Journal: Immunology

Article Title: Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen‐presenting cells and promotes resistance to Salmonella infection

doi: 10.1111/imm.13169

Figure Lengend Snippet: Human SLAMF9 is expressed on circulating monocytes and monocyte‐derived cells. (a) ELISA detection of antibodies produced by clone FC2 reactive to human SLAMF9‐Fc fusion protein but not human IgG1. (b) Flow cytometry validation of SLAMF9‐reactive hybridomas using HEK‐293T cells transduced with lentivirus encoding FLAG‐tagged human SLAMF9 (red) or untransduced cells (dark grey). (c, d) Surface expression of SLAMF9 (red) compared with isotype control (dark grey) measured by flow cytometry on freshly isolated PBMCs or monocyte‐derived cells. (c) Peripheral blood mononuclear cells (PBMCs) gated on: classical monocytes (CD14 + CD16 − ), intermediate monocytes (CD14 + CD16 + ), non‐classical monocytes (CD14 low CD16 + ), B cells (CD19 + FSC/SSC lymphocytes) natural killer (NK) cells (CD14 − CD16 + , FSC/SSC lymphocytes), and T cells (CD3 + , FSC/SSC lymphocytes). (d) Surface expression of SLAMF9 on CD14 + monocyte‐derived macrophages and dendritic cells differentiated for 7 days with the indicated cytokine(s). (e) Change in SLAMF9 transcript expression during monocyte differentiation measured by quantitative RT‐PCR and normalized to UBC.

Article Snippet: Western blot detection of immunoprecipitated SLAMF9 was achieved using purified anti‐mouse SLAMF9 (9318) and anti‐rabbit IgG‐horseradish peroxidase (Cell Signaling Technologies, Danvers, MA).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Produced, Flow Cytometry, Biomarker Discovery, Transduction, Expressing, Control, Isolation, Quantitative RT-PCR

Journal: Immunology

Article Title: Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen‐presenting cells and promotes resistance to Salmonella infection

doi: 10.1111/imm.13169

Figure Lengend Snippet: SLAMF9‐dependent activation of inflammatory cytokine production. (a) Quantitative RT‐PCR measurement of SLAMF9 expression in THP‐1 cells before and after differentiation with PMA. (b) Quantitative PCR measurement of SLAMF9 mRNA interference using stable expression of SLAMF9‐specific shRNAs (434 and 530) compared with a non‐targeting control shRNA in PMA‐differentiated THP‐1 cells. Transcript levels in (b) and (c) are normalized to UBC . (c) Pro‐inflammatory cytokine production by PMA‐differentiated THP‐1 cells with and without stimulation for 24 hr with lipopolysaccharide, measured by BD Cytometric Bead Array. Error bars indicate standard deviation from the mean. Statistical tests for differences between vector control and SLAMF9 knockdowns were performed using two‐way analysis of variance. **** P < 0·0001. (d) Analysis of cytokine expression in control and SLAMF9 knockdown (shRNA 434) THP‐1 cells across multiple independent experiments from (c). Ratio paired t ‐tests show differences in cytokine production at * P < 0·05 and ** P < 0·01.

Article Snippet: Western blot detection of immunoprecipitated SLAMF9 was achieved using purified anti‐mouse SLAMF9 (9318) and anti‐rabbit IgG‐horseradish peroxidase (Cell Signaling Technologies, Danvers, MA).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Control, shRNA, Standard Deviation, Plasmid Preparation, Knockdown

Journal: Immunology

Article Title: Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen‐presenting cells and promotes resistance to Salmonella infection

