invivomab anti mouse mhc class ii  (Bio X Cell)

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 , H&E stain of kidneys isolated from 8-month-old wild-type (WT) and Er1 Lyz2 /− mice ( n = 7−8, P = 0.0040). The magnification in each image is indicated, and black arrows point to large activated lymphocytes and plasma cells (inflammation score: 0: no/focal, 1: moderate, 2: intense). b , Antinuclear autoantibody (ANA) detection in the sera of 8−10-month-old WT and Er1 Lyz2 /− mice by ELISA (n = 8−10, P = 0.0449). Autoantibody patterns of 8-month-old WT and Er1 Lyz2 /− and 24-month-old WT (aged) mice are shown in Extended Data Fig. . c − f ,Flow cytometry analysis of 8-month-old WT and Er1 Lyz2 /− splenocytes stained for plasma cell (CD19, CD138), monocyte-derived cell (CD11b + Ly6G − ), CD4 + T cell (CD4) and regulatory T cell (CD4, CD25, FOXP3) markers. All bar plots show the total number of CD19 + CD138 + plasma cells ( n = 7, P = 0.0201). Myeloid cells ( n = 4−5, P = 0.04) ( c ), CD4 + cells ( n = 9−10, P = 0.0469) ( d ), regulatory T (T reg ) cells ( n = 3, P = 0.0483) ( e ) and natural killer ( f ) and CD8 + T ( g ) cells ( n = 4, P = 0.0004 and P = 0.0395). Representative scatter plots and gating strategies are shown in Extended Data Fig. . h ,Flow cytometry analysis of activated macrophages in spleens of 8-month-old mice using CD11b and Ly6G for gating in cells of monocytic origin (gating strategy in Extended Data Fig. ) and MHC-II and CD86 as antigen presentation markers. Representative scatter plots (top) as well as bar plots (bottom) depict the percentage of MHC-II + CD86 + macrophages. MFIs are shown in Extended Data Fig. ( n = 10, P = 0.0014). i , Graphs showing the percentage of memory CD4 + T cells in the spleens of 2-month-old, 6-month-old and 10-month-old WT and Er1 Lyz2 /− mice as well as 24-month-old (aged) WT mice, quantified by flow cytometry analysis. Representative scatter plots for the activation status of T cells isolated from 10-month-old mice are included in the same figure, and representative plots for cells isolated from 2-month-old and 24-month-old mice are shown in Extended Data Fig. ( n = 3−7, P = 0.0311 and P < 0.0001). j , ELISA analysis for the detection of IFNγ in the supernatants of PMA and ionomycin-stimulated CD4 + T cells ( n = 3, P = 0.0124). k , Bar plot displaying the expression levels of T-bet (MFI) in CD4 + T cells in 8-month-old WT and Er1 Lyz2 /− spleens. A representative histogram plot can be found in Extended Data Fig. ( n = 4−5, P = 0.0182). l , Histogram showing the MFI of PD-1 inhibitory marker in the FOXP3 − CD44 + population of WT and Er1 Lyz2 /− CD4 + T cells in the spleen, at 10 months of age ( n = 4, P = 0.0063). m , Adoptive transfer of WT or Er1 Lyz2 /− BMDMs in young WT mice for a time period of 8−12 weeks, every 2 weeks (experimental scheme shown in top legt). The percentage of activated (CD44 hi CD62L lo ) CD4 + cells in the inguinal lymph nodes (LN) and the levels of antinuclear autoantibodies in the sera of WT mice receiving WT or Er1 Lyz2 /− BMDMs are plotted (bottom left and right, respectively). Plasma cells (CD19 + CD138 + ) in the spleens of WT mice receiving WT or Er1 Lyz2 /− BMDMs are shown in Extended Data Fig. ( n = 5−8, P = 0.0374 and P = 0.0320). Representative scatter plots are shown in Extended Data Fig. . Error bars indicate s.e.m. among replicates. * P ≤ 0.05 and ** P ≤ 0.01 (two-tailed Student’s t -test). NS, not significant.

