Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: NK cells are a main source of XCL1 but not a critical one upon MCMV infection (A and B) Analysis of mTFP1 expression in splenocytes of Wt (dark grey) and Xcl1-mTfp1 fl/fl (white) mice at steady state (A) or 40 h after infection (B). Cells were gated as follow: NK cells (TCRβ - CD19 - NK1.1 + ), ILC1 (NK1.1 + TCRβ - CD19 - CD127 + ), NKT cells (CD19 - CD1d + ), CD8 + T (CD1d - NK1.1 - TCRβ + CD19 - CD8 + ), CD4 + T (CD1d - NK1.1 - TCRβ + CD19 - CD4 + ), γδ T cells (CD1d - NK1.1 - CD3ε + TCRβ - TCRγδ + ) and B cells (NK1.1 - TCRβ - CD19 + ). One representative experiment of 4 independent ones with at least three mice per group is shown. (C and D) Proportion of immune populations within mTFP1-positive cells at steady state (C) and 40 h after MCMV infection (D). Others: sum of all the other cell subsets not detailed in the pie charts. One representative experiment of 4 independent ones with at least three mice per group is shown. (E) IFN-γ production by NK cells in Wt, Xcr1 −/− , Xcl1 fl/fl , and Nkp46 Cre ;Xcl1 fl/fl mice 40 h after infection. One representative experiment of two independent ones with at least four mice per group is shown. A one-way ANOVA statistical analysis with a Tukey's multiple comparison test was applied. ∗, p < 0.05; ∗∗, p < 0.01, n.s., nonsignificant; ANOVA, analysis of variance. See also Figure S3 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Infection, Expressing, Comparison

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: CCR7-mediated cDC1 relocalization into the T cell zone promotes MCMV-specific CD8+ T cell responses and host resistance to MCMV infection (A) Analysis of m45-specific CD8 + T cell response in Xcr1 −/− mice and littermate controls 6 days after MCMV infection. One representative experiment of two independent ones with at least 4 mice per group is shown. ∗, p < 0.05. (B) Analysis of m45-specific CD8 + T cell response in Wt : Xcr1 - Dta, Wt : Ccr7 −/− and Xcr1 - Dta : Ccr7 −/− BM chimera mice 6 days after MCMV infection (10 4 PFU). Two independent experiments with at least 4 mice per infected group were pooled. A one-way ANOVA statistical analysis with a Tukey's multiple comparison test was applied. ∗∗, p < 0.01; ∗, p < 0.05, n.s., nonsignificant. Statistical analysis between all other groups is nonsignificant. (C) MCMV titers in spleens and livers of Wt and Xcr1 −/− mice 5 days after MCMV infection (10 4 PFU). Two independent experiments with at least 3 mice per infected group were pooled. NI, noninfected. ∗∗∗, p < 0.001. See also Figure S6 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Infection, Comparison

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: Dermal cDC1 make cell/cell contacts with Xcl1 -expressing DETCs (A) Analysis of the lymphocyte heterogeneity within mTFP1 + cells in the skin of Xcl1 mTfp1 fl/fl mice at steady state. One representative experiment of 2 independent ones with at least three mice per group is shown. (B) DETC staining in the skin at steady state. DETCs are CD3ε + TCRγδ + lymphocytes localized in the epidermis. The dotted line delineates the basal layer separating the dermis (D) from the epidermis (EP). C, cartilage. Scale bar: 50 μm (C–E) Analysis of cDC1 localization and contacts with DETCs in the skin, at steady state in Karma Cre ;Rosa26 tdRFP ;Xcr1 +/− mice (C), and at 40 h after MCMV infection in Karma Cre ;Rosa26 tdRFP ;Xcr1 +/− (D) and Karma Cre ;Rosa26 tdRFP ;Xcr1 −/− (E) mice. The dotted line delineates the basal layer separating the dermis from the epidermis. Skin sections (scale bar: 50 μm) were stained for tdRFP (red; cDC1), CD3ε (cyan), TCRγδ (yellow), IE-1 (purple), and Avidin (green; mast cell). EP, epidermis; D, dermis; C, cartilage. (F) 3D visualization of DETC/cDC1 contacts identified in (D). (G) Kinetics of cDC1/DETC contacts occurring in the epidermis and through the epidermis-dermis border per mm of skin length, during MCMV infection. Data are represented as mean (+/− SEM). p.i., postinfection. (H) Quantification of cDC1 nuclear bodies inside the epidermis per mm of skin length 40 h after skin infection. For (G-H), two independent experiments with at least 4 mice per infected group were pooled. n.s., nonsignificant; ∗∗, p < 0.01; ∗∗∗, p < 0.001. (I) Proportion of CD103 + migcDCs in the ear-draining LN of Xcr1 −/− and littermate controls 48 h after skin infection. (J) Analysis of CCR7 expression on migcDCs in ear-draining LN 48 h after skin infection of Xcr1 −/− and Wt mice. Two independent experiments with at least 3 mice per infected group were pooled. ∗∗, p < 0.01. (K and L) Analysis of m45-specific CD8 + T cell response in Xcl1 −/− mice (K), Xcr1 −/− mice (L) and their respective controls 6 days after MCMV infection of the ear. CD8 + T cells from ear-draining LN were stimulated in vitro for 4 h with m45 peptide. Two independent experiments with at least 3 mice per infected group were pooled for (K), and one experiment with at least 4 mice per group is shown for (L). ∗, p < 0.05∗∗, p < 0.01. See also Figure S7 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Expressing, Staining, Infection, Avidin-Biotin Assay, In Vitro