doi: 10.1111/imm.13169

Figure Lengend Snippet: Mouse SLAMF9 is expressed on circulating plasmacytoid dendritic cells (pDCs) and non‐classical monocytes. (a) Flow cytometry screening of mouse SLAMF9 reactive hybridomas against HEK‐293T cells lentivirally transduced to express mouse SLAMF9 (red line) or untransduced (shaded) cells. (b) Validation of mSLAMF9‐reactive hybridoma clone M349 on mouse bone marrow‐derived macrophages from wild‐type C57BL/6 (red shaded) or Slamf9 −/− mice (black line). (c–e) Flow cytometry staining of SLAMF9 on circulating peripheral blood leucocytes using anti‐SLAMF9 clone M349 with cells from Slamf9 −/− mice used as a negative staining control. (c) Staining of WT C57BL/6 (red) or Slamf9 −/− (grey) B cells, T cells and natural killer cells shows an absence of SLAMF9 on resting lymphocytes. (d) SLAMF9 staining is found on CD11b – Siglec‐H + CD11c low pDCs and (e) CD19 − CD11b + Ly6C − non‐classical monocytes. (f,g) Flow cytometry staining of mouse splenocytes using anti‐SLAMF9 antibody M349 finds expression of SLAMF9 on (f) wild‐type (red) but not knockout (grey) CD19 − Siglec‐H + pDCs and (g) a fraction of CD11b + CD11c + cDCs.

Article Snippet: Western blot detection of immunoprecipitated SLAMF9 was achieved using purified anti‐mouse SLAMF9 (9318) and anti‐rabbit IgG‐horseradish peroxidase (Cell Signaling Technologies, Danvers, MA).

Techniques: Flow Cytometry, Biomarker Discovery, Derivative Assay, Staining, Negative Staining, Control, Expressing, Knock-Out

Journal: Immunology

Article Title: Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen‐presenting cells and promotes resistance to Salmonella infection

doi: 10.1111/imm.13169

Figure Lengend Snippet: SLAMF9 in mouse tissues is found on niche antigen‐presenting cells. Staining of live, single cells from peritoneal lavage for SLAMF9 expression. Cells from Slamf9−/− mice are used as a negative staining control for SLAMF9 expression found on wild‐type C57BL/6 cells. SLAMF9 is restricted to (a) CD19 − CD11b int MHC‐II + F4/80 low small peritoneal macrophages and (b) CD19 + CD11b + B1 cells, but is absent on the more abundant CD11b high F4/80 high large peritoneal macrophages and CD19 + CD11b − B2 cells. (c) Expression of SLAMF9 on leucocytes from perfused and collagenase‐disaggregated liver is also restricted to PDCs (not shown) and a subset of CD11b + Ly6C − mononuclear phagocytes. Gating on SLAMF9 + (red histogram) and SLAMF9 − (black histogram) CD11b + Ly6C − cells shows a surface phenotype for SLAMF9 + cells as CD11c + F4/80 low CX 3 CR1 + MHC‐II high.

Article Snippet: Western blot detection of immunoprecipitated SLAMF9 was achieved using purified anti‐mouse SLAMF9 (9318) and anti‐rabbit IgG‐horseradish peroxidase (Cell Signaling Technologies, Danvers, MA).

Techniques: Staining, Expressing, Negative Staining, Control

Journal: Immunology

Article Title: Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen‐presenting cells and promotes resistance to Salmonella infection

doi: 10.1111/imm.13169

Figure Lengend Snippet: SLAMF9 promotes resistance to Salmonella . C57BL/6 and Slamf9 −/− mice were infected with Salmonella enterica serovar Typhimurium M525 by intravenous injection. Spleen and liver were harvested at day 14 post‐infection and CFU/g was quantified. Each dot represents a single mouse. P ‐values were determined using Student's t ‐test. Results are representative of two independent experiments.

Article Snippet: Western blot detection of immunoprecipitated SLAMF9 was achieved using purified anti‐mouse SLAMF9 (9318) and anti‐rabbit IgG‐horseradish peroxidase (Cell Signaling Technologies, Danvers, MA).

Techniques: Infection, Injection

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

Article Title: Dependence of Glomerulonephritis Induction on Novel Intraglomerular Alternatively Activated Bone Marrow-Derived Macrophages and Mac-1 and PD-L1 in Lupus-Prone NZM2328 Mice a