Article Snippet: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

Techniques: Staining, Isolation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Derivative Assay, Immunopeptidomics, Activation Assay, Expressing, Marker, Adoptive Transfer Assay, Two Tailed Test

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.) Flow cytometry analysis of bone marrows derived from 8-month-old wt and Er1 Lyz2/− animals for the detection of myeloid granulocyte-monocyte progenitors. The scatter plots show the gating sequence and representative images. The bar plot shows the quantification of the Lin − c-kit + Sca-1 − CD34 + CD16/32 + cell population (n = 5). (B.) Gating sequence for the flow cytometry analysis of splenic macrophages and representative plots for the quantification of the MHC-II MFIs (n = 3, pval = 0.0474). (C.) MHC-II (left) and CD86 (right) protein expression levels in wt and Er1 Lyz2/− splenic macrophages. The overlaying histograms represent the fluorescence intensities of wt, Er1 Lyz2/− or isotype (iso) control samples (in the dashed black line, continuous black line and in gray color respectively). Mean fluorescence intensities are plotted in the bar graphs (n = 4-6, pval = 0.0283 and pval = 0.0261). (D.) Numbers of memory CD4 + T cells (CD44 hi CD62L l °) in wt and Er1 Lyz2/− spleens (n = 5, pval = 0.026). (E.) Representative scatter plots and corresponding bar charts depicting the percentage of activated CD4 + T cells (CD44 hi CD62L l °) in 8- and 10-month-old wt and Er1 Lyz2/− inguinal lymph nodes (n = 3, pval=0.031 and pval=0.0276). (F.) Plots showing the percentages of naïve CD4 + T cells (CD44 l °CD62L hi ) in 2- and 10-month-old spleens (n = 4-6, pval = 0.0131). (G.) Representative scatter plots of CD44 and CD62L expression in 2- (young) and 24-month-old (aged) wt spleens (n = 4). (H.) Representative histogram plots of T-bet protein levels in CD4 + T cells after staining for T-bet and CD4 in 10-month-old wt and Er1 Lyz2/− lymph node single cell suspensions (shown in the dashed black line and continuous black line respectively). (I.) Histograms overlaying the expression of ki67 protein in CD4 + CD44 + cells in splenocytes isolated from 10-month-old wt and Er1 Lyz2/− animals and stained for the ki67 proliferation, CD4 T cell and CD44 activation marker (shown in gray and in the continuous black line respectively) (n = 4, pval = 0.0487). Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05 (two-tailed Student’s t-test).

Article Snippet: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

Techniques: Flow Cytometry, Derivative Assay, Sequencing, Expressing, Fluorescence, Control, Staining, Single Cell, Isolation, Activation Assay, Marker, Two Tailed Test

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 analysis for the detection of DNA damage levels in wt untreated, Er1 Lyz2/− untreated and Er1 Lyz2/− anti-oxidant-treated (mito-TEMPO and N -acetylcysteine (NAC)) BMDMs. The percentage of cells with 3 or more γ-H2A.X + 53BP1 + co-localized foci is plotted (n = 3-4, pval = 0.0036, pval = 0.0204 and pval = 0.0093). Single-channel images of Extended Data Fig. 5A are shown in Supplementary file (B.) Immunofluorescence analysis for the detection of 8-oxoguanine (8-oxoG) lesions in wt and Er1 Lyz2/− BMDMs. The plot shows the MFI of 8-oxoG in each condition (n = 4, pval = 0.0086). (C.) Western blot analysis for the quantification of phosphorylated ATM and total ATM levels in whole-cell extracts. Beta tubulin was used for normalization (n = 4-5, pval = 0.0307). (D.) Gating strategy for the flow cytometry analysis of single cell suspensions of BMDMs and MHC-II levels in wt and Er1 Lyz2/− untreated macrophages and Er1 Lyz2/− cells treated with an ATR or a DNA-PK inhibitor (n = 3). (E.) Flow cytometry analysis of the quantification of γ-H2A.X levels in cells of monocytic origin isolated from lupus prone NZB/W 11-month-old mice (SLE) or from their age-matched controls (cont). An isotype control histogram is shown in gray (n = 4, pval = 0.0365). (F.) Quantification of the MHC-II levels in control, lupus prone untreated (SLE) and lupus monocyte-derived cells treated with an ATM kinase inhibitor (SLE+ATMi). The MFI in each corresponding condition is plotted (n = 5-6, pval = 0.0037 and pval = 0.0074). (G.) Histograms showing the expression of MHC-II protein in wt BMDMs treated with camptothecin (wt CPT , left), tunicamycin or underwent nutrient starvation (wt tun and wt starv respectively, right), as indicated, and their corresponding plots for their quantification (n = 3-4, pval = 0.0138). 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: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

Techniques: Immunofluorescence, Western Blot, Flow Cytometry, Single Cell, Isolation, Control, Derivative Assay, Expressing, Two Tailed Test