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet:

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Purification, Avidin-Biotin Assay, Virus, Recombinant, High Molecular Weight, Cell Isolation, Microarray, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 1. SCARF1 profiling in immune cells. (A) SCARF1 is highly expressed on endothelial cells. CRISPR-Cas9 deletion of SCARF1 was per- formed on the human endothelial cell line, TIME. The histogram is repre- sentative of three independent experiments. (B) Uptake of ACs in SCARF11 (sgControl) and SCARF1−(sgSCARF1) endothelial cells. Cells were stimulated with ACs for 30, 60, or 90 min. AC uptake was measured by flow cytometry. Results shown are the average percent uptake ± SD of two independent experiments performed in triplicates. p 5 0.03 by Student t test analysis. (C) SCARF1 is expressed on phagocytes. PBMCs were iso- lated from the blood of healthy donors (n 5 5) and stained for flow cytome- try against SCARF1 and immune cell markers. TIME cells were used as a positive control. Data represent the mean (± SEM) of two independent experiments with two to three replicates. (D) Representative flow cytometry plot of total SCARF1 expression in the presence or absence of ACs. PBMCs gated on CD451CD11c1BDCA11 DCs were incubated in the presence or absence of Texas Redlabeled ACs for 4 h. Intracellular and extracellular stains were performed for SCARF1 to measure the total amount of SCARF1 interacting with ACs. Data shown are representative of three independent experiments (n 5 3) with similar results. (E) SCARF1 is upregulated after stimulation with ACs. BDCA11 DCs (red), CD141

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: CRISPR, Flow Cytometry, Staining, Positive Control, Expressing, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 2. SCARF1 is a nonredundant efferocytosis receptor on BDCA11 DCs. (A) SCARF1 is internalized after interacting with ACs. PBMCs (1 × 106 cells/ml) from healthy control subjects (n 5 5) were stimulated with ACs (2 × 106 cells/ml) for 1 h. After 1 h, media were removed, and new media were added. Cells were incubated for 3 h, and PBMCs were surface stained for flow cytometry. Data represent the mean (± SEM) of three independent experiments with one to two independent donors. Percent reduction is shown on the graph. ***p < 0.001. (B) SCARF1 is a phagocytic receptor for ACs in conventional DCs. MUTZ-3 DC line confocal images of SCARF1 (green), DNA (blue, DAPI), and ACs (red) 2 h poststimulation. Representative images from two independent experi- ments with three replicates. Original magnification, ×40 (left and middle); ×20 (right). Arrows show the colocalization of ACs and SCARF1. (C and D) Blocking SCARF1 reduces AC uptake. PBMCs (1 × 106/ml) were incubated with anti-SCARF1 for 30 min and then stimulated with pHrodo redlabeled ACs (2 × 106/ml). Cells were incubated for 3 h and then stained and analyzed by flow cytometry against CD451CD11c1BDCA11 (BDCA11 DCs) or CD451CD11cintCD11b1CD141