doi: 10.4049/jimmunol.1601565

Figure Lengend Snippet: Blocking Mac-1 and ICAM-1 binding inhibits anti-GBM-induced proteinuria and myeloid infiltration into the glomerulus in NZM mice. (A) NZM mice (10 – 12 weeks old) were injected on day 0 with anti-GBM to induce nephritis. Anti-CD11b (M1/70), anti-ICAM-1 (YN1), and rat isotype control (LTF-2) mAb were injected (100 µg/injection) on alternate days beginning on day 0. Mice were sacrificed on days 3 and 7. Urine was collected daily and assayed for albumin and creatinine. The albumin/creatinine ratio were normalized to the highest value in each experiment which is set at 100% with the range for individual experiments at 3.7 – 4.9 mg/mg. The % maximum for replicates were averaged and displayed as mean ± SEM. The experiments were performed 4 times and a total of 10, 6, and 8 mice were used for LTF-2-, M1/70-, and YN1- injected groups respectively. (B) Confocal microscopy of kidneys from control LTF-2-injected mice at day 7 after anti-GBM injection. The Ag visualized are as shown in the labeling and the micrographs show low (a) and high power (b, c) magnifications. Glomeruli (a,b) are shown by mesangial cell labeling (blue). CD11b+F4/80 macrophages are shown by arrows (b, c, red). F4/80+ macrophages marked by asterisk (c, blue or magenta) showed varying degrees of CD11b expression (red). Circles demarcates glomeruli (b, c). g, glomeruli. Bars, 50 µm in a and 10 µm in b, c. (C) Single cell suspensions of purified glomeruli from anti-GBM-injected mice on day 3 immediately preceding proteinuria onset were analyzed by flow cytometry as described in Fig. 3. Representative analyses of mice from LTF-2-, M1/70-, and YN1- injected group are shown in a, b, and c respectively and the mean ± SEM of various cell types with 4 mice in each group and havested on day 3 is plotted and shown in (E). (D) The direct comparison of intraglomerular myeloid cell population numbers for young NZM mice, NZM with cGN (3+ proteinuria, Fig. 3D) and NZM mice injected with anti-GBM plus control LTF-2 mAb and harvested on day 3 (from Fig. 4E) and on day 7 (10 mice) are plotted. *, p<0.05; ***, p<0.001; ****, p<0.0001.

Article Snippet: The following goat affinity-purified polyclonal Ab against recombinant mouse proteins were from R&D (Minneapolis, MN, USA): CD103, Itgα8, MMR, nephrin, Mgl1/2, NKp46, PD-L1, and SLAMf9 and they were conjugated with Pacific Blue (PacBlue) or Alexa Fluors (A) with mAb labeling kits (Invitrogen).

Techniques: Blocking Assay, Binding Assay, Injection, Confocal Microscopy, Labeling, Expressing, Purification, Flow Cytometry

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

Article Title: Dependence of Glomerulonephritis Induction on Novel Intraglomerular Alternatively Activated Bone Marrow-Derived Macrophages and Mac-1 and PD-L1 in Lupus-Prone NZM2328 Mice a

doi: 10.4049/jimmunol.1601565

Figure Lengend Snippet: PD-L1 expression by intraglomerular CD11b+F4/80−I-A− macrophages in NZM mice with cGN and with anti-GBM-induced nephritis and anti-PD-L1 mAb blocking of proteinuria. (A) Glomerular CD11b+ macrophages are the predominant PD-L1 expressing cell population in NZM with severe proteinuria (4+ proteinuria by dipstick; a – h) and anti-GBM-induced nephritis (i – p). Kidney sections were stained with mAb against PD-L1, CD11b, and Mac-2 or F4/80 as labeled and analyzed by confocal microscopy as described in Fig. 1. Low (a – d, i – l) and high magnification (e – h, m – p) micrographs in which glomeruli are shown by arrows and circles respectively are presented. *, Ly6Ghi PMN (m – p). Bars, 50 µm in low magnification and 10 µm in high magnification micrographs. Mac-2 intensity in A-c and A-d were highly attenuated because of the bright Mac-2 staining of tubular cells. (B) Inhibition of anti-GBM-induced proteinuria by anti-PD-L1. mAb (200 µg/injection) were administered on alternate days beginning on day 0 and proteinuria were measured as described in Fig. 4. Proteinuria results are expressed as mean ± SEM of values normalized to the maximal albumin/creatinine value in each experiment. Eight mice were used in each group and the experiment has been performed 4 times. (C) Intraglomerular infiltrating leukocytes were analyzed on day 3 as described in Fig. 3 by flow cytometry and the cell numbers in myeloid populations and T cells are presented as mean ± SEM for 3 mice in each group. The experiment has been performed twice.