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 , Flow cytometry analysis of WT and Er1 Lyz2 /− BMDMs and Er1 Lyz2 /− BMDMs treated or not with an ATM inhibitor (ATMi), stained with the antigen presentation protein MHC-II. The histograms show the surface MHC-II expression levels of BMDMs (MFI). Isotype controls are shown in gray. Gating strategy is indicated in Extended Data Fig. ( n = 3, P = 0.0029 and P = 0.0015). b , Western blotting of MHC-II protein levels in whole-cell extracts from WT and Er1 Lyz2 /− DMSO-treated controls (Ctrl) or ATMi-treated or ATR kinase inhibitor (ATRi)-treated BMDMs. Actin was used as a loading control ( n = 4−5, P = 0.0244, P = 0.0441 and P = 0.0133). NEL, normalized expression levels. c , Flow cytometry analysis of the MHC-II expression levels of WT control and WT etoposide (ETO)-treated BMDMs. An overlay of representative histograms is shown, and MFIs are plotted. An isotype control is shown in gray ( n = 3, P = 0.0189). d , e , Co-culture of WT or Er1 Lyz2 /− BMDMs and 10-month-old Er1 Lyz2 /− CD4 + T cells in a 1:1 or 1:4 ratio ( n = 5, P = 0.0316). d , 1:1 ratio. The rectangle gate and the corresponding bar plots mark the percentage of activated CD4 + T cells in culture (CD69 + CD4 + cells). e , 1:4 ratio. ELISpot assay measuring the secretion of T-cell-derived IFNγ. Representative images and corresponding bars in the plot show the number of spots per 2 × 10 5 cells plated. Er1 Lyz2 /− BMDMs were untreated or treated with anti-IFNβ (aIFNβ) or anti-MHC-II (aMHC-II) neutralizing antibodies ( n = 4−5, P = 0.0006, P = 0.0354 and P < 0.0001). f − h , In vivo MHC-II blockade. f , The experimental scheme indicates that mice were treated with an aMHC-II or an isotype control antibody for a total of 6 weeks, receiving intraperitoneal injections once per week. m, months. g , The graph shows the percentage of activated CD4 + T cells (CD44 hi CD62L lo ) in the spleens of WT and Er1 Lyz2 /− mice treated with an aMHC-II or an isotype control antibody ( n = 5, P = 0.0213 and P = 0.0265). Representative plots are shown in Extended Data Fig. . h , ELISA analysis. The plot depicts the secreted IFNγ protein levels in the PMA and ionomycin-stimulated CD4 + T cells isolated from Er1 Lyz2 /− mice receiving either an MHC-II-blocking or an isotype control antibody ( n = 4−5, P = 0.0447). i , Experimental scheme for a 6-week CD4 + T cell depletion in Er1 Lyz2 /− mice. Mice received injections once per week intravenously. j , CD4 + T cell depletion experiment. Representative scatter plots and graphs of the plasma cell percentages and numbers in spleens of Er1 Lyz2 /− mice receiving isotype an isotype control or an anti-CD4 (aCD4) antibody ( n = 5−6, P = 0.0020 and P = 0.0357 for percentages and P = 0.0002 and P = 0.0007 for counts). Error bars indicate s.e.m. among replicates.* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (two-tailed Student’s t -test).

Article Snippet: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

Techniques: Flow Cytometry, Staining, Immunopeptidomics, Expressing, Western Blot, Control, Co-Culture Assay, Enzyme-linked Immunospot, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Blocking Assay, Clinical Proteomics, Two Tailed Test

