Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) ICN1-transduced human ETPs were treated with either blocking anti-CD44 mAb (515; IgG1) or control IgG1 before transplantation into RAG-2–/– × γc–/– mice. Absolute ICN1+ cell numbers infiltrating the BM of 5 mice/group were analyzed at 3 weeks after transplant. (B) Schematic representation of experiment design (left): RAG-2–/– × γc–/– mice transplanted with ICN1-transduced ETPs received 3 weekly i.p. injections of either blocking anti-CD44 (HP2/9; IgG1) mAb or control IgG1, starting at day 5 after transplant. (Right) Absolute numbers of human ICN1+ cells infiltrating the BM of 4 mice/group analyzed at day 15 after transplant. (C) Human cell numbers infiltrating the BM of RAG-2–/– × γc–/– mice (5/group) after 3 weeks of transplant with ETPs transduced with either ICN1 or CD44. Data in A–C were normalized to 105 transduced input cells (n = 3). (D) Mean percentages ± SEM of human cells infiltrating the BM of RAG-2–/– × γc–/– mice (4/group) transplanted with ETPs cotransduced with ICN1 along with either a CD44-encoding or a control retrovirus (n = 4). (E) Cells obtained from the BM of a T-ALL patient (T-ALL1) were pretreated with either blocking anti-CD44 mAb 515 or control IgG1 and then transplanted into RAG-2–/– × γc–/– mice. Relative T-ALL1 cell numbers infiltrating the BM, PB, spleen, and thymus of 4 mice/group were analyzed at the indicated times after transplant (n = 3). *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, Transplantation Assay, Transduction

Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) RAG-2–/– × γc–/– mice transplanted with primary human T-ALL1 or T-ALL2 cells (Supplemental Figure 4) received 3 weekly i.p. injections of either blocking anti-CD44 mAb (HP2/9) or control IgG1 during 4 weeks, starting at 1 week (B/C) or 5 weeks (D/E/F) after transplant. When indicated, T-ALL cells recovered from the BM of transplanted mice at the end of treatment were transplanted into secondary hosts. (B) Percentages of T-ALL1 and T-ALL2 cells infiltrating the BM, PB, and spleen of mice treated from weeks 1 to 5 after transplant as shown in A. Mean values from 3 independent experiments with a total of 13 to 18 mice for T-ALL and 3 to 8 mice for T-ALL2 are shown. (C) Image of representative spleens obtained at the end of treatment from mice shown in B. (D) Thorough analysis of anti-CD44 in vivo treatment showing percentages of human T-ALL2 cells infiltrating the BM (left) and PB (right) of 5 mice/group treated from weeks 5 to 9 after transplant as shown in A. Empty symbols represent cell percentages before the onset of Ab treatment. Boxes identify individual donors for secondary transplantations shown in F. (E) Kaplan-Meier survival curve of mice treated with anti-CD44 mAb (HP2/9) or control IgG1 in D. (F) Kaplan-Meier survival curve of secondary recipients transplanted with T-ALL2 cells (33 cells/mouse) obtained from the BM of individual donors represented as boxed in D. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, In Vivo

Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) NSG mice transplanted with primary T-ALL2 cells received 3 weekly i.p. injections of either blocking anti-CD44 mAb (HP2/9) or control IgG1 during 7 weeks, starting at 1 week after transplant. (B) Image of representative spleens obtained at the end of treatment from mice shown in A. (C) Percentages of T-ALL2 cells infiltrating the BM, PB, and spleen of 5 to 10 mice treated as shown in A. (D) Representative FACS analysis showing persistence of anti-CD44 HP2/9 mAb on the surface of T-ALL cells recovered from the BM of NSG mice represented in A at the end of treatment with either anti-CD44 mAb or IgG1. Bound HP2/9 mAb was detected by reactivity with PE-labeled anti-mouse IgG1 (n = 3). (E) FACS analysis showing levels of HP2/9 anti-CD44 mAb persisting on the surface of T-ALL2 cells that infiltrate the BM of anti-CD44–treated mice shown in A at the indicated weeks after transplant. Shown are MFI values from a total of 7 mice, determined as in D. (F) BM and PB infiltration of T-ALL1 cells transduced with a lentiviral vector encoding GFP and either CD44-specific shRNA (28% transduction efficiency) or a scramble control shRNA (36%). Data show percentages of GFP+ cells within infiltrating CD45+ T-ALL1 cells of 5 NSG mice/group at 11 weeks after transplant. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, Labeling, Transduction, Plasmid Preparation, shRNA

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: IgG reactivity to (A) purified protein derivative (PPD) from Mtb , (B) Mtb cytosolic protein, (C) Mtb culture filtrate, (D) Ag85A and Ag85B, (E) Mtb cell wall, (F) ESAT-6 and CFP-10, and (G) control respiratory syncytial virus (RSV) was determined by customized multiplex Luminex for each individual patient sample with TB over three serial dilutions. Each dot represents relative reactivity for one individual patient sample determined by the area under the curve (AUC), summarizing the median fluorescence intensity (MFI) from serial dilutions. Bars represent median and 95% confidence intervals for latent ( n = 18) and active ( n = 19) TB groups. The p values determined by a Mann-Whitney U test are marked by ^ for significance after adjustment for multiple comparisons by Benjamini-Hochberg.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Purification, Control, Virus, Multiplex Assay, Luminex, Fluorescence, MANN-WHITNEY