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Control, Incubation, Staining, Flow Cytometry, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 3. SCARF1 is involved in the inflammatory response. (A) NanoString gene profiling of SCARF1lo and SCARF1hi BDCA11 DCs. PBMCs were isolated from healthy control subjects and treated with ACs for 4 h. Live BDCA11 DCs (CD11c1BDCA11Live/Dead-UV) were sorted into SCARF1 negative (SCARF1lo) or SCARF1 positive (SCARF1hi). mRNA was extracted, and genetic profiling was performed using a Nano- String immunology panel. Heatmap showing the top 35 differentially expressed genes (log 2) after normalization using Morpheus software. Rep- resentative heatmap of single NanoString assay (n 5 4). p values by Stu- dent t test. (BD) Expression of IL-10 on BDCA11 DCs. BDCA11 sorted DCs (1 × 106/ml) were blocked or not with anti-SCARF1 blocking Ab for 30 min; then they were stimulated with Live/Dead-labeled ACs (2 × 106/ ml) for 4 h. (B) Cells were stained and analyzed for flow cytometry CD11c1CD11b1BDCA11SCARF11 and intracellular stain for IL-10. Data represent the mean (± SEM) of three independent experiments. p < 0.002 by two-way ANOVA. (C) Cells were lysed, and mRNA was measured using qPCR. Data are the mean (± SEM) of two independent experiments with n 5 3. p 5 0.01 by MannWhitney U test. (D) Cells were lysed, and mRNA was measured using qPCR. Data are the mean (± SEM) of two independent experiments with n 5 5. p 5 0.003 by MannWhitney U test. MFI, mean fluorescent intensity; U, unstimulated.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Isolation, Control, Software, Expressing, Blocking Assay, Labeling, Staining, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 4. SCARF1 regulates AC- induced phosphorylation of MAPK and STAT kinases. (A) Upregulation of STAT genes in SCARF11 BDCA11 DCs. PBMCs were isolated from healthy control subjects and treated with ACs for 4 h. Live CD11c1BDCA11 DCs were sorted into SCARF1-negative (SCARF1lo) or SCARF1- positive (SCARF1hi) groups. mRNA was extracted, and the genetic profiling was mea- sured by NanoString immunology panel. Unbiased heatmap depicting the regulation of kinases. Gene expression as log2 after normalization using Morpheus software. Representative heatmap of single NanoString assay (n 5 4). p values by Student t test. (BI) SCARF1 is partly responsible for phosphorylation of kinases. PBMCs from healthy control subjects (1 × 106/ml) were treated with SCARF1-blocking Ab for 30 min or left untreated; then Live/Dead- UVlabeled ACs were added for 15 or 30 min. Cells were immediately fixed and stained for flow cytometry. Cells were gated on CD11c1BDCA11SCARF11 in the pres- ence or absence of ACs. Left panel, MFI quantification; right panel, representative his- tograms. (B and C) p-MAPK p38 Thr180/ Tyr182, (D and E) p-STAT3 Tyr705, (F and G) p-STAT1 Tyr701, (H and I) p-ERK p44/ 42 Thr202/Tyr204. Data represent the mean (± SEM) of two independent experiments; n 5 4 independent individuals. *p < 0.02 by the MannWhitney U test. MFI, mean fluorescent intensity; ns, not significant; U, unstimulated.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Phospho-proteomics, Isolation, Control, Gene Expression, Software, Blocking Assay, Staining, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 5. SCARF1 expression is dysre- gulated after AC uptake on BDCA11 DCs. (A) The basal expression of SCARF1 is comparable between patients with SLE and healthy control subjects. PBMCs were iso- lated from healthy control subjects (n 5 7) or patients with SLE (n 5 17). PBMCs were stained for SCARF1, BDCA11 DCs, plas- macytoid DCs, and monocytes. Data are shown as MFI of SCARF1 surface expres- sion. Data are not significant by Man- nWhitney U test. (B) Representative flow cytometry for healthy control subjects and patients with SLE. Scheme for selection of CD11c1BDCA11 cells. Histogram of basal SCARF1 expression on BDCA11 DCs. Gray: healthy; black: SLE. (CF) SCARF1 contributes to the removal of ACs in patients with lupus. (C and D) PBMCs (1 × 106/ml) were incubated with anti-SCARF1 for 30 min and then stimulated with pHrodo redlabeled ACs (2 × 106/ml). Cells were incubated for 4 h. Cells were stained and analyzed by flow cytometry for SCARF1 and BDCA11 DCs (C) or CD141 mono- cytes (D). Data are the mean (± SEM) of three independent experiments with n 5 3 per group. (E and F) BDCA11 sorted DCs or CD141 monocytes (1 × 106/ml) were incu- bated with anti-SCARF1 for 30 min and then stimulated with ACs (2 × 106/ml) for 4 h. Cells were lysed and mRNA levels of Scarf1 (E) or Il10 (F) were measured using qPCR. Data are the mean (± SEM) of three independent experiments. *p < 0.01, **p < 0.005 by two-way ANOVA with Geissler- Greenhouse correction; ***p < 0.0001 by two-way ANOVA. MFI, mean fluorescent intensity; ns, not significant.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Expressing, Control, Staining, Flow Cytometry, Selection, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 6. Increased levels of anti-SCARF1 autoantibodies in the sera of patients with lupus show defects in removal of AC. (AC) Soluble SCARF1 or blocking buffer-coated ELISA plates were incubated with sera from patients with SLE or healthy individuals. IgG SCARF1 Abs (healthy: n 5 100; SLE: n 5 145) (A), IgM SCARF1 Abs (healthy: n 5 9; SLE: n 5 62) (B), and IgA SCARF1 Abs (healthy: n 5 9; SLE: n 5 62) (C). Data are shown as anti- SCARF1 minus antiblock data to reduce the level of binding to the blocking buffer. The OD values of each individual are represented as a single point. Dashed lines indicate the OD values that exceed the mean controls by >3 SDs. Horizontal bars represent the mean values. p 5 0.0009, p 5 0.0003, by Man- nWhitney U test. (DF) Lupus serum reduces AC uptake in SCARF11BDCA11 DCs. PBMCs (1 × 106/ml) were incubated with autologous serum, lupus serum, or serum starved for 2024 h. Cells were incubated for 3 h with pHrodo redlabeled ACs (2 × 106/ml). Cells were stained and analyzed by flow cytometry. (D) Representative histogram of pHrodo expression in SCARF11 BDCA11 DCs. (E) Percentage of ACs phagocytosed by BDCA11 DCs treated with the specified serum and measured as pHrodo-positive cells. (F) MFI quantification of SCARF1 expression from BDCA11 DCs that have phagocytosed ACs. (E and F) Data represent the mean (± SEM) of three independent experiments; n 5 5, by two-way ANOVA. (G and H) Levels of autoantibodies to SCARF1 directly correlate with deficiency in efferocytosis. PBMCs (1 × 106/ml) were incubated with autologous serum, lupus serum, or serum starved for 2024 h. Cells were incubated for 3 h with pHrodo redlabeled ACs (2 × 106/ml). Cells were stained and analyzed by flow cytometry. (G) Representative histogram of pHrodo expression in SCARF11 BDCA11 DCs. (H) Percentage of pHrodo-labeled ACs phagocytosed by BDCA11 DCs treated with specified serum and measured as pHrodo-positive cells. Data represent the mean (± SEM) of two independent experiments; n 5 12, by two-way ANOVA. (I) Autoanti- bodies block SCARF1 expression on BDCA11 DCs. PBMCs (1 × 106/ml) were incubated with autologous serum, lupus serum, or serum starved for 2024 h. Cells were incubated for 3 h with pHrodo redlabeled ACs (2 × 106/ml). Cells were stained and analyzed by flow cytometry. Data represent the mean (± SEM) of two independent experiments; n 5 12, by two-way ANOVA. ns, not significant.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Staining, Flow Cytometry, Expressing, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

