Journal: eLife

Article Title: IFNγ induces epigenetic programming of human T-bethi B cells and promotes TLR7/8 and IL-21 induced differentiation

doi: 10.7554/elife.41641

Figure Lengend Snippet: Figure 2. ASC development from BN precursors is enhanced in Th1 containing co-cultures. Cartoon (a) depicting day 6 paired co-cultures containing Th1 (Be1 co-cultures) or Th2 (Be2 co-cultures) effectors generated from the same HD, BN cells from a second allogeneic HD and exogenous IL-21 and IL-2. Flow cytometric analysis showing T-bet expression (b) on gated HD B cells (non-ASCs) from Be1 and Be2 co-cultures. Phenotyping (c) of day 6 B cell-gated Be1 cells showing T-bet expression in combination with other surface markers. (d–g) ASC development in HD day 6 paired Be1 and Be2 co-cultures showing representative flow plots (d) and frequencies (e) of CD38hiCD27+ ASCs in CD19+/lo-gated B lineage cells. Frequencies of IgM+ (f) or IgG+ (g) ASCs in day 6 paired Be1 and Be2 co-cultures. See Figure 2—figure supplement 1 for BN isolation strategy and characterization of polarized Th1 and Th2 effectors. See Figure 2—figure supplement 2 for gating strategy to identify IgG+ and IgM+ ASCs. See Figure 2—figure supplement 3 for proliferation analysis of B cells in paired day 6 HD Be1 and Be2 co-cultures. Analyses in (b–c) are from representative co-cultures (n > 30). Experiments (e–g) performed on 15 (e), 8 (f) or 6 (g) independent paired Be1 and Be2 co-cultures. Statistical analyses were performed using a non- parametric Wilcoxon paired t test (e) or paired Student’s t test (f–g). P values *<0.05, **<0.01, ****<0.0001. DOI: https://doi.org/10.7554/eLife.41641.005 The following figure supplements are available for figure 2:

Article Snippet: DOI: https://doi.org/10.7554/eLife.41641 23 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody PercP/Cy5.5 Mouse Anti-Human CD4 (clone OKT4) eBioscience 45-0048-42 (1:200) Antibody BV510 Mouse Anti-Human CD4 (clone OKT4) Biolegend 317444 (1:100) Antibody Fitc Mouse Anti-Human CD11c (clone Bu15) Biolegend 337214 (1:200) Antibody PE Mouse Anti-Human CD11c (clone Bu15) Biolegend 337205 (1:400) Antibody PercP/Cy5.5 Mouse Anti-Human CD14 (clone HCD14) Biolegend 325621 (1:200) Antibody Fitc Mouse Anti-Human CD19 (clone LT19) Miltenyi 302256 (1:100) Antibody PE Mouse Anti-Human CD19 (clone HIB19) Biolegend 302208 (1:200) Antibody PercP/Cy5.5 Mouse Anti-Human CD19 (clone HIB19) Biolegend 302230 (1:100) Antibody APC Mouse Anti-Human CD19 (clone HIB19) BD Pharmingen 555415 (1:200) Antibody APC-H7 Mouse Anti-Human CD19 (clone HIB19) BD Pharmingen 560727 (1:100) Antibody BV421 Mouse Anti-Human CD19 (clone HIB19) Biolegend 302234 (1:200) Antibody V500 Mouse Anti-Human CD19 (clone HIB19) BD Horizon 561121 (1:100) Antibody PercP/Cy5.5 Mouse Anti-Human CD21 (clone Bu32) Biolegend 354908 (1:100) Antibody Fitc Mouse Anti-Human CD23 (clone M-L23.4) Miltenyi 130-099-365 (1:100) Antibody PE Mouse Anti-Human CD23 (clone EBVCS-5) Biolegend 338507 (1:200) Antibody APC Mouse Anti-Human CD23 (clone M-L233) BD Pharmingen 558690 (1:200) Antibody Fitc Mouse Anti-Human CD27 (clone M-T271) Biolegend 356404 (1:100) Antibody PercP/Cy5.5 Mouse Anti-Human CD27 (clone M-T271) Biolegend 356408 (1:100) Antibody APC Mouse Anti-Human CD27 (clone M-T271) Biolegend 356410 (1:200) Continued on next page Zumaquero et al. eLife 2019;8:e41641.

