Journal: bioRxiv

Article Title: Macrophage extracellular traps promote maladaptive cardiac remodelling and heart failure via PAD4-dependent mechanisms

doi: 10.64898/2026.03.15.711858

Figure Lengend Snippet: A , Schematic overview of the experimental design. WT-BMT and PAD4KO-BMT mice received either IgG isotype control or anti-Ly6G antibody (100 μg per mouse) by intraperitoneal injection every 3 days for 28 days, as indicated. B , Peripheral blood cell counts, and neutrophil and monocyte fractions (n=4-5). Neutrophils were determined by CD45 high Gr-1 high CD11b low and monocytes were determined by CD45 high Gr-1 low CD11b high by FACS analysis. C , Echocardiographic assessment 28 days after TAC (n=6 in each group). D , Representative fluorescent immunohistochemistry images of left ventricular tissue stained for neutrophil elastase (NE, green), citrullinated histone H3 (CitH3, red), and troponin I (yellow), with nuclear counterstaining by DAPI (blue). Arrowheads indicate NET-positive cells (NE + CitH3 + ). Scale bars, 50 μm. E , Quantification of the numbers of neutrophils (NE + ) and NETs (NE + CitH3 + ) per myocardial tissue area (n=4). F , Representative left ventricular sections stained for CD68 (green), CitH3 (red), troponin I (yellow) and DAPI (blue). Arrowheads indicate MET-positive cells (CD68 + CitH3 + ). Scale bars, 50 μm. G , Quantification of CD68 + macrophages and METs (CD68 + CitH3 + ) per tissue area (n=3-4). All data are presented as mean ± SEM. *P < 0.05 versus the corresponding IgG-treated group, † P < 0.05 versus IgG-treated WT-BMT mice, and ‡ P < 0.05 versus anti-Ly6G-treated WT-BMT mice as determined by one-way analysis of variance with Tukey’s post hoc analysis.

Article Snippet: Magnetic-activated cell sorting was performed on bone marrow cells using MACS MS columns (Miltenyi Biotec GmbH) with CD11b MicroBeads (130-097-142, Miltenyi Biotec GmbH) according to the manufacturer’s instructions.

Techniques: Control, Injection, Immunohistochemistry, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: Overexpression of SP140 induces the production of inflammatory cytokines and chemokines and reprogram macrophages to induce TAM-mediated tumor cytotoxicity. (A, B) Control CRISPR/dCAS9 (control) or SP140 CRISPR/dCAS9 (SP140 overexpression) was transfected in naïve macrophages, and the expression of CD80 and CD86 was quantified by flow cytometry. (C–E) SP140 CRISPR/dCAS9 (SP140 OE) or scramble control (control CRISPR/dCAS9) were introduced to the naïve macrophages, and a multiplex fluorescence BioLegend assay was used to identify the levels of CXCL10, IFN-γ, and IL-12 (P70) in the supernatant after 48 hours. LPS (100 nM) was administered after 24 hours. (F–I) Correlation of SP140 expression and IFNG, CXCL10, IL-12B, and IL-12A levels in over 500 HNSCCs in the TCGA dataset. (J) Control CRISPR/dCAS9 or SP140 CRISPR/dCAS9 was introduced in TAMs isolated from syngeneic HNSCC tumor and cocultured with murine SCC7 cells. Cell viability after 48 hours of coculture was quantified with a quantitative viability assay kit. Data are presented based on the fold change of non-treated control. *P<0.05, ***P<0.001, ****P<0.0001. HNSCC, head and neck squamous cell carcinoma; IFN-γ, interferon gamma; IL, interleukin; SP140, speckled protein 140; TAM, tumor-associated macrophage; TCGA, The Cancer Genome Atlas.

Article Snippet: A flow cytometry panel consisting of human CD80 (PE, BioLegend), human CD86 (BV711, BioLegend), and human CD206 (PerCP-Cy5.5, BioLegend) was used for the characterization of macrophages.

