Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left , Representative immunostaining of macrophages in the splenic marginal zone expressing CD169 (red) and Tim4 (green); DAPI nuclear staining (blue). Right , FACS contour plots for CD169 and Tim4 expression in splenic CD45 + CD11b low F4/80 low cells and quantitation of Tim4 expression in CD169 + MMMs; n=4. B . Top Left , FACS pseudocolor plots of circulating Ly6C monocytes with histograms identifying CD169 + and CD169 − populations within Ly6C low monocytes, and corresponding cell quantitation; NTM, normalized to mode. Top Right , principal component analysis of top 500 differentially expressed genes (DEGs) from bulk RNAseq of sorted Ly6C low CD169 − and Ly6C low CD169 + cells, and heat maps with dendrograms for 334 DEGs (q<0.05) between the same sub-populations. Bottom , FACS pseudocolor plots of circulating Ly6C low CD169 + Tim4 + macrophages in naïve and spx mice blood with flow histograms identifying CD64 and MHCII surface expression (in black), together with quantitation of frequency or absolute number of the populations shown; n=5-7, statistics: unpaired t test. C. Top, Representative blood FACS dot plots and quantitation of tdTomato (Tdt) expression in blood Ly6C + monocytes from CX3CR1 CreERT ; Rosa26 tdTomato mice after a tamoxifen (TAM) pulse to induce Cre recombination. Shown are data from several time points after TAM and 14 d after splenectomy (Spx) performed at 26 d post-TAM; *p<0.05, n=3, statistics: unpaired t-test. Bottom , representative FACS plots showing CD169 expression in Tdt+ cells 26 d post-TAM pulse and overlay of the CD169 + Tdt + cells (green) on blood Ly6C + monocytes. D . Top , UMAP plots derived from single cell RNA sequencing of blood leukocytes from 3 naïve mice (∼400K total cells, 11 identified cell clusters), heat map demonstrating CD169 ( Siglec1 ) expression primarily in the monocyte cluster, and quantitative expression of select macrophage genes in cells with and without CD169 expression (adjusted FDR p values are shown). E . Left, FACS plots identifying CD169 + Tim4 + cardiac macrophages in intact and Spx mice, overlay of these macrophages on contour plots of CCR2 and LYVE1 expression (in intact mice), and quantitation of overall LYVE1 and CCR2 expression (n=6). Right , quantitation of frequency and number of cardiac CD169 + Tim4 + LYVE1 low macrophages naïve and Spx C57BL/6 mice; n=5-7/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Immunostaining, Expressing, Staining, Quantitation Assay, Derivative Assay, RNA Sequencing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and group quantitation for circulating Ly6C low CD169 + Tim4 + macrophages in sham-operated and MI wild type (WT) C57BL/6 mice 1 d post-MI; n=4/group, statistics: unpaired t test. B. Left , Flow histograms demonstrating surface expression of CCR3 and CCR4 in Ly6C low CD169 + Tim4 + macrophages from the same groups; statistics: unpaired t test, NTM, normalized to mode, y-axis represents cell counts. Right , chemokine gene expression by RT-PCR (normalized to 18s) in the myocardial border zone (BZ) 1 d after MI or sham operation; n=4-5/group, statistics: unpaired t test. *p<0.05, **p<0.01, ***p<0.001 versus sham. C . Left , Circulating Ly6C low CD169 + Tim4 + cell frequency prior to and 1 d after MI in Spx mice; n=9, statistics: paired t test. NS, not significant. D. FACS density plots, histograms, and quantitation of cardiac Ly6C low CD169 + Tim4 + macrophages in WT and Spx mice 1 d post-MI or sham operation; n=4-7/group, statistics: unpaired t test. E . Immunostains and