Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Schematic of differentiation heterogeneity in the OCI-AML8227 cell line model. This cell line produces leukemic blasts expressing immature myeloid markers (CD34) as well as differentiated monocytic markers (CD64 and CD14). CD38 is a transient myeloid differentiation marker, which is initially expressed in early myeloid progenitor cells. b. OCI-AML8227 cells were immunomagnetically fractionated by CD34 expression and cultured in triplicate for 96 hours along an 8-point dose curve with venetoclax. Cell viability was assessed by CellTiter Aqueous colorimetric assay. c. AUC values from the dose-response curves for each immunophenotypic population. Significance was evaluated using a two-tailed t-test. d–i. Cell surface expression of CD34, CD38, CD64, and CD14 in OCI-AML8227 cells cultured in triplicate for 72 hours with 1 μM venetoclax or an equivalent volume of DMSO. Cells were analyzed either as immunomagnetically fractionated populations (CD34-enriched and CD34-depleted) or as unfractionated cells. Significance was evaluated using ordinary one-way ANOVA followed by Holm-Šidák post-test correction. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Expressing, Marker, Cell Culture, Colorimetric Assay, Two Tailed Test

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a, b. OCI-AML8227 cells were immunomagnetically fractionated by CD34 expression and cultured in triplicate for 30 minutes or 6 hours following treatment with 1 μM venetoclax or an equivalent volume of DMSO. Proteins were extracted from cell pellets and either separated by liquid chromatography and analyzed by tandem mass spectrometry for protein identification and quantification or enriched for phosphorylated species prior to LC–MS/MS analysis to profile phosphorylation-dependent signaling. Relative (a) global protein and (b) phosphoprotein abundance at 6 hours following venetoclax treatment in CD34-enriched and CD34-depleted populations were analyzed using proteomic network analysis.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Expressing, Cell Culture, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Predicted transcription factor activity was evaluated in single-cell RNA-seq data presented in . Significantly enriched transcription factors were identified and denoted as enriched in progenitor (blue), monocytic (gold), or both (green) populations. Gray dots represent transcription factors that did not reach statistical significance (FDR < 0.05). b. Predicted activity of PU.1 visualized by each time point and drug condition. c. Pseudotime values generated in were used to determine each cell’s relative position along the myeloid differentiation trajectory. Predicted activity of PU.1 is visualized along its pseudotime trajectory in each drug condition. d. OCI-AML8227 cells were cultured with DMSO or 1 μM venetoclax and processed at either 0 or 72 hours for single-cell ATAC-seq. Differential peaks were identified from open chromatin regions in each condition. Significant peaks were compared between monocytic and progenitor clusters. Gray dots represent motifs that did not reach statistical significance (FDR < 0.05). e, f. Transcription factor motif analysis was performed on (e) upregulated and (f) downregulated regions in monocytic cells relative to progenitor cells as described in panel d. Gray dots represent motifs that did not reach statistical significance (FDR < 0.05). g. Venn diagram displaying the overlap of significantly dysregulated transcription factors identified from single-cell RNA-seq analysis in panel a compared to enriched motifs identified in panels e and f.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Activity Assay, RNA Sequencing, Generated, Cell Culture

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Schematic of the targeted CRISPR screen. A stable Cas9-expressing OCI-AML8227 cell line was generated by electroporation and sorted for GFP expression. Cells were transduced with three guide RNAs per transcription factor target by electroporation, then cultured in triplicate with DMSO or 1 μM venetoclax for 72 hours before flow cytometry analysis of CD34, CD38, CD64, and CD14 surface expression. Image was created with BioRender. b. Live cell counts determined by forward/side scatter gating, excluding DAPI-stained cells. Counts were normalized to the average of their respective DMSO-treated controls. Blue lines and asterisks indicate comparisons with the safe-harbor locus AAVS1 (control) whereas black lines and asterisks indicate intra-sample comparisons. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. c–h. Live cell counts for each immunophenotypic population, normalized as described in panel b. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. i–j. Representative flow cytometry plots showing CD64-FITC and CD38-APC-Fire surface expression in OCI-AML8227 cells transduced with guide RNAs targeting AAVS1 or SPI1 following treatment with DMSO or 1 μM venetoclax. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: CRISPR, Expressing, Generated, Electroporation, Transduction, Cell Culture, Flow Cytometry, Staining, Control