Techniques: Over Expression, Control, CRISPR, Transfection, Expressing, Flow Cytometry, Multiplex Assay, Fluorescence, Isolation, Viability Assay

Journal: bioRxiv

Article Title: MAFB surrogates the glucocorticoid receptor ability to induce tolerogenesis in dendritic cells

doi: 10.1101/2021.07.27.453975

Figure Lengend Snippet: (A) Schematic representation of the experimental approach, comparing dendritic cell (DC) with tolerogenic dendritic cell (tolDC) differentiation. (B) DC and tolDC were cocultured with CD8+ cells for 5 days. The final CFSE signal of CD8+ cells is shown (left panel). CD8+ with only CD3/CD28 T-activator beads (C+) or alone (C-) are also shown. In the right panel, the average proliferation of the quadruplicate is shown (mean ± standard error of the mean (SEM)). (C) IL-10, TNFα, IL-12p70 and IL1-β production of DC and tolDC, after 5 days of differentiation and 24 h of LPS (10 ng/μL) and IFNg (20 ng/μL) stimuli. P-values of paired t-tests are shown. (D) Box-plots of CD80, CD83, CD86 and HLA-DR surface expression (Median Fluorescence Intensity) in DCs and tolDCs in steady-state or stimulated with LPS (10 ng/μL) and IFNg (20 ng/μL) (ns p > 0.05, ** p < 0.01, *** p ≤ 0.001). (E) Gene expression heatmap of differentially expressed genes comparing tolDCs with DCs and also displaying the gene expression values of the precursor cell type (MO) (logFC > 0.5, FDR < 0.05). Scaled fluorescence values of expression arrays are shown, ranging from -2 (lower gene expression, green) to +2 (higher gene expression, orange). (F) Gene ontology (GO) over-representation of GO Biological Process categories. Fold change of tolDC induced genes over background and -log10(FDR) of Fisher’s exact tests are shown. (G) Discriminant regulon expression analysis (DoRothEA) of tolDC compared with DC. Only transcription factors with FDR < 0.05 are shown. NES and logFC of transcription factor expression are depicted. (H) T-distributed stochastic neighbor embedding (t-SNE) plot of the aggregated and batch-corrected gene expression data from our study (MO, DC and tolDC) and two additional public datasets (GSE40484 (moMAC, moDC, cDC2, CM (Classical MOs) and NCM (Non-Classical MOs) and GSE99056 (M-MAC (M2 Macrophages) and GM-MAC (M1 Macrophages)). The 4 different groups obtained using k-means clustering are represented with grey ellipses of multivariate t-distributions.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were performed, following the manufacturer’s instructions: Human IL-10, Human IL-12p70, and Human TNFα from BioLegend, and Human IL-1β from ThermoFisher.

Techniques: Expressing, Fluorescence, Gene Expression

Journal: bioRxiv

Article Title: MAFB surrogates the glucocorticoid receptor ability to induce tolerogenesis in dendritic cells

