Journal: bioRxiv

Article Title: Imaging the mechanisms of anti-CD20 therapy in vivo uncovers

doi: 10.1101/2020.05.26.116806

Figure Lengend Snippet: ( A ) Experimental set-up. Eµ-Myc lymphomas were established by intravenous injection of 1×10 6 fluorescent-CD20 + tumor cells in C57BL/6 mice. Recipient mice were analyzed three to four weeks later. Fluorescent CD20-expressing tumors cells were generated by retroviral transduction of B lymphoma cells isolated from Eµ-myc transgenic mice. ( B ) Representative FACS plots showing cells from the bone marrow and the blood of a tumor-bearing mouse gated on CD45.2 + cells. Data are representative of 4 independent experiments with n=12 mice analyzed. ( C ) Representative 2-photon time-lapse images of the liver and the bone marrow of tumor-bearing mice. Images illustrate that circulating tumors (white arrows) are detected in the liver sinusoids but that tumor cells stably reside in the bone marrow. Tumor cells appear in magenta and F4/80 + macrophages in green. Scale bar, 20µm. Representative of 3 and 5 independent experiments for liver and bone marrow imaging, respectively ( D-E ) Different outcomes for tumor-bearing mice treated early or late with anti-CD20 mAb. ( D ) Schematic of the survival study and the kinetics of treatments. ( E ) Survival curve for mice treated early or late with anti-CD20 Ab or left untreated. Results are compiled from 2 independent experiments with 8 to 17 mice per group. Log-rank test was used for statistical analysis. ***, p<0.001; **, p<0.01; *, p<0.5.

Article Snippet: Stainings were performed with the following mAb: CD45.2-BUV737 (Clone 104, BD), Ly6G-BUV395 (Clone 1A8, BD), Ly6C-BV785 (Clone HK1.4, BioLegend), CD11b-PerCp5.5 (Clone M1/70, BioLegend), F4/80-AlexaFluor594 (BM8, BioLegend), F4/80-APC (BM8, BioLegend), F4/80-APCCy7 (BM8, Biolegend), mCD20-Alexa647 (Clone SA275A11, BioLegend).

Techniques: Injection, Expressing, Generated, Transduction, Isolation, Transgenic Assay, Stable Transfection, Imaging

Journal: bioRxiv

Article Title: Imaging the mechanisms of anti-CD20 therapy in vivo uncovers

doi: 10.1101/2020.05.26.116806

Figure Lengend Snippet: ( A ) Representative two-photon image of the bone marrow comparing macrophage density in tumor-rich area (zone 1) and a tumor-free zone (zone 2) within the same imaging field. Scale bar, 100µm; inset scale bar, 50µm. ( B ) The density of bone-marrow associated macrophages in tumor-bearing or tumor-free mouse is shown by immunofluorescence on frozen bone-marrow sections ( C ) Bar graph showing the percentage of macrophages (CD11b + Ly6G - Ly6C low F4/80 + cells) in the bone-marrow within total CD45.2 + cells or within YFP - cells (excluding tumor cells). Representative of two independent experiments. A Mann-Whitney statistical test was performed for statistical significance. **, p<0.01; *, p<0.5. ( D ) Bone marrow-associated macrophages are mostly sessile. Representative time lapse images of F4/80 + macrophages in the bone-marrow. A color was attributed to each time point and overlaid to visualize the migratory phenotype of macrophages. Superimposed signals appear in white and reflect the lack of macrophage motility ( E-F ) Quantification of the distance of macrophage survey. ( E ) Schematic illustrating the distance separating the targeted tumor cell from the macrophage center and ( F ) compilation of distances recorded for individual ADP events.

Article Snippet: Stainings were performed with the following mAb: CD45.2-BUV737 (Clone 104, BD), Ly6G-BUV395 (Clone 1A8, BD), Ly6C-BV785 (Clone HK1.4, BioLegend), CD11b-PerCp5.5 (Clone M1/70, BioLegend), F4/80-AlexaFluor594 (BM8, BioLegend), F4/80-APC (BM8, BioLegend), F4/80-APCCy7 (BM8, Biolegend), mCD20-Alexa647 (Clone SA275A11, BioLegend).