Article Snippet: The following goat affinity-purified polyclonal Ab against recombinant mouse proteins were from R&D (Minneapolis, MN, USA): CD103, Itgα8, MMR, nephrin, Mgl1/2, NKp46, PD-L1, and SLAMf9 and they were conjugated with Pacific Blue (PacBlue) or Alexa Fluors (A) with mAb labeling kits (Invitrogen).

Techniques: Expressing, Blocking Assay, Staining, Labeling, Confocal Microscopy, Inhibition, Injection, Flow Cytometry

Journal: Cell Discovery

Article Title: Single-cell spatiotemporal analysis of the lungs reveals Slamf9 + macrophages involved in viral clearance and inflammation resolution

doi: 10.1038/s41421-024-00734-4

Figure Lengend Snippet: a Cell fate inference analysis by CellRank based on monocyte/macrophage scRNA-seq data at d7 and d14. b Boxplot showing the proportion of Trem2 + AMs, Fbp1 + AMs and proliferating Fbp1 + AMs in total Stereo-seq bin80-bins of nine lung slides at each timepoint. Data are represented as mean ± SEM ( n = 9 slides per timepoint). Center line, median; box bounds, first and third quartiles; whiskers, 1.5 times the interquartile range. Kruskal–Wallis test. c RNA velocity analysis of AM subpopulations. Arrows indicate the potential directions of state transitions. d Boxplot showing the spatial correlation between different cell types and Fbp1 + AMs of nine lung slides at d14. Data are represented as mean ± SEM ( n = 9 slides per timepoint). Center line, median; box bounds, first and third quartiles; whiskers, 1.5 times the interquartile range. Kruskal–Wallis test was performed to calculate the P value across different groups. Pearson correlation was performed to calculate the P value of all spots within each slide. Black dots: P < 0.05; gray dots: P ≥ 0.05. Red dotted line represents the correlation value of 0. e , f Representative images of the lung sections of hACE2 mice before and after SARS-CoV-2 infection detected by immunofluorescent staining. TREM2 and CD68 at d0 and d14 ( e ); FBP1 and CD68 at d0 and d14 ( f ). TREM2 + or FBP1 + macrophages were indicated by arrows. Scale bars, 50 μm. g Boxplot showing the proportion of SLAMF9 + SPP1 + , TREM2 + , FBP1 + , FBP1 + MKI67 + , and MARCO + CD36 + TREM2 – FBP1 – macrophages in CD68 + macrophages in scRNA-seq data of human lung autopsy samples of control ( n = 8) and COVID-19 patients ( n = 5). Center line, median; box bounds, first and third quartiles; whiskers, 1.5 times the interquartile range. Data are represented as mean ± SEM. Two-sided Wilcoxon test.

Article Snippet: Different primary antibodies were sequentially applied to examine specific cell markers, including anti-CD68 Polyclonal antibody (28058-1-AP, 1:800; Proteintech) and TREM2 Polyclonal antibody (13483-1-AP, 1:200; Proteintech), or FBP1 Polyclonal antibody (12842-1-AP, 1:100, Proteintech), or Osteopontin Polyclonal antibody (22952-1-AP, 1:200; Proteintech) and SLAMF9 polyclonal antibody (LM-2205R, 1:200; LMAI BIO), followed by HRP-conjugated secondary antibody incubation and tyramide signal amplification (TSA).

Techniques: Infection, Staining, Control

Journal: Cell Discovery

Article Title: Single-cell spatiotemporal analysis of the lungs reveals Slamf9 + macrophages involved in viral clearance and inflammation resolution