Techniques: Generated, Expressing, Isolation

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: A) This diagram depicts the dual‐targeting strategy of biHSNPs in facilitating tumor cell elimination. Each biHSNP features two functional arms: one specifically binds to a tumor‐associated antigen on lymphoma cells (CD19/CD20), while the other engages and activates immune effector cells (CD3/CD16). By bridging tumor cells and immune cells, this approach enhances immune‐mediated tumor cell lysis, leveraging the immune system's inherent ability to identify and eliminate abnormal cells for precise and efficient cancer cell killing. B) Schematic illustration of the T cell activation and tumor cell death mechanism mediated by biHSNPs.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Functional Assay, Lysis, Activation Assay

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: Schematic illustration of the chemical synthesis and characterization of antibody‐conjugated hollow silica nanoparticles. A) Synthetic pathway of PEGylated hollow silica nanoparticles. B) Fourier transform‐infrared spectroscopy (FT‐IR) characterization of HSNP@PEG (2). C) Proton nuclear magnetic resonance ( 1 H NMR) characterization of HSNP@PEG. D) DLS and TEM images of HSNP@PEG. E) DLS and TEM images of HSNP@PEG@NH 2 (3). F) Fluorescence spectrum of HSNP@PEG@NH 2 @FITC (9.62 µ m ), confirming successful FITC conjugation. G) DLS and TEM images of HSNP@PEG@NH 2 @FITC (4). H–K) DLS and TEM results for monospecific nanoparticles (HSNP αCD3 , HSNP αCD16 , HSNP αCD19 , HSNP αCD20 ). L–O) DLS and TEM results for bispecific nanoparticles (HSNP αCD3 + αCD19 , HSNP αCD3 + αCD20 , HSNP αCD16 + αCD19 , HSNP αCD16 + αCD20 ). All scale bars in the TEM images represent 100 nm. HSNP αCD3 , HSNP αCD16 , HSNP αCD19 and HSNP αCD20 : Monospecific HSNPs conjugated with a single type of antibody (anti‐CD3, anti‐CD16, anti‐CD19, or anti‐CD20, respectively). HSNP αCD3 + αCD19 , HSNP αCD3 + αCD20 , HSNP αCD16 + αCD19 and HSNP αCD16 + αCD20 : Bispecific HSNPs conjugated with two different types of antibodies (e.g., anti‐CD3 and anti‐CD19). Antibody types are indicated using lowercase Greek letters (e.g., αCD3).

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Fourier Transform Infrared Spectroscopy, Spectroscopy, Nuclear Magnetic Resonance, Fluorescence, Conjugation Assay

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: Binding specificity and cell–cell linkage induced by biHSNPs. Target cells were incubated with FITC‐labeled, antibody‐coated HSNPs at 4 °C for 30 min, followed by flow cytometry analysis to confirm binding specificity. A–C) Depict cell–cell linkage induced by biHSNPs: A) Schematic illustration of cell‐cell complex formation mediated by biHSNPs. B) Confocal microscopy images showing stable junctional complexes between immune effector cells and tumor cells facilitated by HSNP αCD3 + αCD19 and HSNP αCD3 + αCD20 . C) Flow cytometry analysis of cell–cell complexes between pre‐stained Jurkat (green, CellTracker Green CMFDA) and Raji cells (red, CellTracker Red CMTPX), demonstrating effective linkage by biHSNPs. D) Verification of biHSNPs binding to CD3 + cells using PBMCs as the CD3 + cell line. E) Verification of biHSNPs binding to CD19 + /CD20 + cells using Raji cells as the target. F) Verification of biHSNPs binding to CD16 + cells using PBMCs as the CD16 + cell line. G) Verification of biHSNPs binding to CD19 + /CD20 + cells using Raji cells as the target.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry, Confocal Microscopy, Staining