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 detection of the colocalized DNA damage markers γ-H2A.X and 53BP1 in wt untreated and wt etoposide-treated BMDMs. The percentage of positive cells for over 2 colocalized γ-H2A.X + 53BP1 + foci/cell is plotted. (At least 4 independent optical fields were counted from n = 3 biological replicates, pval = 0.0018) Single-channel images of Extended Data Fig. 6A are shown in Supplementary file (B.) Quantitation of the mRNA levels of chemokine genes in Er1 Lyz2/− BMDMs, as shown. The mRNA levels of these genes in wt BMDMs are indicated with the red dotted line (n = 3-6, exact pvalue provided in Source Data file). (C.) (Top) Western blotting for the detection of interferon beta (IFN-β) protein levels secreted in the supernatants of wt and Er1 Lyz2/− BMDMs. The splice point is indicated by a vertical black line. Equal volumes of concentrated supernatants were loaded on the gel. For the normalization of the secreted protein levels, equal volumes of cells lysed with RIPA buffer were loaded and the membranes were probed with beta tubulin. (Bottom) Blots for the quantification of interferon regulatory factor 5 (IRF5) and phosphorylated signal transducer and activator of transcription 1 (pSTAT1) versus total STAT1 protein levels. Actin was used for normalization (n = 3-4, pval = 0.0011 for IFN-β, pval= 0.009 for STAT1, pval = 0.048948 for IRF5). (D.) Western blot analysis of ERCC1 protein levels in CD4 + T cells purified from wt or Er1 Lyz2/− mice at the age of 10 months (n = 3). (E.) Gating strategy for the flow cytometry analysis of CD4 + T cells in a BMDM-CD4 + T-cell co-culture. (F-G.) Flow cytometry analysis of splenocytes isolated from wt and Er1 Lyz2/− anti-MHC-II-treated mice and from wt and Er1 Lyz2/− isotype control-treated mice and stained for (F.) the CD44 and CD62L activation T-cell markers and (G.) the CD11b myeloid cell marker and MHC-II. MHC-II levels exhibit a drop in the CD11b + population (n = 3, pval = 0,0384). (H.) Immunofluorescence analysis of C3 complement protein localized in the kidney glomeruli of Er1 Lyz2/− isotype control-treated and Er1 Lyz2/− anti-MHC-II-treated animals. Representative images and plots with the MFI quantification of C3 are shown (n = 3, pval = 0.0343). (I.) H&E staining of kidneys derived from Er1 Lyz2/− anti-CD4-and isotype control-treated animals. The arrows point to inflammatory foci (n = 6-8, pval = 0,0452). Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.

Article Snippet: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

Techniques: Immunofluorescence, Quantitation Assay, Western Blot, Purification, Flow Cytometry, Co-Culture Assay, Isolation, Control, Staining, Activation Assay, Marker, Derivative Assay, Two Tailed Test

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: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

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

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 , Experimental outline for the preparation and mass spectrometry analysis of MHC-II-bound peptides from WT and Er1 Lyz2 /− untreated and LPS-treated BMDMs. IP, immunoprecipitation. Created in BioRender. Arvanitaki, E. (2025) https://BioRender.com/m68w132 . b , Histogram displaying the range of the peptide amino acid (aa) length. All peptides from all samples (WT, Er1 Lyz2 /− and LPS-treated WT) were used for this graph. c ,Gibbs clustering depicting the predominant MHC-II core amino acids. The peptides that were classified as low or high binders of MHC-II from all samples were included for this analysis. NetMHCIIpan (version 4.3; H-2-IAb and H-2-IAq alleles) was used for the identification of peptides with any binding affinity. d , Volcano plot of differentially presented peptides in WT (downregulated, blue) and Er1 Lyz2 /− (upregulated, red) cells. log 2 (fold change) of −6 or +6 represents peptides uniquely identified in the WT or Er1 Lyz2 /− MHC ligandome. Statistical significance was set at P ≤ 0.05 (ANOVA analysis) (horizontal black dashed line) and peptide enrichment at log 2 (fold change) ≥ 0.3 (upregulated in Er1 Lyz2 /− ) or log 2 (fold change) ≤ −0.3 (downregulated in Er1 Lyz2 /− ) (vertical black dashed line). Nuclear and ribosomal as well as uniquely identified proteins are labeled with their corresponding gene symbol. e , Bar charts showing the intracellular origin of peptide antigens significantly enriched in the WT, Er1 Lyz2 /− and LPS-treated WT samples. WT overrepresented peptides were compared to Er1 Lyz2 /− ones, whereas Er1 Lyz2 /− and LPS-treated WT enriched peptide sequences were both compared to WT controls. Bold numbers correspond to the percentages of peptides derived from nuclear proteins. ECM, extracellular matrix. f , Venn diagram for the comparison of Er1 Lyz2 /− overrepresented peptides with the previously characterized peptides in the IEDB. g , Bubble plot of the Gene Ontology (GO) term enrichment analysis (Cellular Component, Mann−Whitney U -test) of significantly overrepresented Er1 Lyz2 /− peptides, when compared to WT controls ( P ≤ 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. h , IFNγ ELISpot analysis of splenocytes isolated from either 8-month-old WT or Er1 Lyz2 /− mice and pulsed with peptides derived from the indicated source proteins in the presence of IL-2. Representative images are shown in this figure or in Extended Data Fig. . Er1 Lyz2 /− unpulsed, IL-2-exposed splenocytes were used as a negative control (red dotted line) ( n = 4; exact P value is provided in the Source Data file). Error bars indicate s.e.m. among replicates. * P ≤ 0.05 and ** P ≤ 0.01 (two-tailed Student’s t -test).