FACS dot plots of splenic CD169 + MMMs (red) 24 h after MI or sham operation, and quantitation of MMM frequency by FACS and spleen weight (Wt); n=5-7/group, statistics: unpaired t test. TL, tibia length. F . Left , FACS dot plots and histograms, and corresponding quantitation, of blood and heart Ly6C low CD169 + Tim4 + Bioparticle + cells from sham and MI mice given 10 mg/kg Texas Red-conjugated bioparticles i.v. 3 h before sacrifice; n=3-4/group, statistics: unpaired t test for blood and non-parametric Mann-Whitney U test for heart (non-normal distribution). Right, quantitation of splenic BioParticle + MMMs in the same experimental mouse groups; statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A Left , Parabiosis schema joining CD45 isotype-mismatched host (spleen-intact or after splenectomy [Spx]) and donor parabiont mice, with MI induced in the host. Middle and Right , FACS plots and quantitation of donor chimerism in host mouse blood (total CD45 + leukocytes) and heart (Ly6C low CD169 + macrophages) in spleen-intact and Spx host mice 48 h after MI. n=4-7/group, statistics: unpaired t-test. B Top Left , Parabiosis schema joining CD169 DTR host and donor parabiont MaFIA mice, with host mice given either vehicle or diphtheria toxin (DT) at the time of MI. Right , Representative FACS dot plots of donor GFP + CD169 + macrophages in 48 h post MI hearts from host mice and flow histograms of CD169 expression in GFP + CD64 + MHCII + Tim4 + cells delineated as NTM or cell counts. Bottom Left , quantitation of GFP + frequency in host cardiac CD169 + Tim4 + macrophages and total CD169 + Tim4 + cells as a percentage of all autofluorescent(Auto) + macrophages in vehicle and DT treated host MI mice; n=3-4/group, statistics: unpaired t test. C. Left , Parabiosis schema joining CD169 DTR host mice and either spleen-intact or Spx MaFIA donor parabionts, with host mice given DT at the time of MI to deplete CD169 + macrophages. Right , Example FACS dot plots and quantitation of donor CD45 + Auto + CD64 + MHCII + GFP + CD169 + Tim4 + macrophages in the host MI heart 48 h post MI; n=3-4/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left, FACS pseudocolor plots of cardiac CD169 + Tim4 + macrophages and separation based on LYVE1 surface expression in naïve and 1 d post-MI mice. Right , quantitation of LYVE1 hi and LYVE1 low CD169 + Tim4 + macrophages in hearts from naïve and 1 d post-MI mice. n=4-6/group; statistics: unpaired t test. B . Heat map of 462 significant (p adjusted<0.05) DEGs by RNAseq analysis in sorted LYVE1 hi and LYVE1 low CD169 + Tim4 + cardiac macrophages 1 d post-MI. C. PCA plots using the top 500 DEGs after rlog transformation of RNAseq data from these macrophages sorted from the indicated sites in naïve and 1 d post-MI mice. D. Left , Flow histograms depicting LYVE1 surface expression on cardiac CD169 + Tim4 + macrophages (green) and total Autofluorescence + CD64 + MHCII + macrophages (brown) in WT and Spx mice, 1 d post-MI. Right , FACS quantitation of LYVE1 hi and LYVE1 low CD169 + Tim4 + macrophages in the hearts of WT and Spx mice, 1 d post-MI; n=5-6/group. Statistics: unpaired t test. E. Representative confocal micrograph of border zone (BZ) myocardium immunostained for CD169 (red) and Tim4 (green) 1 d post-MI in WT and Spx mice; nuclear staining with DAPI (blue). Scale bar, 200 μm. Inset shows magnified images of CD169 and Tim4 staining. Yellow arrows indicate double positive cells; scale bar 10 μm.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Expressing, Quantitation Assay, Transformation Assay, Staining