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: A) Cytotoxicity and cytokine release assays of biHSNPs in luciferase‐transfected Raji cells. B) In vitro cytotoxicity of HSNP@PEG, HSNP αCD3 , HSNP αCD19 , and bispecific HSNP αCD3 + αCD19 . C) In vitro cytotoxicity of HSNP@PEG, HSNP αCD3 , HSNP αCD20 , and bispecific HSNP αCD3 + αCD20 . D,E) Cytokine secretion levels (IFN‐γ, IL‐2, and TNF‐α) for HSNP αCD3 , HSNP αCD19 , and HSNP αCD3 + αCD19 ; and HSNP αCD20 and HSNP αCD3 + αCD20 . F) In vitro cytotoxicity of HSNP@PEG, HSNP αCD16 , HSNP αCD19 , and bispecific HSNP αCD16 + αCD19 . G) In vitro cytotoxicity of HSNP@PEG, HSNP αCD16 , HSNP αCD20 , and bispecific HSNP αCD16 + αCD20 . H,I) Cytokine secretion levels (IFN‐γ, IL‐2, and TNF‐α) for HSNP αCD16 , HSNP αCD19 , HSNP αCD16 + αCD19 , HSNP αCD20 , and HSNP αCD16 + αCD20 . J) Cytotoxicity of combined bispecific nanoparticle treatments: HSNP αCD3 + αCD19 with HSNP αCD16 + αCD19 , and HSNP αCD3 + αCD20 with HSNP αCD16 + αCD20 . K) In vitro cytotoxicity of HSNP αCD3 + αCD19 combined with HSNP αCD16 + αCD19 , and HSNP αCD3 + αCD20 combined with HSNP αCD16 + αCD20 . Data are presented as mean ± SD ( n ≥ 3), Statistical differences in B–I) were analyzed by One‐way ANOVA and K) were analyzed by Student's t‐test and the statistical significance is indicated as ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Luciferase, Transfection, In Vitro

Journal: Advanced Science

Article Title: Bispecific Nanosystems Enable Multieffector Immune Cell Retargeting for Hematologic Malignancy Therapy

doi: 10.1002/advs.202509103

Figure Lengend Snippet: Enhanced T‐cell activation mediated by biHSNPs. A) Representative Quantitative analysis of CD19 + and CD8 + cell populations based on IHC. D,F) Representative immunofluorescence (IF) images showing the distribution of CD19 + Raji cells and CD8 + T cells. E–G) Quantitative analysis of CD19 + and CD8 + cells based on IF staining. H–J) Representative flow cytometry plots illustrating; I) Ki67 + expression in Raji cells and K) CD107a expression in CD8 + T cells across treatment groups. Statistical differences are analyzed by using One‐way ANOV and statistical significance is indicated as * P ≤ 0.05, ** P ≤ 0.01 and **** P ≤ 0.0001.

Article Snippet: FITC anti‐human CD19 antibody[CB19] (catalog No.: E‐AB‐F1004C, species reactivity: human), FITC anti‐human CD20 antibody[BCA/B20] (Catalog No.: E‐AB‐F1045C, species reactivity: human) and FITC anti‐human CD3 antibody[OKT‐3] (catalog No.: E‐AB‐F1001C, species reactivity: human) were obtained from Elabscience Biotechnology Co.,Ltd. (China, Wuhan).

Techniques: Activation Assay, Immunofluorescence, Staining, Flow Cytometry, Expressing

Journal: Cell Transplantation

Article Title: Adipose-derived stem cells inhibit dendritic cell migration by secreting tumor necrosis factor-α-stimulated gene 6 to improve the allogeneic skin transplantation survival rate in mice

doi: 10.1177/09636897251376125

Figure Lengend Snippet: Characteristics of adipose-derived stem cells. (a) Positive expression for CD90 and CD105 and negative expression for CD19 and CD34. (b), (c) Differentiation of ADSCs into adipocytes and osteoblasts. After adipogenic differentiation, newly differentiated adipocytes had lipid droplets identified by Oil Red O staining. Osteogenic differentiation was confirmed by Alizarin Red S staining. ADSCs: adipose-derived stem cells; CD: cluster of differentiation.

Article Snippet: Passage 3 cells were stained with antibodies against CD90 (E-AB-F1283C) (Elabscience, Wuhan, China), CD105 (E-AB-F1233UE) (Elabscience, Wuhan, China), CD19 (E-AB-F1004E) (Elabscience, Wuhan, China), and CD34 (E-AB-F1284D) (Elabscience, Wuhan, China).