doi: 10.1038/s41421-024-00734-4

Figure Lengend Snippet: a Cell fate inference analysis by CellRank based on monocyte/macrophage scRNA-seq data at d7 and d14. b Boxplot showing the proportion of Trem2 + AMs, Fbp1 + AMs and proliferating Fbp1 + AMs in total Stereo-seq bin80-bins of nine lung slides at each timepoint. Data are represented as mean ± SEM ( n = 9 slides per timepoint). Center line, median; box bounds, first and third quartiles; whiskers, 1.5 times the interquartile range. Kruskal–Wallis test. c RNA velocity analysis of AM subpopulations. Arrows indicate the potential directions of state transitions. d Boxplot showing the spatial correlation between different cell types and Fbp1 + AMs of nine lung slides at d14. Data are represented as mean ± SEM ( n = 9 slides per timepoint). Center line, median; box bounds, first and third quartiles; whiskers, 1.5 times the interquartile range. Kruskal–Wallis test was performed to calculate the P value across different groups. Pearson correlation was performed to calculate the P value of all spots within each slide. Black dots: P < 0.05; gray dots: P ≥ 0.05. Red dotted line represents the correlation value of 0. e , f Representative images of the lung sections of hACE2 mice before and after SARS-CoV-2 infection detected by immunofluorescent staining. TREM2 and CD68 at d0 and d14 ( e ); FBP1 and CD68 at d0 and d14 ( f ). TREM2 + or FBP1 + macrophages were indicated by arrows. Scale bars, 50 μm. g Boxplot showing the proportion of SLAMF9 + SPP1 + , TREM2 + , FBP1 + , FBP1 + MKI67 + , and MARCO + CD36 + TREM2 – FBP1 – macrophages in CD68 + macrophages in scRNA-seq data of human lung autopsy samples of control ( n = 8) and COVID-19 patients ( n = 5). Center line, median; box bounds, first and third quartiles; whiskers, 1.5 times the interquartile range. Data are represented as mean ± SEM. Two-sided Wilcoxon test.

Article Snippet: Different primary antibodies were sequentially applied to examine specific cell markers, including anti-CD68 Polyclonal antibody (28058-1-AP, 1:800; Proteintech) and TREM2 Polyclonal antibody (13483-1-AP, 1:200; Proteintech), or FBP1 Polyclonal antibody (12842-1-AP, 1:100, Proteintech), or Osteopontin Polyclonal antibody (22952-1-AP, 1:200; Proteintech) and SLAMF9 polyclonal antibody (LM-2205R, 1:200; LMAI BIO), followed by HRP-conjugated secondary antibody incubation and tyramide signal amplification (TSA).

Techniques: Infection, Staining, Control

Journal: bioRxiv

Article Title: Spatiotemporal landscape of SARS-CoV-2 pulmonary infection reveals Slamf9 + Spp1 + macrophages promoting viral clearance and inflammation resolution

doi: 10.1101/2022.05.03.490381

Figure Lengend Snippet: a. Phenotypic linkage between AM and mono/macrophage subpopulations from d7 and d14. b. Temporal changes of AM-Trem2, AM-Fbp1 and AM-Fbp1-Mki67. c. RNA velocity analysis of AM subpopulations. Arrows indicate the potential directions of state transitions. d. Density of different cell types in the neighborhood of AM-Fbp1 of nine lung sections at d14 (HD group). Neighborhood: <7.5 μm. Boxplots: center line, median; box bounds, first and third quartiles; whiskers, 1.5 times the interquartile range. e-g. Immunofluorescence analyses of the lung sections of hACE2 mice before and after SARS-CoV-2 infection. Staining of SLAMF9, SPP1 and CD68 at d0 and d7 ( e ); TREM2 and CD68 at d0 and d14 ( f ); FBP1 and CD68 at d0 and d14 ( g ). Scale bar, 50 μm. h. Identification of SLAMF9 + SPP1 + , TREM2 + , FBP1 + , FBP1 + MKI67 + , and MARCO + CD36 + TREM2 - FBP1 - macrophages in scRNA-seq data of human lung autopsy samples of COVID-19 patients. Control: pulmonary bulla or lung cancer, and these samples were to be inflammation-free pathologically confirmed. Two-sided Wilcoxon test.

Article Snippet: Different primary antibodies were sequentially applied to examine specific cell markers, including anti-CD68 Polyclonal antibody (28058-1-AP, 1:800; proteintech) and TREM2 Polyclonal antibody (13483-1-AP, 1:200; proteintech), or FBP1 Polyclonal antibody (12842-1-AP, 1:100, proteintech), or Osteopontin Polyclonal antibody (22952-1-AP, 1:200; proteintech) and SLAMF9 polyclonal antibody (LM-2205R, 1:200; LMAI BIO), followed by HRP-conjugated secondary antibody incubation and tyramide signal amplification (TSA).

Techniques: Immunofluorescence, Infection, Staining, Control