SARS-CoV-2-infected HeLa-hACE2 (Inf cells) in the presence (+TW) or absence (–TW) of a transwell insert. Inf cells, infected cells. Data shown are mean ± SEM from independent experiments with seven donors. Each dot depicts a donor. Mann-Whitney U test. ** P < 0.01. ( B ) (Upper panel) Design of an experiment aimed at determining whether cell-free virus can trigger residual IFN-I production in the absence of physical contact (created with BioRender.com). (Lower panel) IFN-I was measured in supernatants of PBMCs either co-cultured with infected HeLa-hACE2 (MOI 0.1) in the presence or absence of transwell inserts or exposed to cell-free virus (MOI 0.1, based on PBMC number) in the same conditions. The number of PBMCs used for the co-cultures or exposed to cell-free viruses was the same. Data shown are mean ± SEM from independent experiments with four donors. Mann-Whitney U test. * P < 0.05. In both panels: LOD, limit of detection of the IFN-I assay as described in legend. See also .

Techniques: Infection, Cell Culture, Membrane, MANN-WHITNEY, Virus

Journal: Journal of Virology

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

doi: 10.1128/jvi.01235-24

Figure Lengend Snippet: CD54/CD11a adhesion complex is involved in the recognition and sensing of SARS-CoV-2 infected cells by pDCs in a potentially bidirectional manner ( A ) Design of a CD54 blocking experiment (created with BioRender.com). PBMCs, enriched pDCs, or SARS-CoV-2-infected cells were pretreated with various concentrations of αCD54 Ab for 30 min at room temperature before the 24 h co-cultures. Cells treated with an isotype Ab control served as a negative control. ( B ) IFN-I in the supernatant of a co-culture between infected HeLa-hACE2 and αCD54 Ab-pretreated PBMCs. The IFN-I level in the Ab-untreated condition was set at 100%, six donors. ( C ) IFN-I production in the supernatant of a 24 h co-culture whereby infected HeLa-hACE2 were pretreated with αCD54 Ab and then exposed to PBMCs. The IFN-I level in the untreated condition was set at 100%, six donors. ( D ) IFN-I release from the supernatant of SARS-CoV-2-infected Calu-3 co-cultured with anti-CD54 Ab-pretreated PBMCs. Data analysis was as described for panel B, four donors. ( E ) IFN-I from a co-culture supernatant of PBMCs with infected Calu-3 that were treated with αCD54 Ab. Data analysis was as described for Panel B, four donors. ( F ) Experimental design of a CD11a blocking experiment (created with BioRender.com). Either PBMCs were pretreated with increasing concentrations of αCD11a Ab or the αCD11a Ab was added to a co-culture of PBMCs and infected Calu-3. IFN-I was determined 18–24 h post-co-culture. ( G and H ) IFN-I production in the supernatant of infected-Calu-3 co-cultured with PBMCs. ( G ) PBMCs were pretreated with the indicated concentrations of αCD11a Ab or isotype Ab control, and IFN-I release from a co-culture with untreated PBMCs was set at 100%. ( H ) αCD11a Ab was added to the co-culture of PBMCs and infected Calu-3. IFN-I production from the Ab-untreated co-culture was set as 100%, six donors. In all relevant panels, data are shown as mean ± SEM. Statistical analysis: Mann-Whitney U test; * P < 0.05, ** P < 0.01, ns, not significant. See also .

Techniques: Infection, Blocking Assay, Control, Negative Control, Co-Culture Assay, Cell Culture, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 α L integrin

doi: 10.1128/jvi.01235-24

Figure Lengend Snippet: Formation of conjugates between pDCs and SARS-CoV-2-infected cells is dependent on receptor engagement with CD54. ( A ) Schematic representation of the experimental design (created with BioRender.com). Mock- and SARS-CoV-2-infected HeLa-hACE2 were pre-labeled with cell-membrane dye eFluo 670 before co-culturing with PBMCs. Cell conjugates were analyzed by flow cytometry and defined as CD3 - CD14 - CD19 - ILT7 + BDCA2 + eFluor670 + population. Analysis was done 18–24 h after the co-culture. ( B ) (Upper panel) Dot plots showing the gating strategy to identify cell conjugates between pDCs (gated from PBMCs) and mock- or SARS-CoV-2-infected HeLa-hACE2. Numbers shown in the dot plots are cell percentages. (Lower panel) Frequency of cell conjugates from different experiments done with five donors. ( C ) (Upper panel) Representative dot plots showing the frequency of conjugates formed between enriched pDCs (CM-Dil + ) and mock- or SARS-CoV-2-infected HeLa-hACE2 (eFluor 670 + ). (Lower panel) Overall data from different experiments done with 7 PBMC donors. ( D ) (Upper panel) Representative dot plots showing the effect of blocking CD54 receptor engagement on conjugate formation between pDCs and infected HeLa-hACE2. Numbers indicated represent the percentage of conjugates. (Lower panel) Frequencies of conjugates between pDCs and eFluor670-labelled mock- and SARS-CoV-2-infected HeLa-hACE2 in the presence of αCD54 Ab or isotype control (5 µg/mL). Analysis was done with four donors. In all relevant panels, each line depicts a donor. Statistical analysis Mann-Whitney U test. * P < 0.05. See also .

Techniques: Infection, Labeling, Membrane, Flow Cytometry, Co-Culture Assay, Blocking Assay, Control, MANN-WHITNEY

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