doi: 10.1101/2021.07.27.453975

Figure Lengend Snippet: (A) Volcano plot comparing tolDCs treated with control siRNA (siCTL) and MAFB siRNA (siMAFB). Dashed lines indicate significance thresholds (FDR < 0.05, absolute logFC > 0.5). tolDC-induced and tolDC-repressed genes are shown in blue and orange, respectively. (B) Gene set enrichment analysis (GSEA) of tolDCs (siCTL) vs. tolDCs (siMAFB), using tolDC-induced and tolDC-repressed gene sets. The running enrichment score is represented and the normalized enrichment score (NES) is shown above (FDR < 0.01). (C) DNA methylation heatmap of previously obtained differentially methylated CpGs (C1-CpGs and C2-CpGs) in tolDCs (siCTL) and tolDCs (siMAFB). Scaled β-values are shown (lower DNA methylation levels in blue and higher methylation levels in red). On the right side, violin plots of Cluster 1 (C1) and Cluster 2 (C2) depict β-values (ns p > 0.05, *** p ≤ 0.001). (D) Methylated CpG set enrichment analysis (mCSEA) of tolDCs (siCTL) vs. tolDCs (siMAFB), using MAFB-only CpGs, GR/MAFB CpGs and GR-only CpGs as CpG-sets (depending on the overlap of CpGs with GR or MAFB peaks). The running enrichment score is represented and the normalized enrichment score (NES) and FDR are shown above. (E) Box-plots of median fluorescence intensity (MFI) of CD14, CD16, CD163 and CD1a flow cytometry data from DCs (siCTL), tolDCs (siCTL) and tolDCs (siMAFB) (n = 7) (ns p > 0.05, * p < 0.05, ** p ≤ 0.01). (F) Box-plots of supernatant concentration from DCs (siCTL), tolDCs (siCTL) and tolDCs (siMAFB) (n = 7) of IL-10 in steady-state and stimulated conditions (LPS 10 ng/μL and IFNγ 20 ng/μL) and IL-12p70 and TNFα under stimulated conditions (pg/mL). TNFα and IL-12p70 in steady state were not detected. (ns p > 0.05, * p < 0.05, ** p ≤ 0.01) (G) DC (siCTL), tolDC (siCTL) and tolDC (siMAFB) were cocultured with CD8+ cells for 5 days (n = 4). The final CFSE signal of CD8+ cells is shown (left panel). CD8+ with only CD3/CD28 T-activator beads (C+) or alone (C-) are also shown. On the right panel, the average proliferation of the quadruplicate is shown (mean ± standard error of the mean (SEM)) (** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were performed, following the manufacturer’s instructions: Human IL-10, Human IL-12p70, and Human TNFα from BioLegend, and Human IL-1β from ThermoFisher.

Techniques: Control, DNA Methylation Assay, Methylation, Fluorescence, Flow Cytometry, Concentration Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Binding affinities to human HER2 and CD3 at 37 °C by surface plasmon resonance. Data are K D ( n = 12 technical replicates of a single experiment for HER2-XPAT protein and HER2(1x-N); n = 8 for HER2(1x-C) and uTCE). Surface plasmon resonance sensorgrams for these data are provided in Supplementary Figs. – . b – d , In vitro tumor cytotoxicity of HER2-XPAT protein and its metabolites following a 48-h incubation with co-cultures of huPBMCs and the high HER2-expressing human tumor cell lines (1:1 effector–target ratio) SKOV3 ( b ), BT-474 ( c ) or the medium-low HER2-expressing MCF7 cell line ( d ). e , In vitro cytotoxicity of HER2-XPAT protein and its metabolites against BT-474 cells co-cultured with huPBMCs (1:1 effector–target ratio). f , Impact of the protease-cleavable linker on in vitro cytotoxicity versus BT-474 cells co-cultured with huPBMCs. g , h , CD69-positive T cells ( g ) and IL-2 secretion ( h ) following 72-h incubation of huPBMC/SKOV3 co-cultures with HER2-XPAT protein or its unmasked form (uTCE). i , Target-dependent T-cell activation with HER2-XPAT protein and its metabolites. CD3-expressing Jurkat reporter T cells were incubated with BT-474 cells at a 5:1 effector–target ratio for 6 h, followed by quantification of NFAT -induced luciferase activity and measured in relative luminescence units (RLUs). Mean data for n = 2 technical replicates within one single experiment ( b – i ). Extended Data Table provides a summary of EC 50 values for the different forms of the HER2-XPAT proteins in the cytotoxicity and reporter T-cell activation assays.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Binding Assay, SPR Assay, In Vitro, Incubation, Expressing, Cell Culture, Activation Assay, Luciferase, Activity Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: CD3 + Jurkat reporter T cells were incubated with or without BT-474 cells at a 5ː1 effector-target ratio for 6 hours, followed by quantification of NFAT -induced luciferase activity. The graph shows individual data points from a single representative experiment. There were n = 2 biological repeats at each concentration tested within the same experiment for each cell line. HER2, human epidermal growth factor receptor 2; RLU, relative luminescence unit; uTCE, unmasked T-cell engager; XPAT proteins, TCE fused to XTEN polypeptides.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Incubation, Luciferase, Activity Assay, Concentration Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , TGI ± s.e.m. ( n = 8 mice for each concentration tested within one single experiment) promoted by the i.v. administration of equimolar doses (every week (QW) for 3 weeks) of HER2-XPAT protein (2.1 mg kg −1 ) or uTCE to NOG mice bearing established (maximum tolerated volume (MTV) ~185 mm 3 ) BT-474 human tumors. The dependence of tumor-resident proteases for activity was demonstrated by the lack of significant TGI in mice treated with HER2-XPAT-NoClvSite. The average body weight of the mice bearing tumor xenografts remained generally stable for the duration of the experiment following dosing with the HER2-XPAT proteins or the vehicle control. b ,TGI ± s.e.m. ( n = 8 mice for each concentration tested within one single experiment) in established human HT-55 xenografts (MTV ∼150 mm 3 ) following i.v. administration of HER2-XPAT protein (QW for 4 weeks) and HER2-uTCE (0.9 mg kg −1 three times a week (TIW) for 4 weeks). HER2-XPAT-NoClvSite (QW for 4 weeks) had no impact on tumor growth. The average body weight ± s.e.m. of the mice bearing tumor xenografts remained generally stable for the duration of the experiment following dosing with the HER2-XPAT proteins or the vehicle control. c , T-cell activation in BT-474 human tumor xenografts and peripheral blood evaluated by flow cytometry on day 18 following equimolar TIW i.v. dosing with HER2-XPAT protein and HER2-uTCE (day 40 following tumor inoculation). HER2-XPAT and HER2-uTCE induced robust and comparable activation of intratumoral CD4 + and CD8 + T cells, whereas no trends for T-cell activation were apparent in blood samples in which HER2 was not present. Statistical differences in TGI and T-cell activation for test compounds versus vehicle were assessed using mixed-effects multiple comparison analyses followed by Tukey’s post hoc test.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Concentration Assay, Activity Assay, Control, Activation Assay, Flow Cytometry, Comparison

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Fluorescent dye-labeled HER2-XPAT or EpCAM-XPAT protein was used to track proteolytic cleavage in PDX tumor-bearing mice. b , A representative SDS–PAGE gel showing XPAT protein cleavage forms arising in a single PDX-bearing mouse, after imaging by LI-COR Biosciences. Four bands representing HER2-XPAT protein and its three unmasked forms are visible in the tumor sample (green channel) while the other (red channel) predominantly shows the XPAT protein form. c , Concentration of XPAT protein forms in tumors and healthy tissues. Results are the mean ± s.d. from 20 mice within one single experiment, with tumors consisting of ten different PDX (Supplementary Table ); mice were injected with fluorescent dye-labeled HER2-XPAT or EpCAM-XPAT protein. Note that tissues for which ≥14 samples were below the limit of quantification (BLQ) were reported as ‘BLQ.’ Tissues with averages calculated using some samples with BLQ readings are marked with an asterisk.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Labeling, SDS Page, Imaging, Concentration Assay, Injection

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: HER2-XPAT protein was administered via short i.v. infusions (∼1–3 h), whereas the unmasked counterpart, due to its short half-life, was administered via 48-h continuous i.v. infusion to provide prolonged systemic exposure. a , A dose-escalation study with a single dose of HER2-XPAT protein administered to each cynomolgus monkey. *At doses below 21 mg kg −1 , a HER2-XPAT prototype was used. b , Dose de-escalation study of uTCE in NHPs administered as a 48-h continuous infusion due to its short half-life. c , Plasma concentrations in NHPs following administration of a single i.v. dose of HER2-XPAT protein (42 mg kg −1 , n = 2 NHPs; 25 mg kg −1 , n = 4 NHPs; 6 mg kg −1 , n = 12 NHPs; 2 mg kg −1 , n = 12 NHPs) or a 48-h continuous infusion of uTCE (0.2 mg kg −1 d −1 , n = 1 NHP; 0.1 mg kg −1 d −1 , n = 1 NHP; 0.06 mg kg −1 d −1 , n = 1 NHP). Data represent mean plasma concentration (±s.d. when n > 3 NHP treated) for the NHPs treated at each dose within one single experiment. d , e , Activation of circulating CD4 + T cells ( d ) and CD8 + T cells ( e ) 24 h after drug administration (HER2-XPAT protein, n = 16 NHPs; HER2-uTCE, n = 5 NHPs). f – h , Highest plasma levels of TNF-α ( f ), IL-6 ( g ) and IFN-γ ( h ) measured 0–24 h following drug administration (HER2-XPAT protein, n = 23 NHPs; HER2-uTCE, n = 8 NHPs). Note that the normal ranges for cytokine levels in NHP are as follows: IL-6 0.3–1.2 pg ml −1 , TNF-α 1–10 pg ml −1 , IFN-γ 1.1–8.0 pg ml −1 (ref. ). Data in d – f represent a single sample from each NHP receiving the test material within each single experiment. TNF, tumor necrosis factor; IFN, interferon.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Concentration Assay, Activation Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Mean plasma concentrations (±s.d.) following a single i.v. administration of HER2-XPAT protein (single plasma samples from n = 6 NHPs) and HER2-XPAT-NoClvSite ( n = 4 NHPs) within one single experiment. b , Plasma concentrations of HER2-XPAT(1x−N) and HER2XPAT(1x−C) relative to the entire HER2-XPAT protein, following a single i.v. dose of HER2-XPAT protein (25 mg kg −1 , n = 4 NHPs; 42 mg kg −1 , n = 4 NHPs) within one single experiment. c , Quantification of cleavage products with a similar molecular weight to the uTCE generated following incubation of fluorescent-labeled HER2-XPAT protein in plasma from cancer patients ( n = 11), patients with inflammatory diseases ( n = 27), healthy donors ( n = 4) and NHPs (with ( n = 6) and without ( n = 4) drug-induced inflammation). The plasma incubation experiment represents a closed system with no clearance mechanisms and will therefore overestimate the accumulation of cleavage products occurring in vivo. Horizontal bars represent median values; dots represent individual observations. P values are based on unpaired t -tests.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Molecular Weight, Generated, Incubation, Labeling, In Vivo

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , An XPAT protein comprises a TCE core with two scFvs, one targeting CD3 and the other, a TAA. Each scFv is masked by a protease-releasable XTEN mask, unstructured, hydrophilic polypeptides that act as modular, tunable masks, in addition to extending the half-life of the TCE. Each XTEN mask connects to the TCE core via a protease-cleavable linker, designed to be cleavable by any of eight proteases from three different classes (matrix metalloproteinases, serine proteases and cysteine proteases) involved in cancer progression . b , Predicted structure of HER2-XPAT protein visualized using AlphaFold2 v.2.0, a machine-learning-based computational method for predicting protein structures with reasonable accuracy . Colors indicate anti-HER2 domain, pale green; anti-CD3 domain, light orange; XTEN masks, blue; protease-cleavable linker, red; linkers, gray. The model represents a static picture showing a plausible conformation of the unstructured XTEN masks and the length of unstructured XTEN relative to the folded antibody domains in an XPAT protein. c , XPAT proteins are expected to remain largely intact in healthy tissues, where protease activity is well controlled by protease inhibitors. XPAT protein unmasking occurs in two steps via one of two potential paths to the fully unmasked state. The two requisite cleavage events can occur in either order and each sequence (either the top or bottom paths shown) is equally likely. In aggregate, both 1x-N and 1x-C partially unmasked forms will exist, depending on the cleavage path. Removal of both XTEN masks liberates the unmasked HER2-TCE (uTCE). d , XPAT proteins are designed to exploit the dysregulated protease activity present in tumors versus healthy tissues and expand the therapeutic index of TCEs through preferential unmasking in the TME. The active uTCE promotes the formation of immunologic synapses between tumor and T cells, resulting in potent cytotoxicity. Notably, the uTCE has a short half-life and should be rapidly cleared, thereby sparing healthy tissues when the uTCE diffuses away from the TME. By design, the molecular weight of the uTCE (∼59 kDa) is sufficiently small to allow rapid kidney filtration .

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Activity Assay, Sequencing, Molecular Weight, Filtration