Journal: Nature Communications

Article Title: An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses

doi: 10.1038/s41467-024-49825-6

Figure Lengend Snippet: a Representative H&E and IF staining of cytokeratin 19 (CK19), F4/80 + macrophages and CD8 + T cells for the corresponding 4T1 tumor tissues after different treatments. Scale bars are marked in the figures. b Representative flow cytometry plots depicting CD45 + CD11b + F4/80 + macrophages phagocytosis, CD11b + F4/80 + CD206 hi macrophages, CD11b + F4/80 + CD86 hi macrophages, CD45 + CD3 + CD4 + /CD8 + T cells, and CD45 + CD3 + CD4 + Foxp3 + T cells in 4T1 tumors after different treatments. c – g Quantification analysis of CD45 + CD11b + F4/80 + macrophages phagocytosis, CD11b + F4/80 + CD206 hi macrophages, CD11b + F4/80 + CD86 hi macrophages, CD45 + CD3 + CD4 + /CD8 + T cells, and CD45 + CD3 + CD4 + Foxp3 + T cells in 4T1 tumors after different treatments. The error bars represent the mean ± SD ( n = 4 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; the p value was analyzed by a two-tailed unpaired Student’s t test). h Identification of differentially expressed genes in 4T1 tumors after treating with IgG Control or PAC-SABIs ( n = 5 mice). i Number of differential genes enriched in GO term of 4T1 tumors treated with PAC-SABIs versus IgG Control ( n = 5 mice). j GO enrichment analysis of the differential pathways in 4T1 tumors treated with PAC-SABIs versus IgG Control ( n = 5 mice). k Volcano plot for the transcriptome sequencing of 4T1 tumors treated with PAC-SABIs versus IgG Control. Differentially expressed genes were calculated using a two-sided limma moderated t -test with Benjamini–Hochberg correction for multiple testing ( n = 5 mice). Source data are provided as a Source Data file.

Article Snippet: Flow cytometry: PerCP/Cy5.5 anti-mouse CD45 antibody (1:200, Elabscience, #E-AB-F1136J, clone 30-F11), PE/Cyanine5 anti-mouse CD11b antibody (1:200, Biolegend, #101209, clone M1/70), APC anti-mouse F4/80 antibody (1:200, Biolegend, #123116, clone BM8), FITC anti-mouse CD206 antibody (1:200, Biolegend, #141703, clone C068C2), FITC anti-mouse CD86 antibody (1:200, Biolegend, #159219, clone A17199A), FITC anti-mouse CD3 antibody (1:200, BDbioscience, #555274, clone 17A2), APC anti-mouse CD4 antibody (1:200, BDbioscience, #553051, clone RM4-5), PE anti-mouse CD8a antibody (1:200, BDbioscience, #553033, clone 53-6.7), PE anti-mouse Foxp3 antibody (1:200, BDbioscience, #563101, clone R16-715).

Techniques: Staining, Flow Cytometry, Two Tailed Test, Control, Sequencing

Journal: Nature Communications

Article Title: An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses

doi: 10.1038/s41467-024-49825-6

Figure Lengend Snippet: a Scheme of the combination therapeutic strategy. Figure was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 international license. b Spider plots of individual tumor growth curves for 4T1-tumor-bearing BALB/c mice in each treatment group. c Average tumor growth curve of 4T1-tumor-bearing BALB/c mice in each treatment group. The error bars represent the mean ± SD ( n = 7 mice; **** p < 0.0001; the p value was analyzed by a two-tailed unpaired Student’s t test). d Kaplan–Meier survival curves of 4T1 tumor-bearing BALB/c mice treated with the indicated formulation. ( n = 7 mice; ** p < 0.01, *** p < 0.001; the p value was analyzed by log-rank test). e Representative H&E staining of lungs harvested from the indicated treatment group. Scale bar: 100 μm. Three independent experiments were performed. f Quantitative analysis of phagocytosis in CD45 + CD11b + F4/80 + macrophages flow cytometry results. The error bars represent the mean ± SD ( n = 4 independent experiments; *** p < 0.001, **** p < 0.0001; the p value was analyzed by a two-tailed unpaired Student’s t test). g Representative IF images of tumor-infiltrated CD8 + T cells. Scale bar: 100 μm. Three independent experiments were performed. Source data are provided as a Source Data file.

Article Snippet: Flow cytometry: PerCP/Cy5.5 anti-mouse CD45 antibody (1:200, Elabscience, #E-AB-F1136J, clone 30-F11), PE/Cyanine5 anti-mouse CD11b antibody (1:200, Biolegend, #101209, clone M1/70), APC anti-mouse F4/80 antibody (1:200, Biolegend, #123116, clone BM8), FITC anti-mouse CD206 antibody (1:200, Biolegend, #141703, clone C068C2), FITC anti-mouse CD86 antibody (1:200, Biolegend, #159219, clone A17199A), FITC anti-mouse CD3 antibody (1:200, BDbioscience, #555274, clone 17A2), APC anti-mouse CD4 antibody (1:200, BDbioscience, #553051, clone RM4-5), PE anti-mouse CD8a antibody (1:200, BDbioscience, #553033, clone 53-6.7), PE anti-mouse Foxp3 antibody (1:200, BDbioscience, #563101, clone R16-715).