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Cells were transduced with three guide RNAs per transcription factor target by electroporation, then cultured in triplicate with DMSO or 1 μM venetoclax for 72 hours before flow cytometry analysis of CD34, CD38, CD64, and CD14 surface expression as described in . Quantification of total cell counts in additional knockout models of OCI-AML8227 cells. Total cell counts were normalized to the average of their respective DMSO-treated controls. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. Sensitivity to venetoclax was unchanged by the experimental knockouts compared to control AAVS1 . b–g. Live cell counts for each immunophenotypic population were calculated by multiplying their proportions of live single cells from flow cytometric analysis by their normalized values in panel a. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. Few changes were observed in knockouts relative to control. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Transduction, Electroporation, Cell Culture, Flow Cytometry, Expressing, Knock-Out, Control

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. OCI-AML8227 cells were cultured in triplicate for 72 hours along an 8-point dose curve of DB2313. Cell viability was assessed by CellTiter Aqueous colorimetric assay. b. OCI-AML8227 cells were treated in triplicate with an 8×8 dose matrix of DB2313 and venetoclax for 72 hours prior to viability assessment by CellTiter Aqueous colorimetric assay. Zero interaction potency (ZIP) synergy scores were calculated on the average values for each drug dose. The white box indicates the DB2313 and venetoclax concentrations corresponding to maximal synergy. c–e. Live cell counts in OCI-AML8227 cells following 72 hours of treatment with 0.1 μM venetoclax, 0.5 μM DB2313, both drugs in combination, or an equivalent volume of DMSO. Live cell counts were determined by forward/side scatter gating and exclusion of DAPI-stained cells, then normalized to DMSO-treated controls. Cells were analyzed by flow cytometry for CD34, CD38, CD64, and CD14 surface expression. Quantification of live cell counts for the remaining cell surface markers is shown in . Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. f–h. Live cell counts in OCI-AML8227 cells following 72 hours of treatment with 1 μM venetoclax, 5 μM DB2313, both drugs in combination, or an equivalent volume of DMSO. Analysis was performed as described in panels c–e. i. Transcriptional signatures of nine primary AML samples selected for drug sensitivity evaluation are shown. One sample was excluded from downstream analyses due to widespread cell death (18-00105). j. Primary AML blasts from eight patients with stem-monocytic AML were cultured in triplicate for 72 hours along a 7-point dose curve with venetoclax, DB2313, or equimolar amounts of the drug combination. Viability was assessed using the Guava/EMD Millipore platform after a short incubation with Guava Nexin Reagent (Annexin V–PE + 7-AAD). ZIP synergy scores were calculated from averaged viability data across replicates for each drug dose in primary AML blasts shown in panel i. The white box indicates the DB2313 and venetoclax concentrations corresponding to maximal synergy. k. Venetoclax dose-response curves for each patient at a fixed dose of 1.25 μM DB2313, which corresponds to maximal synergy in panel j. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Cell Culture, Colorimetric Assay, Staining, Flow Cytometry, Expressing, Incubation

Journal: European Journal of Immunology

Article Title: LILRA5 Functions to Induce ROS Production on Innate Immune Cells

doi: 10.1002/eji.70079

Figure Lengend Snippet: A highly specific anti‐LILRA5 antibody with cross‐linking capacity. (A) SDS‐PAGE analysis of rLILRA5‐His. (B) Binding of anti‐LILRA5 P4‐11A mAb to magnetic beads coated with rLILR or control protein, detected using anti‐IgG mAb and flow cytometric analysis. Mean ± SD of n = 3 independent experiments. Paired t ‐test. (C) Binding of anti‐LILRA5 clone P4‐11A mAb to U937 cell lines, detected using anti‐IgG mAb and flow cytometric analysis. One representative experiment from n = 3 independent experiments. (D) A schematic of the LILRA5CD3ζ reporter 2B4 T cell line, expressing a surface protein composed of extracellular and transmembrane LILRA5 domains fused to the cytoplasmic tail of CD3ζ. Cross‐linking of CD3 or the fusion LILRA5CD3ζ protein induces ITAM phosphorylation, NFAT activation and GFP expression. (E) Reporter cells stimulated with anti‐CD3 mAb, anti‐LILRA5 P4‐11A mAb, or isotype control. Mean ± SD of n = 4 independent experiments. (F) Detection of rLILRA5‐His by ELISA using anti‐LILRA5 P4‐11A mAb or isotype control. Mean and SD of n = 3 experiments. In all, * p < 0.05 and **** p < 0.0001.

Article Snippet: After transduction into 2B4 NFAT‐GFP T cells, the selected transductant cells were tested for surface LILRA5 expression using anti‐LILRA5 pAb (16059‐RP01; Sino Biological) and flow cytometry analysis.

Techniques: SDS Page, Binding Assay, Magnetic Beads, Control, Expressing, Phospho-proteomics, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: European Journal of Immunology

Article Title: LILRA5 Functions to Induce ROS Production on Innate Immune Cells

doi: 10.1002/eji.70079

Figure Lengend Snippet: LILRA5 is expressed on human phagocytes and stimulates ROS production. (A) Normalised transcripts per million (nTPM) of LILRA5 in immune cells from the HPA and Monaco datasets. Mean ± SD are shown. (B) Representative example showing the gating strategy used to identify monocytes in human whole blood using anti‐CD14 and anti‐LILRA5 (clone P4‐11A). A first gate was set on physical parameters of SSC‐A vs. FSC‐A, then on SSC‐A and SSC‐H to eliminate doublets, then monocytes and granulocytes were gated on CD14+ and CEACAM8+ (not shown), then on CD14+ events to identify monocytes. (C) Representative flow cytometry staining indicating LILRA5 expression in neutrophils and monocytes from a healthy donor. (D, E) Expression of LILRA5 on human monocytes (D, n = 8) and neutrophils (E, n = 12), determined using anti‐LILRA5 P4‐11A or isotype control. Paired t ‐test. (F, G) Reactive oxygen species (ROS) production by PBMCs (F, n = 7) and neutrophils (G, n = 8). One‐way ANOVA of area under the curve (AUC) values, in all, **** p < 0.0001, ** p < 0.01, and * p < 0.05.

Article Snippet: After transduction into 2B4 NFAT‐GFP T cells, the selected transductant cells were tested for surface LILRA5 expression using anti‐LILRA5 pAb (16059‐RP01; Sino Biological) and flow cytometry analysis.

Techniques: Flow Cytometry, Staining, Expressing, Control

Journal: European Journal of Immunology

Article Title: LILRA5 Functions to Induce ROS Production on Innate Immune Cells

doi: 10.1002/eji.70079

Figure Lengend Snippet: LILRA5 expression during sepsis and systemic infections. LILRA5 expression in whole blood of (A) healthy donors ( n = 83), sepsis patients ( n = 156; GSE134364 ). (B) healthy donors ( n = 12) or sepsis patients ( n = 13), septic shock patients ( n = 6; GSE137342 ). (C) of healthy donors ( n = 44) or sepsis patients presenting at emergency rooms (ER) ( n = 266), sepsis patients on intensive care units (ICU; n = 82; GSE185263 ). (D) of healthy donors ( n = 8), sepsis patients ( n = 20; GSE232753 ). (E) of healthy donors ( n = 40), patients with diagnosed but uncomplicated systemic infection ( n = 12), sepsis patients ( n = 20), septic shock patients ( n = 19; GSE154918 ). (F) LILRA5 expression in neutrophils from healthy donors ( n = 8) or sepsis patients ( n = 15; GSE64457 ). LILRA5 expression in CD14+ monocytes from (G) healthy donors ( n = 6), sepsis patients ( n = 8; GSE180387 ), (H) from healthy donors ( n = 4), sepsis patients ( n = 6; GSE136200 ), (I) from healthy donors ( n = 5), ICU patients with sepsis ( n = 4; GSE139913 ). (J) LILRA5 expression in whole blood from healthy donors ( n = 14), patients with bacterial infections ( n = 24), and patients with viral infections ( n = 28; GSE72810 ). (K) LILRA5 expression in whole blood from healthy donors ( n = 43), patients with E. coli infection ( n = 32), patients with S. aureus infection ( n = 19; GSE33341 ). In all panels, mean ± SD are shown. Statistics tested by limma , where adjusted **** p < 0.0001, *** p < 0.001, and * p <0.05.

Article Snippet: After transduction into 2B4 NFAT‐GFP T cells, the selected transductant cells were tested for surface LILRA5 expression using anti‐LILRA5 pAb (16059‐RP01; Sino Biological) and flow cytometry analysis.

Techniques: Expressing, Infection

Journal: European Journal of Immunology

Article Title: LILRA5 Functions to Induce ROS Production on Innate Immune Cells

doi: 10.1002/eji.70079

Figure Lengend Snippet: Surface LILRA5 expression does not increase during infection and inflammation. (A) Whole blood LILRA5 expression after ex vivo infection with E. coli ( n = 5, control n = 8) or S. aureus ( n = 5, control n = 8; GSE65088 ). Mean ± SD are shown. Statistics tested by limma . (B) Expression of LILRA5 on human monocytes after ex vivo infection of whole blood by E. coli or S. aureus , from n = 3 independent experiments. (C) Surface LILRA5 expression on human monocytes from healthy donors ( n = 8) and sepsis patients ( n = 26), upon hospital admission. Mean ± SD are shown. (D) Surface LILRA5 expression on human monocytes from sepsis patients ( n = 8) at the indicated days since hospital admission. (E) Comparison of sLILRA5 in serum from sepsis patients ( n = 128) or healthy donors ( n = 60). Mean ± SD are shown. Student t ‐test. In all, **** p < 0.0001, *** p < 0.001. ** p < 0.01, and * p < 0.05.

Article Snippet: After transduction into 2B4 NFAT‐GFP T cells, the selected transductant cells were tested for surface LILRA5 expression using anti‐LILRA5 pAb (16059‐RP01; Sino Biological) and flow cytometry analysis.

Techniques: Expressing, Infection, Ex Vivo, Control, Comparison

Journal: European Journal of Immunology

Article Title: LILRA5 Functions to Induce ROS Production on Innate Immune Cells

doi: 10.1002/eji.70079

Figure Lengend Snippet: LPS activation reduces LILRA5‐dependent ROS production. (A) LILRA5 expression by monocytes cultured ± LPS for 18 h ( GSE147310 ). Data from n = 5 donors. Statistics tested by limma . (B) Surface LILRA5 expression on human monocytes from healthy donors ( n = 3). Student t ‐test. (C) sLILRA5 in culture supernatants from PBMCs from healthy donors ( n = 4), after 18 h culture ± LPS. Student t‐test. (D, E) Production of ROS by PBMCs in response to LILRA5 ± LPS stimulation. Data from n = 5 independent donors. Raw ROS production values are shown in (D). Student t‐ test. The ROS production induced by anti‐LILRA5 P4‐11A relative to IgG1 is compared through area under the curve (AUC), as shown in (E). In all, ** p < 0.01, * p < 0.05.

Article Snippet: After transduction into 2B4 NFAT‐GFP T cells, the selected transductant cells were tested for surface LILRA5 expression using anti‐LILRA5 pAb (16059‐RP01; Sino Biological) and flow cytometry analysis.

Techniques: Activation Assay, Expressing, Cell Culture

Journal: Diagnostic microbiology and infectious disease

Article Title: Interlaboratory study to assess precision and reproducibility of the meningococcal antigen surface expression (MEASURE) assay to quantify factor H binding protein expression at the surface of meningococcal serogroup B strains

doi: 10.1016/j.diagmicrobio.2025.116920

Figure Lengend Snippet: MEASURE data collected for 42 MenB strains. Geometric mean MFI values from each of the 3 laboratories as well as those collected from Pfizer approximately 2 years before the interlaboratory study (“Pfizer [historic]”) are presented for each strain. CDC = US Centers for Disease Control and Prevention; MEASURE = Meningococcal Antigen Surface Expression; MenB = meningococcal serogroup B; MFI = mean fluorescence intensity; UKHSA = UK Health Security Agency.

Article Snippet: Pfizer therefore developed the flow-cytometry–based Meningococcal Antigen Surface Expression (MEASURE) assay as a reliable method of accurately quantifying fHbp surface expression to establish a predictive hSBA killing threshold [ ].

Techniques: Control, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) Flow cytometric analysis of ADGRE2 surface expression and number of antigens per cell (APC) on primary AML bone marrow samples. Data shown for AML blasts (CD45 dim SSC lo ; n=25) and leukemic stem cell (LSC)-enriched compartments (CD34 + CD38 - ; n=20). Each data point represents an individual patient. Quantification of ADGRE2 expression was performed using a standard curve generated by QuantiBRITE beads and interpolation of geometric mean fluorescence intensity values of positively expressing cells. Data represented as box-and-whisker plots indicating the 25 th , 50 th (median), and 75 th percentiles; whiskers denote minimum and maximum values. (B) EMR2-directed binders were identified by phage display panning against single-chain variable fragment (scFv) and heavy chain variable region (VH) libraries. Binder kinetic and affinity characterization (EC50 and KD) were performed by multipoint flow cytometric, ELISA and Blitz analyses. (C) Schematic representation of ADGRE2 full-length (FL) and splice isoforms, FL CD97, and a chimeric construct in which the native ADGRE2 GAIN and GPS domains were replaced with the corresponding regions from CD97. Illustration created using BioRender. (D) Binding specificity of ADGRE2-directed CAR binders to ADGRE2 isoforms, FL CD97, and the chimeric ADGRE2-CD97 construct (GAIN and GPS replacement) expressed on the surface of HEK293T cells following transient transfection. CAR constructs were tagged with a C-terminal FLAG peptide; binding was assessed by flow cytometry and anti-FLAG secondary detection. (E-F) In vitro cytotoxicity of ADGRE2-directed CAR Ts (ADGRE2-1-scFv, ADGRE2-2-scFv, ADGRE2-5-scFv, ADGRE2-6-VH, ADGRE2-8-VH, or ADGRE2-9-VH) or untransduced T cells (UTD) against MOLM-13 wild-type (WT) and ADGRE2-knockout (KO) targets at an effector-to-target (E:T) ratio of 1:1. Cytotoxicity assessed at 24h and 48h by flow cytometry; viable targets defined as Annexin-V - /LIVE-DEAD - . Each data point represents an individual T cell donor; data shown as mean ± SD from 2-3 donors. Two-way ANOVA with Šidák multiple comparisons test was performed to compare viability of targets in co-culture with CAR T cells to UTD, averaging both timepoints; *p < 0.05, **<0.01, ***p < 0.001, ****p<0.0001. (G) T cell activation measured by CD25 surface expression after 48h co-culture. UTD and effector-alone conditions served as controls. Data shown as mean ± SD from 2-3 donors; each data point represents a donor. (H) Cytokine secretion profile of ADGRE2-directed CAR Ts and UTD controls following 48 h co-culture with MOLM-13 WT or ADGRE2-KO targets at a 1:1 E:T ratio. Supernatants were analyzed for IFNγ, TNFα, IL-2, sFasL, and Granzyme B. Effector-alone controls included. Data shown as mean ± SD from 1-3 donors, each dot represents a donor. Abbreviations: scFv, single-chain variable fragment; VH, heavy chain variable domain; GAIN, G-protein-coupled receptor (GPCR) autoproteolysis-inducing domain; GPS, GPCR proteolytic site; 7TM, 7-pass transmembrane domain; SD, standard deviation; ns, not significant; IFNγ, interferon gamma; TNFα, tumor necrosis factor alpha; IL-2, interleukin-2; sFasL, soluble Fas ligand; N.D., not detected.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: Expressing, Generated, Fluorescence, Whisker Assay, Enzyme-linked Immunosorbent Assay, Construct, Binding Assay, Transfection, Flow Cytometry, In Vitro, Knock-Out, Co-Culture Assay, Activation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) Flow cytometric analysis of ADGRE2 surface expression on MOLM-13 wild-type (WT) and lentivirus engineered clones. ADGRE2 surface quantification was completed using QuantiBRITE bead interpolation. Data shown as mean ± SD from n=3 independent measurements. (B) Representative flow cytometry plots of ADGRE2-5-scFv CAR surface expression (anti-human IgG H+L) from n=3 T cell donors. (C) Geometric mean fluorescence intensity (gMFI) of ADGRE2-5-scFv CAR expression in transduced T cells from panel (B). Data shown as mean ± SD; each data point represents a technical replicate (n=3). (D) In vitro cytotoxicity assessment of untransduced (UTD) or ADGRE2-5-scFv (CAR) T cells against MOLM-13 ADGRE2-KO, Clone 1, Clone 2, or WT targets at a 1:4 effector-to-target (E:T) ratio. Viability was assessed after 48 h by flow cytometry (Annexin-V - /LIVE-DEAD - ). Target-alone cultures served as controls. Data represented as mean ± SD from n=3 T cell donors. (E) ADGRE2-specific killing of MOLM-13 clones and WT at a 1:4 E:T ratio, calculated as ( Viability untreated – Viability treated )/ Viability untreated x 100 followed by subtracting killing observed in KO targets for background correction. Data represented as mean ± SD from n=3 T cell donors. (F) Effector activation measured by CD25 surface expression after 48 h co-culture with indicated MOLM-13 targets; flow cytometry analysis. Effector-alone condition included as a control. Data shown as mean ± SD from n=3 donors. (G) Correlation between ADGRE2-5-scFv T cell activation (CD25 + ) and ADGRE2 surface intensity (antigens per cell, APC) on MOLM-13 targets at a 1:4 effector-to-target (E:T) ratio. Data shown as mean ± SD. Pearson correlation efficient (r) and linear regression analysis were generated using GraphPad Prism. Abbreviations: SD, standard deviation; KO, knockout.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: Expressing, Clone Assay, Flow Cytometry, Fluorescence, In Vitro, Activation Assay, Co-Culture Assay, Control, Generated, Standard Deviation, Knock-Out

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) Flow cytometric analysis of ADGRE2 percent surface expression and antigens per cell (APC) on healthy bone marrow (n=3) and peripheral blood (n=5) hematopoietic cell subsets. Quantification of ADGRE2 expression was performed by interpolation of PE geometric mean fluorescence intensity (gMFI) values using a QuantiBRITE bead-generated standard curve. Data represented as median values; each point represents an individual donor sample. One donor showed undetectable ADGRE2 expression on lymphocytes by QuantiBRITE interpolation. (B) Heatmap summarizing median ADGRE2 APC across healthy hematopoietic and AML disease populations. (C) Schematic of ADGRE2 gene structure (not drawn to scale) with indicated start/stop codons (triangles) and associated coding regions. Positions of reported homozygous predicted loss-of-function (pLOF) variants and the number of affected individuals are indicated (arrows). Exonic regions analyzed by ddPCR are also shown. (D) Summary table of ADGRE2 homozygous pLOF natural variants identified in gnomAD v4.1.0. (E) Schematic of full-length ADGRE2 protein with indicated locations of pLOF variants (A-H; arrows). Wild-type (WT) and variant coding sequences were cloned into pcDNA3.1(+)-IRES-eGFP expression plasmids, with an N-terminal HA tag inserted in each construct. General binding regions for the anti-ADGRE2 (clone 2A1) and anti-HA antibody (clone 16B12) used in flow cytometry and Western blotting are shown. (F) Surface expression (geometric mean fluorescence intensity, gMFI) of ADGRE2 and HA in HEK293T cells transfected with WT and variant (A-H) ADGRE2 plasmids; measured by flow cytometry 24 h post-transfection and normalized to WT. Empty vector (em.) and non-transfected cells served as controls. Data represented as mean ± SD; n=2 biological replicates. Representative Western blot of total ADGRE2 and HA protein expression; β-actin served as a loading control. (G) Transcript expression of ADGRE2 exons 10-12 and exon 6 in healthy bone marrow (BM; n=6), CD34 + hematopoietic stem and progenitor cells (HSPC) day 2 post-thaw (n=9), and AML bone marrow samples at diagnosis (n=10) and relapse (n=10); measured by digital droplet PCR (ddPCR) and normalized to GUSB (glucuronidase beta) for each sample. (H) Base editing efficiency of Variants D and H in CD34 + HSPCs using BE4-PpAPOBEC1 or BE4-PpAPOBEC-SpG guide-RNAs targeting respective variant regions. Editing was assessed on day 5 post-electroporation by next-generation-sequencing (rhAmpSeq). “On-Target” represents reads with the intended C-to-T edit and no bystander conversions; “Bystander” includes reads with off-target C-to-T conversions alone or in combination with the intended on-target edit. ADGRE2 surface expression was assessed in parallel by flow cytometry and normalized to non-edited control HSPC cells; n=1 biological replicate. Abbreviations: HSC, hematopoietic stem cells; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythroid progenitor; CLP, common lymphoid progenitor; cDCs, classical dendritic cell; pDCs, plasmacytoid dendritic cells; NK, natural killer cell; HSPC, hematopoietic stem and progenitor cells; ddPCR, digital droplet PCR; SD, standard deviation.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: Expressing, Fluorescence, Generated, Variant Assay, Clone Assay, Construct, Binding Assay, Flow Cytometry, Western Blot, Transfection, Plasmid Preparation, Control, Biomarker Discovery, Electroporation, Next-Generation Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) CD34 + hematopoietic stem and progenitor cells (HSPCs) were electroporated with ADGRE2-targeting CRISPR-Cas9-ribonucleoprotein (RNP) complex (KO-Cas9) or a non-editing control RNP (Control). Two days post-electroporation (EP), cells were cultured for 14 days under monocytic differentiation conditions (indicated by dashed line and shaded grey). Editing frequency was assessed by ICE analysis and ADGRE2 surface expression was measured by flow cytometry. Data shown as mean ± SD; n=2 donors. (B) Cell viability and concentration throughout the 16-day in vitro culture. Quantified by cell counter; data shown as mean ± SD; n=2 donors. (C) Phenotypic analysis of differentiating cells by flow cytometry using CD11b (pan-myeloid marker), CD33 (myeloid marker), CD14 (monocytic lineage marker), and CD15 (granulocytic lineage marker). Data shown as mean ± SD; n=2 donors. (D) Inflammatory cytokine secretion (IL-6, MIP-1α, TNFα) evaluated in monocytic differentiated cells, collected from supernatant at study end. Data shown as mean ± SD; each dot represents a donor. Statistical analysis was performed using two-way ANOVA comparing basal condition versus LPS stimulation, or basal condition versus R848 (TLR7/8 agonist) stimulation; *p < 0.05, ***p < 0.001. Two-way ANOVA with Šidák multiple comparisons test was performed to compare KO-Cas9 to Control for each stimulation condition. (E) Schematic representation of the lead ABE8.20-m guide-RNA (gRNA) targeting the splice donor site at the end of exon 19 of ADGRE2 . On-target A-to-G editing site is bolded and indicated by an arrow. (F) CD34 + HSPC were electroporated with ABE8.20-m mRNA and either an ADGRE2 -targeting gRNA (KO-ABE) or non-targeting control (Control) and cultured for 5 days. Editing outcomes—splice-site disruption (intended A-to-G edit), missense conversions, and insertions/deletions (InDels)—were quantified by next-generation sequencing (rhAmpSeq). ADGRE2 surface protein expression was measured by flow cytometry. Data represented as mean ± SD; n=4 donors. (G) Frequency of HSPC subpopulations 48 h post-electroporation (KO-ABE and Control), measured by flow cytometry. Subsets included CMPs, MPPs, MLPs, and LT-HSCs, expressed as percentage of total live cells. Cells were bulk sorted and editing frequency of splice-site disruption was assessed by rhAmpSeq. Data shown as mean ± SD; n=2 donors. Statistical analysis by ANOVA with Šidák multiple comparisons test. Abbreviations: KO, knockout; SD, standard deviation; IL-6, interleukin-6; MIP-1α, macrophage inflammatory protein-1 alpha; TNFα, tumor necrosis factor alpha; LPS, lipopolysaccharide; TLR, toll-like receptor; CMP, common myeloid progenitors; MPP, multipotent progenitors; MLP, multi-lymphoid progenitors; LT-HSCs, long-term hematopoietic stem cells.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: CRISPR, Control, Electroporation, Cell Culture, Expressing, Flow Cytometry, Concentration Assay, In Vitro, Marker, Disruption, Next-Generation Sequencing, Knock-Out, Standard Deviation

Journal: bioRxiv

Article Title: Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

doi: 10.1101/2025.08.21.671614

Figure Lengend Snippet: (A) Experimental schema for 16-week xenotransplantation study in NSG mice. (B) KO-ABE and Control (CTR) HSPCs were frozen 48 h post-electroporation and then thawed for same-day injection into NSG mice (n=10 mice per group). Bone marrow (BM) was harvested after 16 weeks. On-target editing (A-to-G conversion at desired splice donor site) was assessed in input cells (pre-freeze) and BM samples by next-generation sequencing (rhAmpSeq). Black square indicates input material; each purple dot represents an individual mouse BM sample and horizontal bar denotes group mean. (C) Chimerism and multilineage reconstitution in BM, 16 weeks post-engraftment; measured by flow cytometry. Human chimerism calculated as hCD45 + / (hCD45 + + mCD45 + ) x 100. Frequencies of CD34 + HSPC, myeloid lineages (Monocytes, pDCs, Neutrophils, cDCs, Basophils, Mast Cells), and lymphoid (T cells, CD3 + ; B-cell, CD19 + ) populations were measured within hCD45 + compartment. CD97 + cells also assessed to confirm editing specificity. Data shown mean ± SD; n=10 mice per group. Each dot represents an individual mouse BM sample. Statistical analysis by two-way ANOVA; ns = not significant (p > 0.05). (D) ADGRE2 surface protein expression in total bone marrow hCD45 + , myeloid, lymphoid, and CD97 + populations in KO-ABE versus CTR mice at study end; measured by flow cytometry. Data represented as mean ± SD; n=10 mice per arm, each dot represents one mouse. Statistical analysis performed using two-way ANOVA; ****p<0.0001. Abbreviations: NSG, NOD-scid IL2Rg null; KO, knockout; HSPC, hematopoietic stem and progenitor cells; hCD45, human CD45; mCD45, murine CD45; pDCs, plasmacytoid dendritic cells; cDCs, classical dendritic cells; SD, standard deviation.

Article Snippet: ADGRE2 surface expression was quantified using a Phycoerythrin (PE)-conjugated anti-ADGRE2 antibody (clone REA301 or 2A1; Miltenyi Biotec Inc., Charlestown, MA, USA) and QuantiBRITE TM beads (BD Biosciences, Franklin Lakes, NJ, USA) via flow cytometry following manufacturer instructions.

Techniques: Control, Electroporation, Injection, Next-Generation Sequencing, Flow Cytometry, Expressing, Knock-Out, Standard Deviation