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: FACS plots and quantitation of Ly6C low CD169 + Tim4 + macrophages and total Ly6C low cells in blood ( A ) and in heart ( B ) 1 d post-MI in wild-type (WT) and splenectomized (Spx) WT mice, and CD169 DTR mice given diphtheria toxin (CD169 DTR /DT) at the time of MI; n=5-7/group, statistics: one-way ANOVA, Bonferroni post-test. Isotype antibody is shown in gray. C . FACS plots and group data for blood CD45 + CD11b + Ly6G + neutrophils and ICAM-1/CD54 + neutrophils 1 d post MI in WT, Spx, and CD169 DTR /DT mice; n=6-8/group; statistics: one-way ANOVA, Bonferroni post-test. D . Left, Representative confocal images of immunofluorescent Ly6G staining in WT, Spx, and CD169 DTR /DT hearts 1 d post-MI demonstrating Ly6G + neutrophil (red) infiltration (arrows); nuclear staining with DAPI (blue). Scale bar 20 μm. Right , FACS plots and corresponding quantitation of cardiac CD45 + CD11b + Ly6G + neutrophils (red) and annexin V + apoptotic neutrophils (blue) in the same groups 1 d post-MI; n=3-4/group, statistics: one-way ANOVA, Dunnett’sT3 post-test. E . FACS density plots for Lin − c-kit + CD34 + CD16/32 + granulocyte monocyte precursors (GMPs) in bone marrow from WT, Spx, and CD169 DTR /DT mice 1 d post MI, together with quantitation. Flow gates were based on isotype antibody control. n=5-7/group; statistics: one-way ANOVA, Tukey’s post-test. F . Left , Representative FACS dot plots identifying cardiac neutrophils as CD11b + Ly6G + cells in WT mice and as Ly6G + tdTomato + cells in Catchup mice at baseline and 1 d post-MI. Right Top , Representative histograms of Ly6G and tdTomato fluorescence intensity in heart mononuclear cells from the same groups. Right Bottom , FACS dot plots gated on cardiac CD169 + Tim4 + macrophages illustrating tdTomato expression in Catchup mice 1 d post-MI. Auto, autofluorescence. G . Representative FACS histograms of intracellular IL4 and IL10 staining in cardiac Ly6C low cells 1 d post MI in WT, Spx and CD169 DTR /DT mice, together with cell quantitation of the Ly6C low subsets. N=6-7/group; statistics: one-way ANOVA, Bonferroni post-test. H . Representative FACS pseudocolor plots for intracellular TGFβ and IL10 staining in cardiac CD169 + macrophages 1 d post MI in WT and Spx mice, with accompanying quantitation; n=4-5/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Staining, Control, Fluorescence, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Kaplan-Meier survival curves over 10 d following MI or sham operation in WT, Spx, and CD169 DTR /DT mice, and after MI in CD45.2 Spx mice with adoptive transfer of naïve splenic CD169 + Tim4 + cells from syngeneic CD45.1 WT mice 24 h post-MI (Spx-MI+AT). Statistical comparisons: log-rank test. B . Gross images of post-MI cardiac rupture with hemothorax or hemopericardium, and Kaplan-Meier curves for freedom from rupture over 10 d post-MI in WT, Spx, CD169 DTR /DT, and Spx-MI+AT mice. Statistical comparisons: log-rank test. C . Left , Representative post-mortem whole hearts and end-diastolic long-axis 2-dimensional echocardiograms from WT-MI, Spx-MI, CD169 DTR /DT-MI and Spx-MI+AT mice (10 d post-MI). Right , Quantitation of LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) at 10 d post-MI; n=4-6/group, statistics: one-way ANOVA, Bonferroni post-test. D . Left , Representative confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the heart infarct border zone (BZ) from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. DAPI (blue) nuclear staining. iNOS + CD206 + cells appear yellow. Higher magnification is shown in the middle panel. Right , quantitation of iNOS + CD206 + and iNOS − CD206 + cells/mm 2 in the hearts. N=3/group, statistics: one-way ANOVA, Bonferroni post-test. NS, not significant. E . Top Left , Confocal images of MMP-9 immunostaining (red) in infarct BZ of hearts from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. Nuclear staining with DAPI (blue). Top Right , Quantitative group data for total MMP-9 mean fluorescence intensity per region of interest (ROI). AU, arbitrary units. N=4/group, statistics: one-way ANOVA, Bonferroni post-test. Bottom , Representative Masson’s trichrome stains of infarcted hearts (10 d post-MI) from the same groups demonstrating MI border zone (BZ) fibrosis, together with BZ fibrosis quantitation. n=4/group; statistics: one-way ANOVA, Bonferroni post-test. F . FACS quantitation of Ly6C hi blood monocytes and serum IL-10 levels in WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice at 10 d post-MI. N=4-7/group, statistics: one-way ANOVA, Bonferroni post-test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Adoptive Transfer Assay, Quantitation Assay, Staining, Immunostaining, Fluorescence