Techniques: Derivative Assay, Expressing, Staining

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet: A proteogenomic atlas of the healthy murine liver (A) Hepatic cells were isolated from healthy C57B/l6 mice by ex vivo (5 mice, 15 samples) or in vivo (5 mice, 19 samples) enzymatic digestion. Alternatively, nuclei were isolated by tissue homogenization (4 mice, 12 samples). Live cells/intact nuclei were FACS-purified. For cells, total live, live CD45 + , live CD45 − , live hepatocytes, or myeloid cells (live CD45 + , CD3 − , CD19 − , B220 − , NK1.1 − ) were sorted. 18 samples (7 ex vivo , 11 in vivo ) were also stained with a panel of 107–161 barcode-labeled antibodies for CITE-seq analysis. All datasets were pooled together and after QC 185,894 cells/nuclei were clustered using TotalVI. (B) UMAP of sc/snRNA-seq data. (C) Tissue and capsule images from Visium analysis with clusters overlaid. (D) UMAP of zonation of Visium spots (left) and origin of the cells (right). (E) Zonation pattern mapped onto tissue slice. (F and G) Indicated cell signatures from sc/snRNA-seq mapped onto the Visium zonation data. (H) mRNA zonation pattern in Visium highly multiplexed protein analysis and VSIG4-ADT expression pattern (left) and zonated expression patterns of indicated antibodies (right). (I) MICS analysis of indicated proteins. (J) Molecular Cartography of indicated genes and cell types. (K) mRNA ( Xcr1, Flt3l, Mafb , and Clec10a ) and protein (MHCII and F4/80) expression in the same tissue slice. Scale bars, 50 μm. PV, portal vein; CV, central vein. Arrows indicate specific cell types, colors correspond to cell type/markers. Images are representative of 2–4 mice. See also Figure S2 and , , , and .

Article Snippet: Anti-Human CD19 (REAL106) PE , Miltenyi Biotec , 130-122-649; RRID: AB_2784034.

Techniques: Isolation, Ex Vivo, In Vivo, Tissue Homogenization, Purification, Staining, Labeling, Expressing

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet: Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables identification of all hepatic cell types including bona fide cell doublets, related to Figure 1 (A and B) Top DEGs (A) and DEPs (B) for cell types from Figure 1 B. (C) Distinct profiles of cells or nuclei within the UMAP depending on isolation protocols; 71,162 cells from ex vivo digestions, 96,066 cells from in vivo digestions, and 18,666 nuclei. Numbers on plots represent numbers of cells/nuclei per population. (D) Correlation plots showing genes captured within the KC, B cell and neutrophil populations with and without addition of CITE-seq antibodies. (E) Expression of VSIG4, CD206, and ESAM (protein, top) and Vsig4 , Mrc1 , and Esam (mRNA, bottom). (F) UMAP showing clusters of cells when only minimal QC for gene number and % mitochondrial genes is performed; 17,669 cells pooled from 3 samples. Expression of Cd5l, Cd19 , and Kdr by the clusters facilitating identification of cell types per annotation. (G) CITE-seq data from (F) in Flow-Jo showing expression of CD206 and ESAM in total KCs (left) and total B cells (middle). Numbers represent % of entire KC or B cell population. Identified populations were then mapped back onto the original UMAP (right). (H) Expression of CD31, CD26, and CD38 by indicated populations. (I) Heatmaps showing expression of top DEGs between KC1s and LSECs (left), KC2s and KC1s + LSECs (middle) and B cell2s and B cell1s + LSECs (right). (J) 3D reconstruction of murine liver following perfusion with antigen fix to inflate endothelial cells and staining with antibodies against CD31, CD206, and F4/80. (K) UMAP showing clusters generated from Visium analysis of liver tissue (4 samples) and liver capsule (1 sample). (L) Top unbiased genes defining zonation trajectory from portal to central vein in Visium. (M) Expression of Glul and Epcam by confocal microscopy (left), annotation of portal, periportal, mid, and central regions on same tissue section (middle) and overlay of both datasets (right). (N) Identification of cholangiocyte (left) and cDC (right) signatures on zonated Visium spots. (P) Molecular Cartography showing expression of indicated zonated hepatocyte mRNAs in liver tissue. Data are representative of 2 mice. (O) Expression of Itgae (encoding CD103) in the UMAP of the total liver (left) and flow cytometric analysis of total cDC1s for CD103 and MHCII expression in the healthy murine liver (right).

Article Snippet: Anti-Human CD19 (REAL106) PE , Miltenyi Biotec , 130-122-649; RRID: AB_2784034.