Article Snippet: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

Techniques: Mass Spectrometry, Immunoprecipitation, Binding Assay, Labeling, Derivative Assay, Comparison, MANN-WHITNEY, Enzyme-linked Immunospot, Isolation, Negative Control, Two Tailed Test

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 early endosome antigen 1 (EEA1) in Er1 Lyz2/− untreated and dynasore-treated BMDMs. White insets indicate a higher magnification on the right (n = 3). (B.) Single- or two-channel images and higher magnifications of Fig. . Magenta arrowheads point to H1 + p62 + LAMP-1 + foci. White arrows point to cytoplasmic chromatin fragments. (C.) Flow cytometry analysis of wt and Er1 Lyz2/− BMDMs stained with annexin V and propidium iodide (PI) for the detection of cell death. Scatter plots are and the percentages of annexin − PI − cells are shown (n = 4). (D.) LAMP-1 protein levels in equally loaded lysosomal extracts isolated from wt and Er1 Lyz2/− BMDMs. Ponceau stain was used for normalization. (n = 4, pval=0.0147) (E.) Quantification of MHC-II in untreated and etoposide-treated THP-1 human monocyte cells. An isotype control histogram is shown in gray (n = 4, pval=0.0006). Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.

Article Snippet: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Isolation, Control, Two Tailed Test

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 , Venn diagram showing the comparative analysis of the lysosomal proteome derived from palbociciclib-induced senescent SK-MEL-103 cells (blue) and of the MHC-II-bound peptidome of Er1 Lyz2 /− BMDMs (green). The numbers indicate the number of proteins in each dataset. b , γH2AX levels in aged monocytes or macrophages. Isotype (Iso.) controls are shown in gray. (i) Flow cytometry analysis of cells isolated from bone marrow of young (2 month (m)) and aged (24 month) mice, stained for Ly6G, CD11b, CD115 surface and γΗ2ΑΧ DNA damage markers. Representative histogram overlay of γH2AX in bone marrow monocytes (Ly6G − CD11b + CD115 + cells) and representative bar plot of the γH2AX MFI ( n = 3−4). (ii) Flow cytometry analysis of splenocytes isolated from young and aged mice, stained for Ly6G, CD11b and γΗ2ΑΧ. A representative histogram overlay of γH2AX levels in cells of monocytic origin (Ly6G − CD11b + cells), with the black bisector gate indicating the γH2AX + population. The bar plot indicates the corresponding percentages of γH2AX + cells ( n = 4). (iii) Immunofluorescence staining of TEMs isolated from young and aged peritonea using a γH2AX antibody. Representative images and quantification of the percentage of γH2AX + cells are shown (5−10 optical fields were counted) ( n = 6). c , Representative images of the SA-β-gal assay using young and aged TEMs. The percentage of β-gal + cells is plotted (three independent optical fields, and over 360 cells were counted per biological replicate) ( n = 4, P < 0.0001). d , Representative images of the immunofluorescence detection of H1, p62 and LAMP-1 in young and aged TEMs. The white arrow points to a cytoplasmic structure stained positive for DAPI and all three proteins. The graphs depict the percentage of cells with chromatin fragments (top, DAPI + H1 + ) and of cells with H1 + p62 + LAMP-1 + foci (4−7 independent optical fields, and n > 67 cells were counted, respectively) ( n = 3−5) P = 0.0093 and P = 0.0133. Single-channel images of d are shown in Supplementary Fig. . e , Western blot analysis of lysosomal preparations derived from young and aged TEMs, treated or not with 3-MA, as indicated. All were treated with chloroquine for the inhibition of lysosomal cargo degradation. LAMP-1 protein levels are shown as a positive control and Ponceau stain as a loading control. In each case, 7 μg of protein was loaded. A quantitative analysis is presented in the bar chart ( n = 3−5). The splice point is indicated by a vertical black line. Error bars indicate s.e.m. among replicates. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (two-tailed Student’s t -test). Scale bars, 10 μm. Exact P values are provided in the .

Article Snippet: Native MHC-II−peptide complexes were purified using InVivoMAb anti-mouse MHC-II (I-A/I-E, M5/114 monoclonal antibody by Bio X Cell) along with Protein G Sepharose beads (Millipore).

Techniques: Derivative Assay, Flow Cytometry, Isolation, Staining, Immunofluorescence, Western Blot, Inhibition, Positive Control, Control, Two Tailed Test