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Protocol for LXRα agonist T0901317 treatment (40 mg/kg i.p.) from 1 d prior to 5 d post-MI, with 10 d post-MI follow-up, in WT and Spx mice. B . FACS contour plots and quantitation of cardiac CD169 + Tim4 + macrophages 1 d post-MI and blood CD169 + Tim4 + macrophages 10 d post-MI in untreated and T0901317-treated WT and Spx mice. N=5-6/group, statistics: one-way ANOVA, Bonferroni post-test. C . Kaplan-Meier survival curves post-MI in untreated and T0901317-treated WT and Spx mice. Statistical comparisons by log-rank test, group sizes as indicated. D . Representative end-diastolic long-axis 2-dimensional echocardiograms and group data for LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) in the same mouse groups at 10 d post-MI; n=5-10/group, statistics: one-way ANOVA, Bonferroni post-test. E . Top , Kaplan-Meier survival curves over 8 w post-MI in WT mice treated with either vehicle or T0901317 from 1 d before MI to 5 d post-MI (statistical comparison by log-rank test, n=12-17/group as indicated) and group data for LVEF, LVEDV, LVESV, and normalized heart and lung weight at 8 w post-MI; n=8-9/group for echocardiography, n=4-7/group for gravimetry. Statistical comparisons: unpaired t test. HF, heart failure; TL, tibia length; NS, not significant. Bottom Left , Representative Masson’s trichrome staining of LV short-axis sections (2x magnification) and infarct border zone (BZ, scale bar 500 μm), along with quantitation of cardiac fibrosis (BZ and remote zone [RZ], blue staining) in vehicle- and T0901317-treated WT HF mice. Also shown are confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the RZ of hearts from vehicle-and T0901317-treated HF mice (8 w post-MI) and quantitation of iNOS + CD206 + and iNOS − CD206 + macrophages (Mφ) per mm 2 . Double positive (CD206 + iNOS + ) cells appear yellow (arrows). DAPI (blue) was used for nuclear staining. N=4-8/group, statistics: unpaired t test. Bottom Right , Representative FACS contour plots to identify Ly6C hi monocytes in vehicle- and T0901317-treated WT HF mice (8 w post-MI), and corresponding quantitation. N=5-10/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Comparison, Staining

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and characterization of human CD45 + CD14 + HLA- DR + CD64 + CD169 + Tim4 + circulating macrophages from a subject with acute STEMI. The accompanying overlaid contour plot illustrates CD14 + HLA-DR + (red) and CD64 + CD169 + Tim4 + (green) subsets superimposed on all CD45 + leukocytes in an SSC-A versus FSC-A gate. B . Top , FACS gating strategy for sorting CD45 + CD14 + HLA-DR + CD169 + cells from human peripheral blood for further characterization using ImageStream analysis. Bottom , ImageStream visualization of FACS-sorted CD45 + CD169 + blood cells from STEMI patients and control subjects undergoing elective percutaneous coronary intervention (PCI), and group data for size distribution of CD169 + cells; scale bar 10 μm. C . FACS contour plots and quantitation of circulating CD45 + CD14 + HLA-DR + CD64 + CD169 + Tim4 + macrophages in STEMI and control PCI subjects. n=11-14/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Control, Quantitation Assay

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD22 , REA340 , 50 , 130-124-223 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Blood Cancer Discovery

Article Title: Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

doi: 10.1158/2643-3230.BCD-21-0087

Figure Lengend Snippet: Alternative splicing of CD22 in human B-ALL. A, Quantification of LSVs across transcripts encoding major B-cell immunotherapeutic targets from pediatric B-ALL samples from the NCI TARGET consortium. B, CD22 splice graph depicting splicing events across the 14 exons of CD22 in B-ALL, with specific depiction of CD22 Δex5–6 and CD22 Δex2* variants. C, Relative frequencies of reads originating in exon 1 (blue numbers) or terminating in exon 7 (green numbers) in normal B-cell precursors (top) and B-ALL (bottom). D and E, Stack plots depicting relative abundance of CD22 isoforms including/skipping exon 5 and 6 across TARGET dataset ( n = 219) and normal B-cell subtypes ( n = 25, from 11 individuals), respectively. BP, datasets corresponding to B-cell precursors obtained through the BLUEPRINT project; ped, pediatric samples. F and G, Stack plots depicting relative abundance of CD22 Δex2* variants across TARGET dataset and normal B-cell subtypes, respectively. H, RT-PCR analysis validating CD22 isoforms in the Nalm6 cell line. I, ONT-based long-read RNA-seq of CD22 transcripts in cells from a TCF3–HLF B-ALL PDX model (ALL1807). CD22 Δex5–6 and Δex2* variant transcripts are highlighted in yellow and purple, respectively.

Article Snippet: For detection of murine and human proteins, primary human anti-CD22 antibodies (Boster Bio, PB9691; R&D Systems, MAB19681) were used in combination with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology) and Amersham Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Life Sciences).

Techniques: Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing, Variant Assay

Journal: Blood Cancer Discovery

Article Title: Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

doi: 10.1158/2643-3230.BCD-21-0087

Figure Lengend Snippet: Biochemical and functional characterization of the CD22 Δex5–6 isoform. A, RT-PCR detection of CD22 Δex5–6 across 18 diagnostic de novo B-ALL samples driven by various genetic alterations. B, Western blotting using N-terminus–directed anti-CD22 antibody performed on CD22 -deleted OCI-Ly8 cells engineered to express CD22 Δex5–6 or FL isoforms. KO, knockout. C, Amino acid sequence of the peptide used for mAb production. D, RT-PCR detection of CD22 Δex5–6 in OCI-Ly8 cell lines from B , human B-ALL cell lines, and B-ALL PDXs. E, Western blotting using the 11F11 mAb performed on cells from D . F, Flow cytometric detection of CD22 KO, CD22 FL, and CD22 Δex5–6. FSC-A, forward scatter area. G, Western blotting demonstrating electrophoretic mobilities of CD22 isoforms treated with deglycosylating enzymes with or without denaturation. H, Western blotting detecting CD22 and total and phosphorylated (p) BLNK in derivatives from B treated with an anti-IgM antibody for indicated time intervals. I, Quantitation of pBLNK bands from H . The experiment was replicated twice with concordant results. J and K, In vitro killing assays performed on cells from B using HA22- and m971-based CD22 CAR T cells (CART22), respectively ( n = 2 technical replicates). Data in both panels are represented as mean values ± SD error bars.

Article Snippet: For detection of murine and human proteins, primary human anti-CD22 antibodies (Boster Bio, PB9691; R&D Systems, MAB19681) were used in combination with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology) and Amersham Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Life Sciences).

Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Diagnostic Assay, Western Blot, Knock-Out, Sequencing, Quantitation Assay, In Vitro

Journal: Blood Cancer Discovery

Article Title: Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

doi: 10.1158/2643-3230.BCD-21-0087

Figure Lengend Snippet: CD22 protein expression in B-cell malignancies is limited by exon 2 inclusion. A, Flow cytometric detection of exogenously expressed CD22 protein in CD22 -deleted OCI-Ly8 cells using anti-CD22 antibodies directed toward either the extreme N-terminus (S-HCL-1) or the C-terminal region (RFB-4) of the extracellular domain. KO, knockout. B, Western blotting detection of CD22 in the same cell lines and parental controls using anti-CD22 antibodies directed toward the N-terminus (R&D Systems, MAB19681) and the C-terminus (Boster Bio, PB9691). C, Schematic annotating the CD22 exon splice junctions (gray arches) assayed using junction-spanning qPCR primers following treatment with Ex2In2 morpholino. Bottom right, morpholino sequence is shown in red, with complementary exon–intron junction sequence shown in blue/white. D, qRT-PCR detection of various CD22 mRNA isoforms in Reh B-ALL cells transfected for 48 hours with Ex2In2 (10 μmol/L and 100 μmol/L). Constitutive expression of ex13–14 junction was used as a measure of total CD22 expression ( n = 6, 2 independent experiments with 3 technical replicates each). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ns, not significant. E and F, Detection of CD22 protein in the morpholino-treated Reh cells by Western blotting and flow cytometric site density assay, respectively ( n = 2 independent experiments). G, Viability assay performed on CD22 -deleted OCI-Ly8 cells reconstituted with indicated CD22 isoforms and treated for 24 hours with indicated concentrations of inotuzumab ( n = 3 technical replicates). IC 50 95% confidence intervals (CI) were as follows: 54–119 ng/mL for CD22 KO, 25–85 ng/mL for CD22 Δex2, and 5–11 ng/mL for CD22 Δex5–6. H, Bar graph representing IC 50 values from G . P values were determined using an unpaired t test. I, Viability assay performed on Ex2In2-treated (48 hours) and inotuzumab-treated (24 hours) Reh cells ( n = 3 technical replicates). Cell viability was assessed using the WST-1 assay. IC 50 95% CIs were 29–72 ng/mL for Ctrl treatment and 119–333 ng/mL for Ex2In2 treatment. J, Bar graph representing IC 50 values from I on the log scale. P values were determined using an unpaired t test. Data in D , F , H , and J are presented as individual and mean values ± SD error bars. Data in G and I are presented as mean values ± SD error bars.

Article Snippet: For detection of murine and human proteins, primary human anti-CD22 antibodies (Boster Bio, PB9691; R&D Systems, MAB19681) were used in combination with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology) and Amersham Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Life Sciences).

Techniques: Expressing, Knock-Out, Western Blot, Sequencing, Quantitative RT-PCR, Transfection, Viability Assay, WST-1 Assay

Journal: Blood Cancer Discovery

Article Title: Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

doi: 10.1158/2643-3230.BCD-21-0087

Figure Lengend Snippet: CD22 protein expression is limited by exon 2 inclusion in B-ALL cells. A, Correlation analysis of CD22 site density versus CD22 mRNA levels in pretreatment primary B-ALL bone marrow or peripheral blood specimens obtained from children enrolled on the COG AALL1621 phase II clinical trial. CD22 mRNA levels were measured by qRT-PCR using primers specific for the exon 13–14 (left) or exon 1–2 (right) junctions. CD22 expression was normalized to that of β-actin. Regression coefficients and P values are shown for each comparison. B, Relative expression of CD2 2 exon 2–containing and exon 2–skipping splice variants within baseline AALL1621 B-ALL specimens. Each stack plot represents a single patient (designated by the COG unique specimen identifier). Yellow arrow highlights PAYYZW as a sample apparently devoid of protein-coding CD22 mRNA isoforms. The legend shows color-coded CD22 splice variants. C, Flow cytometric quantitation of CD22 molecules in paired pre– and post–inotuzumab treatment (pre-ino/post-ino) bone marrow specimens from AALL1621 patient PAWUXD with multiply relapsed B-ALL. D, CD22 mutational analysis of the paired PAWUXD samples. E, CD22 exon 2 splicing analysis of the paired PAWUXD samples. For color coding, refer to legend in B . F, Flow cytometric quantitation of CD22 molecules in paired pre- and posttreatment (pre-ino/post-ino) bone marrow specimens from AALL1621 patient PAVDRV with multiply relapsed B-ALL. G, CD22 mutational analysis of the paired PAVDRV samples. H, CD22 exon 2 splicing analysis of the paired PAVDRV samples. For color coding, refer to legend in B .

Article Snippet: For detection of murine and human proteins, primary human anti-CD22 antibodies (Boster Bio, PB9691; R&D Systems, MAB19681) were used in combination with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology) and Amersham Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Life Sciences).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Quantitation Assay