Techniques: Isolation, Ex Vivo, In Vivo, Expressing, Staining, Generated, Confocal Microscopy

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet: A population of macrophages reside around the bile duct in the healthy murine liver (A) UMAP of murine myeloid cells (71,261 cells/nuclei) isolated from Figure 1 B and re-clustered with TotalVI. (B and C) Top DEGs (B) and DEPs (C) between cell types. (D) Expression of Gpnmb and Cd207 . (E) Expression of VSIG4 and F4/80 (left) or MHCII, CD11c, and DAPI (right) by confocal microscopy. Capsule macs identified by white arrows. Scale bars, 50 μm. (F) Molecular Cartography of indicated genes at liver capsule. (G) Expression of VSIG4, F4/80, GLUL, and DAPI (left) or F4/80 or CCR2 (right, inset) by confocal microscopy. Scale bars, 100 μm. (H) Molecular Cartography of indicated genes at portal triad. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to markers. Images are representative of 2–4 mice. (I and J) Top GO terms for KCs (I) and bile-duct LAMs (J). (K) Representative image showing expression of VSIG4 (red) CD19 (yellow) and CD3E (magenta) by MICS analysis (left) and % of B or T cells found with/without a KC per field of view (right). Data are pooled from multiple fields of view in 2 mice. ∗∗∗ p < 0.001 Student’s t test. (L) Mice (29-week-old) were treated with 3.5 mg/kg LPS or PBS and 2 h later, livers were harvested without the capsule. KCs and LAMs were FACS-purified and expression of Il1b, Tnf, IL10 , and Il18 was examined by qPCR, compared with b-actin . ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way ANOVA with Bonferroni post-test. See also Figure S3 and .

Article Snippet: Anti-Human CD19 (REAL106) PE , Miltenyi Biotec , 130-122-649; RRID: AB_2784034.

Techniques: Isolation, Expressing, Confocal Microscopy, Purification

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet: Validated flow cytometry gating strategy for murine myeloid cells, related to Figure 2 (A) CITE-seq data from the murine myeloid cells in Figure 2 A were exported as an FCS file and an in silico gating strategy identified in FlowJo software. (B) Application of the in silico gating strategy with a 21-color flow cytometry panel. Myeloid cells were pre-gated as live CD45 + lineage cells (Ly6G − CD19 − NK1.1 − B220 − CD3 − ). Data are representative of 3 experiments with 3–6 mice per experiment. (C) cDC1s, cDC2s, migratory cDCs (Mig. cDCs), peritoneal macs (Peri. Macs), KCs, and non-KC macs (non-KCs) were FACS-purified using gating strategy in (B), mRNA was isolated and qPCR performed to examine expression of indicated genes defining each population to validate their identity. Data are representative of 2 experiments with n = 3–6. (D) Putative peritoneal macs were FACS-purified using gating strategy in (B) and expression of Gata6 was examined by qPCR compared with other hepatic myeloid populations. Data are from a single experiment with n = 6. (E) Peritoneal macs as a % of total macs recovered from the liver using different digestion techniques ( in vivo , ex vivo , or capsule) or in supernatants in which livers were washed following removal from the mouse but prior to digestion (wash). Data are from a single experiment with n = 4. ∗ p < 0.05, ∗∗ p < 0.01 one-way ANOVA with Bonferroni post-test compared with wash data. (F) Expression of CD14 and CD207 within the non-KC mac population from (B) (left) and % of CD207 + and CD207 − populations among total macs in livers digested using the ex vivo or in vivo protocols or in dissected and digested liver capsule (right). Data are representative of two experiments with n = 4–5 mice per experiment. ∗∗∗∗ p < 0.0001 mixed effects analysis with Tukey’s multiple comparison test. (G) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy. Insets represent zones featured in E, 2G, and I. (H) Molecular Cartography of indicated genes and cell types. Insets represent zones featured in F, 2H, and J. (I) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy at the central vein. Scale bars, 50 μm. (J) Molecular Cartography of indicated genes and cell types at central vein. (K) Expression of F4/80, EPCAM, CCR2, GPNMB, and DAPI by confocal microscopy at a portal vein (top) or F4/80 or GPNMB alone (bottom). Scale bars, 25 μm. (L) Quantification of % of Gpnmb & Trem2 counts over Adgre1 counts in indicated regions of tissue as assessed using Molecular Cartography data. Each dot represents an individual region. ∗ p < 0.05, ∗∗∗∗ p > 0.0001 one-way ANOVA with Bonferroni post-test. (M) Expression of DESMIN and F4/80 at the liver capsule and underlying parenchyma (left) or EPCAM, DESMIN and F4/80 at the bile duct by confocal microscopy. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to cell type/markers. All images are representative of 2–6 mice.