doi: 10.4049/jimmunol.2100532

Figure Lengend Snippet: FIGURE 7. Ig depletion increases SCARF1-mediated efferocytosis. (A) Decreased SCARF1 binding after IgG depletion. We depleted 20% serum in RPMI, healthy or SLE, using 100 ml of protein A/G agarose beads columns. IgG depletion was confirmed by Western blot (data not shown). SCARF1 bind- ing was analyzed by dot blot. Recombinant protein was transferred to nitrocellulose membrane, and full or depleted serum was used as a primary Ab. Human IgG was used as secondary Ab. Representative blot (SLE: n 5 13; healthy: n 5 8). Anti-SCARF1 was used as a control. (BF) Ig depletion restores efferocy- tosis on BDCA11 DCs in SLE patient serum. PBMCs (1 × 106/ml) were incubated with full or Ig-depleted serum for 2024 h. FBS was used as serum con- trol. After serum treatment, cells were incubated for 3 h with pHrodo redlabeled ACs (2 × 106/ml). Cells were stained and analyzed by flow cytometry. (B) Representative histograms of pHrodo expression to measure phagocytosis. Top panel, CD11c1BDCA11 DCs; bottom panel, CD141 monocytes. Blue line, untreated/full serum; red line, Ig-depleted serum. (C and D) Quantification of phagocytosed ACs measured as percentage of CD11c1BDCA11pHrodo1 cells (C) or CD141 pHrodo1 monocytes. Data represent the mean (± SEM) of two independent experiments, n 5 13 (SLE) and n 5 8 (healthy), by two-way ANOVA. (E and F) Increased BDCA11SCARF11 cells after Ig depletion. Total number of CD11c1BDCA11SCARF11 (E) or CD141 monocytes (F) mea- sured by flow cytometry in the presence of full serum or Ig-depleted serum. Data represent the mean (± SEM) of two independent experiments, n 5 13 (SLE) and n 5 8 (healthy), by two-way ANOVA.

Article Snippet: BDCA11 DCs were sorted using magnetic sorting using the BDCA11 kit from Miltenyi Biotec (Cat#: 130- 119-475) and allowed to recover overnight.

Techniques: Binding Assay, Western Blot, Dot Blot, Recombinant, Membrane, Control, Incubation, Staining, Flow Cytometry, Expressing