Journal: bioRxiv

Article Title: Quantitative extrapolation from single-tags (QuEST) immunofluorescence microscopy to derive TCR signalosome stoichiometries in human primary T cells

doi: 10.64898/2026.03.28.715001

Figure Lengend Snippet: a , Schematic illustrating the principle of QuEST for quantifying molecule numbers and surface densities. b , Isolation and detection of fluorescence-labelled single molecules by TIRFM. c , Workflow for quantifying single-molecule fluorescence intensity. d , e , Intensity distribution within a 5×5-pixel region ( d ) and corresponding mean fluorescence intensity ( e ) of single CD45 AF405, UCHT-1 AF488, ICAM-1 AF568, and UCHT-1 AF647 molecules. f , Workflow for quantifying fluorescence intensity of multiple molecules. g , Quantification of UCHT-1 and ICAM-1 densities on supported lipid bilayers (SLBs) containing different percentages of Ni–NTA using QuEST. h , i , Schematic ( h ) and quantification results ( i ) determining the linear dynamic range of DOPE with different fluorescence levels. j , Linear density ranges of UCHT-1 and ICAM-1 under different fluorescence conditions. k , T cells clustering UCHT-1 and ICAM-1 on SLBs, with dynamic molecular densities quantified. l , Relative errors in quantifying UCHT-1 and ICAM-1 densities using QuEST. Data in g and i are presented as mean ± SD; data in e and k are presented as mean ± SEM. Statistical significance was assessed using an unpaired two-tailed Student’s t -test (**** P ≤ 0.0001; ns, not significant). In e , data were obtained from 16 single molecules. In g and i , data were obtained from three independent TIRF images. The structure of UCHT-1 was derived from PDB entry 3FZU, and the structure of ICAM-1 was generated using AlphaFold3 from the corresponding protein sequence.

Article Snippet: Unlabelled primary antibodies : anti-human CD8α antibody (Cell Signaling Technology, Cat#85336), anti-human CD28 antibody (Cell Signaling Technology, Cat#38774S), anti-human CD45 antibody (Cell Signaling Technology, Cat#13917S), anti-human PD-1 antibody (Cell Signaling Technology, Cat#86163T), anti-human Lck antibody (Cell Signaling Technology, Cat#2787S), anti-human ZAP-70 antibody (Cell Signaling Technology, Cat#3165S), anti-human LAT antibody (Cell Signaling Technology, Cat#45533S), anti-human PLCγ1 antibody (Cell Signaling Technology, Cat#5690S), and anti-human phospho-ZAP-70 (Tyr319) antibody (Cell Signaling Technology, Cat#2701).

Techniques: Isolation, Fluorescence, Two Tailed Test, Derivative Assay, Generated, Sequencing

Journal: bioRxiv

Article Title: Combined Menin and XPO1 inhibition drive synergistic antileukemic activity in KMT2A r and NPM1 -m AML

doi: 10.64898/2026.03.10.710924

Figure Lengend Snippet: (A-C) Survival of MV4;11 cell line-derived xenograft mice treated with metronomic doses of the ziftomenib and selinexor, and in combination. (D-F) Percentage of human CD45 positive cells in the peripheral blood of MV4;11 cell line-derived xenograft mice groups two weeks post last treatment (3 mice/group) with different doses of ziftomenib and selinexor. (G) Survival of GFP/Luciferase positive OCI-AML3 cell line-derived xenograft mice treated with ziftomenib and selinexor, and with combination. (H) Bioluminescence from luciferase in different groups of GFP/Luciferase-positive OCI-AML3 cell line-derived xenograft mice.

Article Snippet: Post-necropsy immunohistochemical analysis (IHC) of mice liver and lung tissues was performed for human CD45 (Rabbit anti-human, Cell Signaling Technology, Catalog no. 13917S).