Article Snippet: Anti-Human CD19 (REAL106) PE , Miltenyi Biotec , 130-122-649; RRID: AB_2784034.

Techniques: Flow Cytometry, In Silico, Software, Purification, Isolation, Expressing, In Vivo, Ex Vivo, Comparison, Confocal Microscopy

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet: Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables generation of a human liver atlas, related to Figure 4 (A) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 27,398 cells) were isolated from Figure 1 B and re-clustered with TotalVI. (B and C) Top DEGs (B) and DEPs (C) for the cell types from Figure S5 A. (D) CITE-seq data from Figure S5 A were exported as an FCS file and an in silico gating strategy identified in FlowJo. (E) Human lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 105,790 cells) were isolated from Figure 4 B and re-clustered with TotalVI. (F and G) Top DEGs (F) and DEPs (G) for the cell types from Figure S5 E. (H) CITE-seq data from Figure S5 E were exported as an FCS file and an in silico gating strategy identified in FlowJo. (I) Proportion of indicated cell types arising from patients with <10% (purple) or >10% steatosis (yellow). (J) Hepatic cells were isolated from 22 C57B/l6 mice fed either a standard diet (SD) or a western diet (WD) for 24 or 36 weeks to induce NAFLD and NASH by ex vivo (10 samples) or in vivo (12 samples) enzymatic digestion. Alternatively, livers were snap frozen and nuclei isolated by tissue homogenization (14 samples). Live cells/intact nuclei were purified using FACS. For cells, total live, live CD45 + , live CD45 − , live hepatocytes or myeloid cells (live CD45 + , CD3 − , CD19 − , B220 − , NK1.1 − ) were sorted. 10 samples were also stained with a panel of 107–161 barcode-labeled antibodies for CITE-seq analysis. All datasets were pooled together and after QC 121,980 cells/nuclei were clustered using TotalVI. (K) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 21,322 cells) from mice fed the SD or WD for 24 or 36 weeks were isolated from Figure S5 J and re-clustered with TotalVI. (L) Proportion of indicated cell types arising from mice fed the SD (purple) or WD (yellow). (M) Top DEGs between CTLs isolated from mice fed the SD (purple) or WD (yellow).

Article Snippet: Anti-Human CD19 (REAL106) PE , Miltenyi Biotec , 130-122-649; RRID: AB_2784034.

Techniques: Isolation, In Silico, Western Blot, Ex Vivo, In Vivo, Tissue Homogenization, Purification, Staining, Labeling

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet: Identification of bona fide Kupffer cells across species (A) Cells/nuclei were isolated from liver biopsies (∼1–2 mm 3 ; 14 cells, 5 nuclei) from patients undergoing either liver resection, cholecystectomy or gastric bypass. Live cells/intact nuclei were FACS-purified. Either total live, live CD45 + , and live CD45 − or live CD45 + , CD3 − , and CD19 − cells were sorted. 7 cell samples were stained with a panel of 198 barcode-labeled antibodies for CITE-seq analysis. All datasets were pooled together and after QC, 167,598 cells/nuclei were analyzed using TotalVI. (B) UMAP of sc/snRNA-seq data. (C) UMAP of Visium data from 4 patient biopsy samples. (D) Split of Visium spots based on % steatosis. (E) Healthy and steatotic Visium liver tissue with clusters overlaid and H+E staining to identify steatotic zones. (F) Zonation of Visium data (top) with zonation pattern mapped onto liver tissue (bottom). (G) Indicated cell signatures from sc/snRNA-seq mapped onto Visium zonation trajectory, healthy (top), steatotic (bottom). (H) Myeloid cells (40,821 cells) were isolated from Figure 4 B and re-clustered with TotalVI. (I) Expression of VSIG4 protein (top) and CD5L mRNA (bottom). (J) Expression of VSIG4, F4/80, FOLRB, and GLUL combined with Cd5l/CD5L on murine (left) and human (H25; right) livers. Scale bars, 50 μm. Inset in bottom panels. Scale bars, 20 μm. Images are representative of 2–4 livers. (K) Livers (2/species) were isolated from healthy macaque, pig, chicken, hamster, and zebrafish. Cells were isolated by ex vivo digestion for CITE-seq (pig; 198 human antibodies) or scRNA-seq (hamster, chicken, and zebrafish), or nuclei were isolated for snRNA-seq (macaque). Total live cells (hamster, chicken, and pig), DsRed + GFP + cells (zebrafish) or nuclei (macaque) were FACS-purified. Following QC, 8,483 nuclei (macaque) or 21,907 (pig), 5,965 (hamster), 7,457 (chicken), and 4,957 (zebrafish) cells were analyzed using TotalVI (pig) or scVI (macaque, hamster, chicken, and zebrafish) (top). KCs were identified using the human-murine KC signature and the signature finder algorithm ( Pont et al., 2019 ) (bottom). See also and and , , , , , , and .