Techniques: Imaging, Immunofluorescence, MANN-WHITNEY

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c + F4/80 + Macrophages through IL-1R8 Regulation

doi: 10.1681/ASN.2019080778

Figure Lengend Snippet: Phenotype and immunostimulatory activity of renal mononuclear phagocytes. (A) Representative contour plot for CD11c (APC-Cy7) and F4/80 (APC) expression on pregated CD45+ renal single cells. (B) Representative contour plots for IRF4 (FITC) and IRF8 (PE) staining on pregated CD11c−F4/80+CD45+, CD11c+F4/80+CD45+, and CD11c+F4/80−CD45+ renal single cells. (C) Frequency of IFN-γ+ and IL-17+ alloreactive Balb/c T cells by ELISpot after 2 days of exposure to CD11c−F4/80+, CD11c+F4/80+, or CD11c+F4/80− cells isolated from C57/BL6 kidneys. Mean±SD of three replicates from one representative experiment, *P<0.05. (D) Frequency of IFN-γ+ and IL-17+ alloreactive Balb/c T cells by ELISpot after 2 days of exposure to renal CD11c+F4/80+ cells either unstimulated or LPS-stimulated; *P<0.05. (E and F) Frequency of IFN-γ+ (E) and IL-17+ (F) OVA-specific OT2-CD4+ (TH1 or TH17) or OT1-CD8+ (TC1 or TC17) T cell clones detected by ELISpot after 2 days of exposure to renal CD11c+F4/80+ cells (OVA-transgenic or wild-type). Wild-type renal CD11c+F4/80+ cells were exposed or not to OVA, either in vitro (100 μg/ml) during MLR or in vivo by intravenous infusion (1mg/500 μl per mouse). OVA-exposed (both in vitro and in vivo) renal CD11c+F4/80+ cells were either stimulated with LPS or left unstimulated during the MLR. Values are mean±SD, n=3–4 independent experiments for each condition; *P<0.05. ND, not detectable.

Article Snippet: Total cells obtained after kidney digestion or microbead-enriched cells, as appropriate, were resuspended in FACS buffer (PBS/FBS 2%) and stained for 30 minutes with fluorochrome-conjugated anti-CD45.1 PE (A20; BD Biosciences), anti-CD45.1 AF488 (A20; BioLegend), anti-CD45.2 PE (104; BioLegend), anti-CD45.2 FITC (104; BD Biosciences), anti-CD45.2 BV510 (104; BD Biosciences), anti-F4/80 APC (CI:A3–1; Bio-Rad), anti-CD11c APC-Cy7 (N418; Tonbo), anti-class II MHC I-A/I-E FITC (2G9; BD Biosciences), anti–Ki-67 PE (SolA15; Thermo Fisher), anti-CD206 BV421 (CO68CZ; BD Biosciences), or anti-CD38 FITC (90; eBioscience).

Techniques: Activity Assay, Expressing, Staining, Enzyme-linked Immunospot, Isolation, Clone Assay, Transgenic Assay, In Vitro, In Vivo

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c + F4/80 + Macrophages through IL-1R8 Regulation

doi: 10.1681/ASN.2019080778

Figure Lengend Snippet: CI injury modifies the phenotype and the immunostimulatory activity of renal CD11c+F4/80+ MΦs. (A) Cell counts of CD11c+F4/80+CD45+, CD11c+F4/80−CD45+, and CD11c−F4/80+CD45+ renal single cells from kidneys subjected or not to 16 hours of CI. Mean±SD, n=4 independent experiments; *P<0.05. (B) Log-fold change of median fluorescence intensity (MFI) of different markers on CD11c+F4/80+ rMΦs from kidneys subjected to CI compared with nonischemic kidneys. Mean±SD, n=3–4 independent experiments for each molecule; *P<0.05 versus pre-CI MFI. (C) T cell proliferation (3H-thymidine incorporation) of alloreactive Balb/c T cells after 2 days of exposure to CD11c+F4/80+ rMΦs isolated from kidneys subjected or not to 16 hours of CI. Values are the three replicates of a representative experiment; *P<0.05. (D) Representative images and results summary of IFN-γ+ and IL-17+ alloreactive Balb/c T cell frequency assessed by ELISpot after 2 days of exposure to renal CD11c+F4/80+ rMΦs isolated from kidneys subjected or not to 16 hours CI and used either unstimulated or LPS-stimulated. Mean±SD, n=7 independent experiments for IFN-γ+ and n=4 independent experiments for IL-17+; *P<0.05. (E and F) Frequency of IFN-γ+ (E) and IL-17+ (F) OVA-specific OT2-CD4+ or OT1-CD8+ T cells by ELISpot after 2 days of exposure to CD11c+F4/80+ rMΦs isolated from kidneys subjected or not to 16 hours CI. Renal CD11c+F4/80+ rMΦs were exposed to OVA in vivo by intravenous infusion and used either unstimulated or LPS-stimulated in vitro during MLR. Mean±SD, n=3–5 independent experiments for each condition; *P<0.05.

Article Snippet: Total cells obtained after kidney digestion or microbead-enriched cells, as appropriate, were resuspended in FACS buffer (PBS/FBS 2%) and stained for 30 minutes with fluorochrome-conjugated anti-CD45.1 PE (A20; BD Biosciences), anti-CD45.1 AF488 (A20; BioLegend), anti-CD45.2 PE (104; BioLegend), anti-CD45.2 FITC (104; BD Biosciences), anti-CD45.2 BV510 (104; BD Biosciences), anti-F4/80 APC (CI:A3–1; Bio-Rad), anti-CD11c APC-Cy7 (N418; Tonbo), anti-class II MHC I-A/I-E FITC (2G9; BD Biosciences), anti–Ki-67 PE (SolA15; Thermo Fisher), anti-CD206 BV421 (CO68CZ; BD Biosciences), or anti-CD38 FITC (90; eBioscience).

Techniques: Activity Assay, Fluorescence, Isolation, Enzyme-linked Immunospot, In Vivo, In Vitro

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c + F4/80 + Macrophages through IL-1R8 Regulation

doi: 10.1681/ASN.2019080778

Figure Lengend Snippet: Donor CD11c+F4/80+ rMΦs cell count and phenotype in transplantation-induced I/RI. (A and B) Cell counts of total donor cells (CD45.2+), donor rMΦs (CD45.2+CD11c+F4/80+), total recipient cells (CD45.1+), and recipient rMΦs (CD45.1+CD11c+F4/80+) in wild-type (IL-1R8+/+) and IL-1R8−/− kidney grafts pretransplant (pre-tx) and at days 1, 3–4, and 7–10 post-transplant. Mean±SD, n=3–7 mice at each time point; *P<0.05 versus pre-tx, ND: not detectable. (C) Percentage of proliferating (Ki67+) cells on donor rMΦs in wild-type kidney grafts pretransplant and at days 1–3 and 7–10 post-transplant. Mean±SD, n=2–3 mice at each time point; *P<0.05 versus pre-tx. (D and E) Post-transplant log-fold change in median fluorescence intensity (MFI) of selected molecules in donor rMΦs compared with pretransplant values, both in wild-type (IL-1R8+/+) and IL-1R8−/− kidney grafts at days 1–3 (D) and 3–7 (E) post-transplantation. Mean±SD, n=2–6 mice for each molecule at each time point; *P<0.05. (F) iNOS, TNFα, and Ym1 mRNA expression in wild-type (IL-1R8+/+) and IL-1R8−/− kidney grafts at day 7 post-transplant. Mean±SD, n=3–4 mice; *P<0.05. (G) Semiquantitative scores (0–3) and representative images of nitro-tyrosine staining in wild-type (IL-1R8+/+) and IL-1R8−/− kidney grafts at day 7 post-transplant. *P<0.05. Original magnification, ×400.