Techniques: Derivative Assay, Luciferase

Journal: bioRxiv

Article Title: Combined Menin and XPO1 inhibition drive synergistic antileukemic activity in KMT2A r and NPM1 -m AML

doi: 10.64898/2026.03.10.710924

Figure Lengend Snippet: (A-B) Survival of vehicle or inhibitor-treated KMT2A- r and NPM1 -m PDX mice. (C) Residual leukemia evaluation on treatment and post-necropsy in NPM1 -m PDX NSG mice. (D) Human (h) CD45 staining in liver biopsy (top panel) and lung biopsy (bottom panel) under the vehicle control, ziftomenib, selinexor, and combination treatment cohorts. (E) hCD45+ cell density score (IHC) in Liver and Lung tissues. (F) Residual Bone marrow hCD45% by FCM during and after treatment in control, ziftomenib, selinexor, and combination cohorts. FCM, flow cytometry. (G) Measured spleen weight post-necropsy/termination in NSG mice in all cohorts

Article Snippet: Post-necropsy immunohistochemical analysis (IHC) of mice liver and lung tissues was performed for human CD45 (Rabbit anti-human, Cell Signaling Technology, Catalog no. 13917S).

Techniques: Staining, Control, Flow Cytometry

Journal: Cell reports

Article Title: Fibroblast-derived alarmin promotes oral wound healing by activating regulatory T cells that relay pro-angiogenic and anti-inflammatory responses

doi: 10.1016/j.celrep.2025.116829

Figure Lengend Snippet: (A) H&E-stained images of oral wounds 2, 4, and 6 day post injury in Il33 f/f mice (control) or Col1a2CreERT + .Il33 f/f mice that received tamoxifen ( ΔIL33 Col1α2 ). (B) Immunofluorescence images of KRT14 (green) and COL3A1 (red) immunopositive signals across wound healing time points as shown in (A). (C) Epithelial gap (left) and area (right) normalized by palatal wound size in control and ΔIL33 Col1α2 mice. (D) Granulation tissue (left) and COL3A1 + area (right) normalized by palatal wound size in control and ΔIL33 Col1α2 mice. (E) CD45 + leukocyte numbers in the wound bed between two groups. (F) Trichrome-stained images of 4-day scalp skin wounds in control and ΔIL33 Col1α2 mice. (G) Epithelial gap (left) and new collagen area (right) from control and ΔIL33 Col1α2 mice 4 day post wounding. (H) Immunofluorescence images stained with antibodies against KRT14 (green) and COL3A1 (red) in day 4 oral wounds from control and Krt14CreERT + .Il33 f/f ( ΔIL33 Krt14 ) mice. (I) Epithelial gap and area, granulation tissue and COL3A1 + area, and CD45 + leukocyte numbers normalized by oral wound size from control and ΔIL33 Krt14 mice. Data represent mean ± SEM. n = 6–7 per group (C–E); n = 5–6 mice per group (G and I). Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001. Arrows demarcate epithelial gaps, and dashed lines encircle subepithelial wound bed. Scale bar, 500 μm.

Article Snippet: Rabbit monoclonal anti-human CD45 (clone D9M8I) , Cell Signaling Technology , Cat. #: 13917T; RRID: AB_2750898.

Techniques: Staining, Control, Immunofluorescence

Journal: Cell reports

Article Title: Fibroblast-derived alarmin promotes oral wound healing by activating regulatory T cells that relay pro-angiogenic and anti-inflammatory responses

doi: 10.1016/j.celrep.2025.116829

Figure Lengend Snippet: (A) Representative flow cytometry plot and gating strategy for determining ST2 + cell identity in day 2 oral wounds. (B) EPCAM + (epithelial), PDGFRA + (fibroblast), CD31 + (endothelial), and CD45 + (leukocyte) cells that express ST2, normalized by total ST2 + cell count per time point. (C) scRNA-seq analysis and UMAP plot of CD45 + sorted cells from day 2 wounds of control Il33 f/f mice (Ctrl) and experimental ΔIL33 Col1α2 mice. 8–10 wounds from 4–5 mice were pooled per condition. (D) Left: UMAP of subset clusters that express Il1rl1 + (encoding ST2). Right: feature plot of Il1rl1 expression in three clusters. (E) Dot plot for genes that are enriched in Treg cells, including Foxp3 . (F) Il1rl1 + cell numbers in each cluster normalized by total number of Il1rl1 + cells. Treg, regulatory T cell; MC, mast cell; ILC2, type 2 innate lymphoid cell. (G) Pseudobulk analysis of DEGs, comparing Treg cells from control versus ΔIL33 Col1α2 groups. (H) GO analysis for the upregulated gene list in control Treg cells. Biological process terms associated with immune responses are shown. (I) CellChat analysis of ligand-receptor interaction pairs. Treg cells are set as the ligand source and other leukocytes as the recipients. Communication probability from the control and ΔIL33 Col1α2 (conditional knockout [cKO]) groups are shown. (J) Violin plots of Mif and Tgfb1 expression in Treg cell cluster from control and ΔIL33 Col1α2 groups. (K) Flow cytometry plots of MIF and TGF-β1 expression in FOXP3 + Treg cells from day 2 wounds of control or ΔIL33 Col1α2 mice. (L) Mean fluorescent intensity (MFI) for MIF or TGF-β1 expression in FOXP3 + Treg cells in day 2 wounds. Data represent mean ± SEM; n = 3–5 mice per group. Student’s t test, * p < 0.05.

Article Snippet: Rabbit monoclonal anti-human CD45 (clone D9M8I) , Cell Signaling Technology , Cat. #: 13917T; RRID: AB_2750898.

Techniques: Flow Cytometry, Cell Characterization, Control, Expressing, Knock-Out

Journal: Cell reports

Article Title: Fibroblast-derived alarmin promotes oral wound healing by activating regulatory T cells that relay pro-angiogenic and anti-inflammatory responses

doi: 10.1016/j.celrep.2025.116829

Figure Lengend Snippet: (A) Top: UMAP of leukocytes from day 2 wounds in control vs. ΔIL33 Col1α2 mice. Bottom: log2fold-ratio change in cell numbers from each cluster comparing control and ΔIL33 Col1α2 mice. (B) GO analysis for the monocyte cluster in control groups truncated to seven biological processes. (C) Violin plots of Ly6c2 and Vegfa expression in each immune cluster. (D) Mif expression levels in Treg cells stimulated with saline, recombinant IL-33, or IL-33 plus soluble ST2. Left: mRNA levels by qPCR. Right: protein levels by ELISA. (E) Cytometry plots (left) and quantification (right) of migrated LY6C + monocyte numbers in Transwell experiments, where Treg cells were primed with saline, IL-33, or IL-33+ST2. (F) Migrated LY6C + monocyte numbers where IL-33-primed Treg cells were treated with isotype immunoglobulin G (IgG) or an anti-MIF (αMIF) antibody. (G) Migrated LY6C + monocytes after monocytes were treated with isotype IgG or an anti-CD74 (αCD74) antibody. (H) Left: flow cytometry plot of cells pre-gated for live CD45 + cells, expressing CD74 and LY6C from day 2 wounds of control and ΔIL33 Col1α2 mice. Right: CD74 + LY6C + cell percentage normalized by total CD45 + cells. (I) Left: immunofluorescence images of 2-day oral wounds stained with antibodies against LY6C (green) and VEGFA (red). Arrows, LY6C + VEGFA + cells; arrowheads, LY6C + VEGFA − cells. Scale bar, 50 μm. Right: LY6C + VEGFA + cells normalized by wound area. (J) Immunofluorescence images of oral wounds stained with CD31 (white) antibody in control and ΔIL33 Col1α2 mice. Dashed lines encircle connective tissues. Scale bar, 500 μm. (K) Blood vessel numbers in oral wounds across healing time points from control and ΔIL33 Col1α2 mice, raw count per wound (left), and normalized by stromal tissue area (right). Data represent mean ± SEM. n = 3 for (D)–(G), in which each N is an independent experiment from individual control mouse; n = 6–10 mice each for (H)–(K). Student’s t test (F–K) or one-way ANOVA and post hoc test (D and E); * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Rabbit monoclonal anti-human CD45 (clone D9M8I) , Cell Signaling Technology , Cat. #: 13917T; RRID: AB_2750898.

Techniques: Control, Expressing, Saline, Recombinant, Enzyme-linked Immunosorbent Assay, Cytometry, Flow Cytometry, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Fibroblast-derived alarmin promotes oral wound healing by activating regulatory T cells that relay pro-angiogenic and anti-inflammatory responses

doi: 10.1016/j.celrep.2025.116829

Figure Lengend Snippet: (A) H&E staining and immunofluorescence images of normal human gingiva and skin. Dashed lines demarcate the boundaries of reticular connective tissue. Scale bar, 500 μm. Inset: respective areas (i–iv) in gingiva and skin, showing immunopositive signals for IL-33, PI16, and CD31. Scale bar, 50 μm. (B) PI16 + IL33 + cell counts per area in the deeper connective tissue of skin dermis and oral lamina propria. (C) Illustrative diagram of inadvertent implant abutment removal and tissue resection for histological assessment. (D) H&E staining and immunofluorescence images of human gingiva that “healed” for 4 days after abutment removal; n = 3 individual patients (56M, 56F, and 54M). Scale bar, 500 μm. Inset: deeper lamina propria showing immunopositive signals for KI67, KRT14, and CD45 antigen. Scale bar, 100 μm. (E) RNAscope in situ hybridization images with specific probes against FOXP3 , MIF , and TGFB1 . The upper and lower aspect of healing gingival connective tissue is shown. Scale bar, 100 μm. Inset: representative Treg cells in the upper (i) and lower (ii) lamina propria (LP). Scale bar, 5 μm. (F) FOXP3 + MIF + (left) and FOXP3 + TGFB1 + Treg cell count per area (right) from the upper versus lower lamina propria in healing gingiva. (G) MIF and TGFB1 puncta in FOXP3 + cells from the lower lamina propria. Data represent mean ± SEM; n = 5–7 individuals per group (B), 3 specimens (F), and 30 fields of view (FOV) (G) from three patients. Student’s t test, * p < 0.05, ** p < 0.01.

Article Snippet: Rabbit monoclonal anti-human CD45 (clone D9M8I) , Cell Signaling Technology , Cat. #: 13917T; RRID: AB_2750898.

Techniques: Staining, Immunofluorescence, RNAscope, In Situ Hybridization, Cell Characterization

Journal: Cell Reports Medicine

Article Title: Clinical and molecular dissection of CAR T cell resistance in pancreatic cancer

doi: 10.1016/j.xcrm.2025.102301

Figure Lengend Snippet: Locally delivered huCART-meso cells are enriched for GzmK + Tem phenotypes and exhibit a dysfunctional T cell phenotype including upregulation of SOX4 and ID3 (A) Summary of the samples collected for flow cytometry and scRNA-seq analyses. (B) Representative flow cytometry plot of patient 04 showing detection of peritoneal fluid infiltrating CD3 + cells with surface expression of the huCART-meso. (C and D) CAR detection performed by qPCR on peripheral blood (left) and peritoneal fluid (right) of patient 04 (C) and patient 05 (D). (E) UMAP projection of scRNA-seq data from sorted CD3 + CD45 + cells infiltrating the peritoneal fluid 7 days post intraperitoneal infusion (patient 04) or 26 days post infusion (patient 05). UMAP plot is labeled with T cell subsets. (F) huCART-meso expression associated with (E). (G) Distribution of huCAR-meso+ (CARpos) and endogenous CAR-negative T cells (CARneg) across cell type clusters defined in (E). Unknown cluster from (E) was excluded. The y axis represents the percent of cells found within each cell type cluster, calculated by taking the number of cells per condition (condition being CARpos or CARneg) within each cell type cluster and dividing by the total number of cells in that condition, calculated for each patient individually. Dots represent the individual patient values, and the bar plot represents the average of the patient values; moderated t test via the propeller method ( n = 2 patients). p values denoted as ∗ p < 0.05 and ∗∗ p < 0.01. (H) Expression of the 30-gene CAR T dysfunction signature in CD3 + CARpos and CARneg T cells of patients 04 and 05 post infusion. (I) Gene expression levels of huCART-meso, SOX4, ID3, SRGAP3, NDFIP2, CTLA4, TNFRSF9, PDCD1, HAVCR2, and TIGIT in CARpos (left, red) and CARneg cells (right, green). CARneg cells were randomly downsampled to have an equal number of CARpos and CARneg cells for analyses in (G)–(I). See also .

Article Snippet: Anti-human CD45 (CD45-LCA) , Cell Signaling Technology , Cat# 13917; RRID: AB_2750898.

Techniques: Flow Cytometry, Expressing, Labeling, Gene Expression

Journal: Cell Reports Medicine

Article Title: Clinical and molecular dissection of CAR T cell resistance in pancreatic cancer

doi: 10.1016/j.xcrm.2025.102301

Figure Lengend Snippet: Mice injected with single KO of SOX4 or ID3 eventually experience tumor recurrence (A) Experimental design for recurrent tumor model. (B) KO efficiency in sorted T cells from relapsing tumors. Each dot represents a technical replicate (independent sequencing for KO assessment from one mouse per group); mean ± SEM. (C) CD3 + CD45 + T cell infiltration in ND561 WT and SOX4KO relapsing tumors (left) and percent of CD3 + CD45 + surface huCART-meso in relapsing tumors and infused products (right). Each dot represents a technical replicate ( n = 1 mouse per group); Mann-Whitney U test (left) and two-Way ANOVA and Sidak post hoc test (right); mean ± SEM. (D) Total number of MSLN + Epcam + cells (#) per gram of tumor (left) and representative flow cytometry plots (right) of MSLN expression in ND561 WT and SOX4KO-treated relapsing tumors ( n = 1 mouse per group). AsPC-1 cells were used as positive control and MSLN minus as negative control for MSLN staining; Mann-Whitney U test. Error bars represent variation between technical replicates; mean ± SEM. (E) UMAP projection of scRNA-seq data from recurrent TILs harvested from mice injected with WT CAR T cells (ND561, n = 1 donor) and SOX4-KO CAR T cells (ND561 and ND539, n = 2 donors). (F) UMAP projection with broad T cell subsets labeled. NK-like T cells clustered on the left (cluster 2) and all other clusters were defined as GZMK + exhausted T cells (Tex), right. (G) huCART-meso expression in recurrent tumor TILs. (H) Cluster distribution depicting the frequency that WT and SOX4KO-injected recurrent tumor TILs were found within the broad clusters defined in (F). y axis represents the percent of cells found within each cell type cluster for each condition (WT or SOX4KO). The percent was calculated by taking the number of cells per condition within each cell type cluster and dividing by the total number of cells in that condition. SOX4KO condition was averaged for the two mice and visualized via bar plot, and circles depict the individual values for each mouse; moderated t test using the propeller method. (I) Gene expression levels of TNFRSF9, TIGIT, LAG3, and HAVCR2 in recurrent tumor CARpos TILs extracted from mice injected with WT and SOX4KO CAR T cells. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ns: not significant. See also .

Article Snippet: Anti-human CD45 (CD45-LCA) , Cell Signaling Technology , Cat# 13917; RRID: AB_2750898.

Techniques: Injection, Sequencing, MANN-WHITNEY, Flow Cytometry, Expressing, Positive Control, Negative Control, Staining, Labeling, Gene Expression

Journal: Frontiers in Cardiovascular Medicine

Article Title: Case Report: A heterozygous loss-of-function variant of the ERG gene in a family with vascular pathologies

doi: 10.3389/fcvm.2025.1550523

Figure Lengend Snippet: Multiple aneurysms in an individual with ERG haploinsufficiency. (A) 3-dimensional reconstruction of the computed tomography angiography of the index patient revealed multiple aneurysm formation. Red arrows indicate aneurysmatic vascular segments. (B) Family pedigree including the index patient with multiple arterial aneurysms (age 52 years) and an affected son (18 years) with cervical artery dissection and the same ERG gene variant. Other family members were not available for Sanger sequencing analysis. WT/WT: wild type homozygous, WT/MUT: heterozygous variant, white coloration: investigated without phenotype, black coloration: affected individual, grey: clinically not investigated. (C) Immunohistochemically and magnification (indicated by black rectangles) stained sections comparing the abdominal aortic aneurysm (AAA) of the index patient and a healthy control aorta. Staining of sequential histological sections with antibodies for ERG, CD31, αSMA and CD45 revealed expression of ERG exclusively in individual endothelial cells (red arrows) of the vasa vasorum in a similar extent and pattern as in healthy aortas. Scale bars: 100 μm (top) and 50 μm (magnification sections). Red arrows indicate ERG-positive cells in the area of the tunica media (index patient) and in the transition zone between the tunica media and tunica adventitia (control sample).

Article Snippet: Immunohistochemical staining was performed overnight with the following primary antibodies: rabbit anti-human ERG (# 97249, Cell Signaling Technology); mouse anti-human CD31 (# 3528, Cell Signaling Technology), rabbit anti-human alpha smooth muscle actin (αSMA) (#19245, Cell Signaling Technology), rabbit anti-human CD45 (# 13917, Cell Signaling Technology).

Techniques: Computed Tomography, Dissection, Variant Assay, Sequencing, Staining, Control, Expressing