Article Snippet: Anti-Human CD19 (REAL106) PE , Miltenyi Biotec , 130-122-649; RRID: AB_2784034.

Techniques: Isolation, Purification, Staining, Labeling, Expressing, Ex Vivo

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet:

Article Snippet: Anti-Human CD19 (REAL106) PE , Miltenyi Biotec , 130-122-649; RRID: AB_2784034.

Techniques: Purification, Recombinant, Staining, cDNA Synthesis, Gene Expression, Software, Microscopy

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Schematic representations of ligand-based conventional and split-design CAR approaches. The extracellular domains of BAFF and APRIL were used as target moieties to generate conventional CAR-T cells, referred to as APRIL CAR and BAFF CAR, respectively. b , d Representative images of cell‒cell conjugates captured at 100× oil objective magnification using a laser scanning confocal microscope (Nikon, A1R). APRIL or 9E10-IgG4m (pre-incubated with Myc-APRIL) CAR-T cells were co-cultured with RPMI8226-GFP cells ( b ), while BAFF or 9E10-IgG4m (pre-incubated with Myc-BAFF) CAR-T cells were co-cultured with IM9-GFP cells ( d ). Fluorescent labels included Hoechst (blue), anti-PKC-θ (red), and GFP (green) and a merged view of all stains. Scale bar = 10 μm. c , e Statistical analysis of the mean fluorescence intensity of PKC-θ at the IS in panels b and d, respectively. In panel c, sample sizes: APRIL CAR, n = 37; 9E10-IgG4m, n = 39. In panel e, BAFF CAR, n = 34; 9E10-IgG4m, n = 44. All n values represent individual cells. P values were determined by paired two-tailed t -tests. f , g Cytotoxicity assays of conventional and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. h , i Inflammatory cytokine release assay. Conventional CAR-T cells or sCAR-T cells along with 1 nM corresponding switches were co-cultured with the specific target cells for 24 h at an E:T ratio of 1:1 in triplicate. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. j , l Schematic representations of ligand-based split-design CAR and FDA-approved CAR, referred to as BCMA CAR ( j ) and CD19 CAR ( l ), respectively. k , m Cytotoxicity assays of FDA-approved CAR-T cells and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. Data in this figure are representative of three independent experiments. Error bars represent mean ± SD. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: Microscopy, Incubation, Cell Culture, Fluorescence, Two Tailed Test, Release Assay

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). b Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). c , d Quantification of the average radiance (p/s/cm /sr) of the luminescence, related to APRIL- ( c ) and BAFF-( d )-based CAR-T-cell therapy. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. e Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Tukey correction were used to assess significance. f , g Survival curves of the mice subjected to the indicated treatments. Survival curves were compared using the log-rank (Mantel‒Cox) test. h Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). i Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). j Quantification of the average radiance (p/s/cm /sr) of the luminescence. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) and CD19/CD22 CAR-T. k Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. l Assessment of the presence of persistent human CD3 + (hCD3 + ) T cells in peripheral blood by flow cytometry over a 3-week follow-up period. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) with BAFF-CAR-T at each time point. m Survival curves of mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. All n represents biological replicates from different mice. Data in this figure are representative of one of two independent experiments. Error bars represent mean ± SEM. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). b Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). c Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. d Quantification of the average radiance (p/s/cm 2 /sr) of the luminescence. Two-way ANOVA multiple comparisons in Sidak correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-APRIL) with BCMA CAR-T. e Survival curves of mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. f Timeline of the in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). g Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). h Evaluation of serum inflammatory cytokine release by ELISA 24 hours after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. i Quantification of the average radiance (p/s/cm 2 /sr) of the luminescence. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) with CD19 CAR-T. j Assessment of the presence of tumor cells (GFP + CD19 + or GFP + CD19 - ) in peripheral blood by flow cytometry on the 18th day of the experiment. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. k Survival curves of the mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. All n represents biological replicates with different mice. Data are in this figure representative of one of two independent experiments. Error bars represent mean ± SEM. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry