Journal: Nature Aging

Article Title: DNA damage in macrophages drives immune autoreactivity via nuclear antigen presentation

doi: 10.1038/s43587-025-01053-3

Figure Lengend Snippet: (A.) Immunofluorescence staining of γ-Η2A.X and 53BP1 in wt untreated, wt LPS-treated (wt LPS ) and Er1 Lyz2/− BMDMs. Cells with over 2 colocalized foci of the two proteins were labeled positive and indicated using white arrows. The percentage of positive cells is plotted. (At least 4 independent optical fields were counted from n = 3 biological replicates.) Single-channel images of Extended Data Fig. 7A are shown in Supplementary file ( B.) Flow cytometry analysis of the MHC-II expression levels in wt untreated, wt LPS and Er1 Lyz2/− BMDMs. Representative histograms and MFIs are plotted (n = 3-5). (C.) Immunoprecipitation and western blot detection of the MHC-II protein in BMDMs using an anti-MHC-II (IP) or isotype control antibody (IgG). (D.) Volcano plot of differentially presented peptides in wt (downregulated, blue) and wt LPS cells (upregulated, red). Log 2 (Fold Change) of -6 or 6 represents peptides uniquely identified in the wt or wt LPS MHC-peptidome. Statistical significance (ANOVA analysis) was set at p-value ≤ 0.05 (horizontal black dashed line) and peptide enrichment at Log 2 (Fold Change)≥0.3 (upregulated in wt LPS ) or Log 2 (Fold Change)≤-0.3 (downregulated in wt LPS ) (vertical black dashed line). Extracellular matrix proteins are labeled with their corresponding gene symbol. (E.) Bubble plot of the Gene Ontology (GO) term enrichment analysis (cellular component, Mann-Whitney U test) of significantly over-represented wt LPS peptides, when compared to wt controls (p-value ≤ 0.05 and Log 2 Fold change≥0.3). The dot size shows the total count of genes per annotated pathway and the blue color scale indicates the statistical significance as per the p-value of the enriched pathways. The x axis indicates the fold enrichment derived from pathway analysis. (F.) IFN-γ ELISpot analysis of splenocytes isolated from either 8-month-old wt or Er1 Lyz2/− mice and pulsed with the indicated peptides. Representative images are shown. Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.

Article Snippet: Antibodies and isotype controls were purchased from BioLegend and Proteintech: anti-CD19 (BioLegend, 152410; clone 1D3/CD19), anti-CD138 (BioLegend, 142503; clone 281-2), anti-CD11b (BioLegend, 101212; clone M1I70), anti-CD4 (BioLegend, 100406, 100412 and 100432; clone GK1.5), anti-CD25 (BioLegend, 102012; clone PC61), anti-FOXP3 (Proteintech, PE-65089; clone 3G3), anti-MHC-II (BioLegend, 107606, 107636 and 107631; clone M5/114.15.2), anti-CD86 (BioLegend, 105026; clone GL-1), anti-Ly6G (BioLegend, 127654 and 127607; clone 1A8), anti-CD62L (BioLegend, 104412; clone MEL-14), anti-CD44 (BioLegend, 103036; clone IM7), anti-T-bet (BioLegend, 644812; clone 4B10), anti-PD-1 (BioLegend, 135214; clone 29F.1A12), anti-CD69 (BioLegend, 104507; clone H1.2F3), anti-F4/80 (BioLegend, 123110; clone BM8), anti-CD115 (BioLegend, 135512; clone AFS98), anti-mouse Lineage Cocktail with Isotype Ctrl (BioLegend, 133305; clones 17A2, RB6-8C5, RA3-6B2, Ter119 and M1/70), anti-mouse CD34 antibody (BioLegend, 119307; clone MEC14.7), anti-mouse Ly-6A/E (Sca-1) (BioLegend, 108123; clone D7), anti-mouse CD16/32 (BioLegend, 101325; clone 93), anti-mouse c-Kit (BioLegend, 105815; clone 2B8), anti-mouse CD170 (Siglec-F) antibody (BioLegend, 155523; clone S17007L), anti-mouse NK-1.1 (BioLegend, 108705; clone PK316), anti-mouse CD3a (Proteintech, PE-65060), anti-mouse FoxP3 (BioLegend, 126409; clone MF-14), i-mouse CD8a (BioLegend, 100712; clone 53-6.7), PerCP rat IgG2a (BioLegend, 400529; clone RTK2758) and FITC rat IgG2a (Proteintech, FITC-65209; clone 2A3) were used as isotype control antibodies.

Techniques: Immunofluorescence, Staining, Labeling, Flow Cytometry, Expressing, Immunoprecipitation, Western Blot, Control, MANN-WHITNEY, Derivative Assay, Enzyme-linked Immunospot, Isolation, Two Tailed Test

Journal: Cancer Immunology Research

Article Title: Tumor-Associated Neutrophils Drive B-cell Recruitment and Their Differentiation to Plasma Cells

doi: 10.1158/2326-6066.cir-20-0839

Figure Lengend Snippet: Figure 5. TANs drive B-cell differentiation independently of T cells. A, Quantification of CD138 expression on splenic B220þ cells following various culture conditions—when cultured alone (B), with T cells (B þ T, ratio 1:1), with TANs (B þ TAN, ratio 1:1), with TAN and T cells (B þ TAN þ T, ratio 1:1:1), with T cells but no contact allowed with TANs (B þT//TAN,ratio 1:1:1), or TANs cocultured with T cells but no contact allowed with B cells (B//T þTAN, ratio 1:1:1). Data represent the mean SEM (n ¼ 6–9; , P < 0.001; n.s., not significant). Groups were compared using one-way ANOVA. B, Representative flow plots displaying CD138 expression out of total B220þ population in the different coculture conditions. C, Quantification of IgG release to the media following overnight coculture of isolated splenic B220þ

Article Snippet: Isotype control antibodies were as follows: APCconjugated rat IgG2a (clone 2A3; Biogems), FITC-conjugated rat IgG2b (clone RTK4530; BioLegend), BGViolet450 rat IgG2a (clone 2A3, Biogems), PE-conjugated rat IgG2a (clone 2A3, Biogems).

Techniques: Cell Differentiation, Expressing, Cell Culture, Isolation

Journal: Cancer Immunology Research

Article Title: Tumor-Associated Neutrophils Drive B-cell Recruitment and Their Differentiation to Plasma Cells

doi: 10.1158/2326-6066.cir-20-0839

Figure Lengend Snippet: Figure 6. TANs express membranal BAFF, but not membranal APRIL, and mediate B-cell IgG production in a BAFF-R–dependent manner. A and B, Expression of membranal BAFF and APRIL in TANs within the whole tumor (A) and following TANs' isolation from the tumors (B). Representative histograms showing BAFF and APRIL expression in TANs (gated as total Ly6Gþ population) are provided (right plots). C, IgG production by splenic B cells cocultured with TANs (ratio 1:5) in the absence or presence of anti–BAFF-R antibody. Data represent the mean SEM (n ¼ 4; , P < 0.01; , P < 0.001). Groups were compared using one-way ANOVA. D, Quantification of CD138 expression on isolated splenic B220þ cells cultured alone or cocultured with TANs (ratio 1:5), without or with blocking of the three potential BAFF receptors, BAFF-R, TACI, or BCMA. Data represent the mean SEM (n ¼ 4–10; , P < 0.001; n.s., nonsignificant). Groups were compared using one-way ANOVA.

Article Snippet: Isotype control antibodies were as follows: APCconjugated rat IgG2a (clone 2A3; Biogems), FITC-conjugated rat IgG2b (clone RTK4530; BioLegend), BGViolet450 rat IgG2a (clone 2A3, Biogems), PE-conjugated rat IgG2a (clone 2A3, Biogems).

Techniques: Expressing, Isolation, Cell Culture, Blocking Assay