Techniques: Two Tailed Test, Formulation, Staining, Flow Cytometry

Journal: JCI insight

Article Title: IRF5 genetic risk variants drive myeloid-specific IRF5 hyperactivation and presymptomatic SLE.

doi: 10.1172/jci.insight.124020

Figure Lengend Snippet: Figure 2. Plasma cells are elevated in the circulation of IRF5 homozygous risk donors. (A–C) Freshly isolated PBMCs were surface-stained and plasma cells gated as CD45+CD19+IgD–CD38+ (PB, plasmablasts). (A) Representa- tive dot plots from flow cytometry are shown from a single round of blood draws from independent homozygous risk (n = 5) and nonrisk (n = 5) donors. A is pregated for CD45 and CD19. (B) The number of circulating PBs from n = 12 risk and n = 11 nonrisk donors is shown as a percentage of the CD19+ gate (unpaired 2-tailed t test). (C) Similar to B except percentage of PBs from homozygous nonrisk, risk, and representative mixed haplotypes from Figure 1A are plotted together (1-way ANOVA with Bonferroni’s multiple-comparisons test using risk [E/E] as control group and excluding groups A/E B/C and E/J because of insufficient sample size. (D) Isolated naive B cells from homozy- gous nonrisk and risk donors were in vitro cultured for 7 days with 150 ng/mL CD40L alone or with 100 ng/mL IL-21, 10 μg/mL anti-IgM antibody, and 2.5 μg/mL CpG-B to drive PB differentiation. Representative dot plots from flow cytometry analysis of a single matched nonrisk and risk donor after 7 days of culture. (E) Summarized data from D of n = 4 risk and nonrisk donors are shown (unpaired 2-tailed t test). Experiments were repeated 4 or more times (A and B), were performed once (C), or were repeated twice (D and E). Single data points represent individual donors. Data are presented as mean ± SEM. ; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: PBMCs were washed and blocked in PBS supplemented with Fc blocker (422302, BioLegend) for 15 minutes and then stained with antibodies against surface makers for 1 hour (BioLegend: CD303-BV421 354212, CD123-BV510 306022, CD14-PE 301806, CD16-APC Cy7 360710, CD40-PE 334308, CD45-PerCP 304026, CD38-BV711 303528, IgD-APC 348222, CD27-BV421 356418, CD19BV510 302242, CD20-BV711 302342, PE secondary 406421; BD Biosciences: CD24-PerCP-Cy5.5 561647; Abcam: cit-H3 ab5103; Novus: MPO-PerCP NB100-64803PCP; eBioscience: CD66b-APC 1706662; 1 5insight.jci.org https://doi.org/10.1172/jci.insight.124020 Molecular Probes, Thermo Fisher Scientific: LIVE/DEAD fixable green stain L23101).

Techniques: Clinical Proteomics, Isolation, Staining, Flow Cytometry, Control, In Vitro, Cell Culture

Journal: JCI insight

Article Title: IRF5 genetic risk variants drive myeloid-specific IRF5 hyperactivation and presymptomatic SLE.

doi: 10.1172/jci.insight.124020

Figure Lengend Snippet: Figure 4. IRF5 homozygous risk donors have elevated numbers of circulating pDCs and spontaneous NETosis. (A and B) Similar to Figure 2, A and B, except freshly isolated PBMCs were surface-stained and pDCs gated as CD45+CD123+BDCA2+. (A) Representative dot plots from flow cytometry are shown from a single round of blood draws. A is pregated for CD45. (B) The number of circulating pDCs from n = 10 risk and n = 12 nonrisk donors is shown as a percentage of CD45+ gate (unpaired 2-tailed t test). (C) Representative dot plots from flow cytometry analysis of myeloperoxidase-positive, citrullinated histone H3–positive (MPO+cit-H3+) NETs in donor samples (n = 5 risk and nonrisk). C is pregated on CD66b+ cells. (D) Quantification of NETs from n = 12 risk and n = 14 nonrisk donors is shown as a percentage from CD66b+ cells (1-way ANOVA with Tukey’s multiple-comparisons test). (E) The presence of NETs was visualized by plating equal numbers of freshly isolated neutrophils from homozygous nonrisk, risk, and patients with SLE on poly-l-lysine–coated coverslips for 4 hours. Representative images are from stain- ing with Sytox green and DAPI or MPO, Cit-H3, and DAPI. PMA was used as a positive control for NET induction on nonrisk neutrophils (original magnification ×20). Experiments were repeated 3 times (A–E). Single data points represent individual donors. Data are presented as mean ± SEM. *P ≤ 0.05.

Article Snippet: PBMCs were washed and blocked in PBS supplemented with Fc blocker (422302, BioLegend) for 15 minutes and then stained with antibodies against surface makers for 1 hour (BioLegend: CD303-BV421 354212, CD123-BV510 306022, CD14-PE 301806, CD16-APC Cy7 360710, CD40-PE 334308, CD45-PerCP 304026, CD38-BV711 303528, IgD-APC 348222, CD27-BV421 356418, CD19BV510 302242, CD20-BV711 302342, PE secondary 406421; BD Biosciences: CD24-PerCP-Cy5.5 561647; Abcam: cit-H3 ab5103; Novus: MPO-PerCP NB100-64803PCP; eBioscience: CD66b-APC 1706662; 1 5insight.jci.org https://doi.org/10.1172/jci.insight.124020 Molecular Probes, Thermo Fisher Scientific: LIVE/DEAD fixable green stain L23101).

Techniques: Isolation, Staining, Flow Cytometry, Positive Control