Article Snippet: Total cells obtained after kidney digestion or microbead-enriched cells, as appropriate, were resuspended in FACS buffer (PBS/FBS 2%) and stained for 30 minutes with fluorochrome-conjugated anti-CD45.1 PE (A20; BD Biosciences), anti-CD45.1 AF488 (A20; BioLegend), anti-CD45.2 PE (104; BioLegend), anti-CD45.2 FITC (104; BD Biosciences), anti-CD45.2 BV510 (104; BD Biosciences), anti-F4/80 APC (CI:A3–1; Bio-Rad), anti-CD11c APC-Cy7 (N418; Tonbo), anti-class II MHC I-A/I-E FITC (2G9; BD Biosciences), anti–Ki-67 PE (SolA15; Thermo Fisher), anti-CD206 BV421 (CO68CZ; BD Biosciences), or anti-CD38 FITC (90; eBioscience).

Techniques: Cell Counting, Transplantation Assay, Fluorescence, Expressing, Staining

Journal: Science Advances

Article Title: Shifting IRES versus Cap-initiated translation during homeostatic stem cell differentiation and stress

doi: 10.1126/sciadv.adz7896

Figure Lengend Snippet: ( A ) Representative gating strategy for sorting low, mid, and high IRES/Cap SLAM-LKS. ( B and C ) Colony formation (B) and size (C, mm 2 ) of Translator SLAM-LKS sorted on the basis of IRES/Cap ( n = 6). ( D and E ) Colony formation (D) and size (E, mm 2 ) of Translator GMP sorted on the basis of IRES/Cap ( n = 9). ( F ) Total protein synthesis rates of Translator GMP based on IRES/Cap measured by AF647-OPP MFI ( n = 3). ( G ) Colony formation of EMCV-IRES reporter-transduced CD34+CD90+EPCR+CD45RA− human cord blood sorted on the basis of IRES/Cap ( n = 3). ( H and I ) Colony formation of SLAM-LKS (H) and GMPs (I) based on RUNX1-IRES/Cap sorted from transplant recipients ( n = 3 and 2, respectively). ( J ) Primary competitive transplant schema. Twenty CD45.2 SLAM-LKS sorted on the basis of IRES/Cap versus 200,000 CD45.1 whole BM cells in lethally irradiated CD45.1 recipients. Peripheral blood chimerism analyzed every four weeks through week 16. Created in BioRender. Li, D. (2026) https://BioRender.com/d7k2abt . ( K ) Total chimerism Translator SLAM-LKS based on IRES/Cap in primary recipients evaluated through week 16 ( n = 12). ( L ) Secondary competitive transplant schema. One million total BM cells were collected from primary recipients (16 weeks posttransplantation) and transplanted to lethally irradiated CD45.1 recipients. Created in BioRender. Li, D. (2026) https://BioRender.com/d7k2abt . ( M ) Total chimerism Translator SLAM-LKS based on IRES/Cap in secondary recipients through week 16 ( n = 12). ( N and O ) Representative image of the culture of a single low (N) or high (O) IRES/Cap SLAM-LKS after 5 days of culture. ( P and Q ) Pie charts depicting the percentage of wells with megakaryocytes (MK) after 5 days of culturing a single low (P) or high (Q) IRES/Cap Translator SLAM-LKS ( n = 20). Data show individual replicates and means ± SEM. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Significance was assessed using a two-way ANOVA with Dunnett’s one-way ANOVA (B to I) or an ANOVA with Tukey’s multiple comparisons test (K and M).

Article Snippet: WT CD45.2 C57BL/6J (strain #000664), CD45.1 (B6.SJL- Ptprc a Pepc b /BoyJ; strain #002014), KH2/iCas9 [B6;129S4- Gt(ROSA)26Sor tm1(rtTA*M2)Jae Col1a1 tm1(tetO-cas9)Sho /J; strain #029415], Rpl24 Bst (C57BLKS-Rpl24 Bst /J; strain #000516), and NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ; strain #005557) were purchased from the Jackson Laboratory.

Techniques: Irradiation