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: (A) Representative chromatograms show patterns of individual glycoforms isolated from the Fc domain of antigen-specific and total bulk IgG in an individual patient with TB, determined by capillary electrophoresis. (B) Violin plots show the relative abundance of sialic acid, galactose, fucose, and bisecting N-acetylglucosamine (GlcNAc) across all individual glycoforms isolated from ESAT-6 and CFP-10 (red) and Mtb cell wall (blue) polyclonal IgG. Each dot represents an individual sample with latent ( n = 18) or active ( n = 19) TB. The median and interquartiles are shown. The dashed lines show the median RSV (green) and total bulk (purple) glycans. The p values determined by a Wilcoxon matched-pairs signed rank test are marked by ^ for significance after adjustment for multiple comparisons by Benjamini-Hochberg.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Isolation, Electrophoresis

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: (A) Luminescence from the virulent Mtb H37Rv luminescent reporter strain relates to colony-forming units (CFUs). The significance was evaluated by Pearson correlation. (B) To test the effect of antibodies on intracellular Mtb , primary human monocyte-derived macrophages were first infected with the virulent Mtb H37Rv luminescent reporter strain, and the extracellular bacteria were washed away. Then, Mtb -infected primary human monocyte-derived macrophages (MOI = 1) were treated with IgG. Finally, the bacterial burden was quantified with >99% of detectable Mtb in the intracellular as compared to the extracellular medium supernatant compartment. The error bars represent the mean ± SEM. Significance was determined by a Wilcoxon matched-pairs signed rank test. (C) Daily Mtb luminescence measurements representing the median of endemic control, active TB, and latent TB samples are shown for one representative healthy donor of human macrophages. (D) Data from n = 3 healthy human macrophage donors in independent experiments are summarized, with each dot representing the Mtb burden for each individual patient with TB relative to control polyclonal IgG. The median and 95% confidence interval (CI) are shown. The dashed line shows the median of endemic IGRA− control individuals. The significance was determined by a Mann-Whitney U test.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Derivative Assay, Infection, Bacteria, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: (A) The relationships between ESAT-6 and CFP-10 IgG levels and subclasses and intracellular Mtb burden within individuals with latent and active TB were evaluated by Spearman correlation. Heatmaps depict the Spearman rank correlation coefficient, ** p ≤ 0.01, and ^ stands for significance after adjustment for multiple comparisons by Benjamini-Hochberg. The scatterplot shows ESAT-6 and CFP-10 IgG1, and the Mtb burden is shown in the scatterplot, with each dot representing each individual with latent TB. (B) As a control, the relationships between Mtb cell-wall IgG levels and subclasses and intracellular Mtb burden are shown.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Control

Journal: Cell reports

Article Title: ESAT-6 and CFP-10 reactive IgG in patients with tuberculosis inhibits intracellular bacteria

doi: 10.1016/j.celrep.2025.116653

Figure Lengend Snippet: (A) Each column in the histogram depicts the intracellular Mtb burden of one individual patient with latent (light gray) and active (dark gray) TB as in . The dashed line represents the intracellular Mtb burden with control IgG. (A and B) The anti- Mtb activity of IgG from each individual patient with TB was determined by the difference in Mtb burden between control and patient IgG (red). (C) For the n = 17 latent and n = 14 active TB samples with detectable anti- Mtb activities relative to ESAT-6 and CFP-10 IgG1, the relationships to ESAT-6 and CFP-10 IgG Fc glycans as determined by Spearman correlations are listed with ^ marking the significance after adjustment for multiple comparisons by Benjamini-Hochberg. (D) N-glycans from anti-ESAT-6 and CFP-10 mAb were enzymatically removed with PNGase F and then used to treat Mtb -infected primary human monocyte-derived macrophages. The intracellular Mtb burden is shown relative to a no-antibody (Ab) control. The graph summarizes data for n = 6 healthy macrophage donors, with each line representing a single donor. (E) An L234A and L235A (LALA) variant of anti-ESAT-6 and CFP-10 mAb was used to treat Mtb -infected primary human monocyte-derived macrophages. Each line represents a single healthy macrophage donor ( n = 9). The significance was determined by a Wilcoxon matched-pairs signed rank test.

Article Snippet: Mouse Anti-Human IgG3 Hinge-PE HP6050 , SouthernBiotech , Cat#9210-09; RRID:AB_2796701.

Techniques: Control, Activity Assay, Infection, Derivative Assay, Variant Assay

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: sWTA probe generation and validation. A) schematic representation of sWTA probe generation for all three glycoforms, i.e. RboP +β-1,4-GlcNAc, +α-1,4-GlcNAc, or + β-1,3-GlcNAc. All sWTA probes were made for detection in two different fluorescence channels using streptavidin conjugated to AF647 or BB515. B) dual sWTA probe labeling of protein A-coated beads coated with anti-β-GlcNAc RboP (clone 4497), anti-α-GlcNAc RboP (clone 4461), and isotype IgG1. Data in dot plots represent geometric mean fluorescence intensity (gMFI) signals (fluorophores: AF647 and BB515) on the beads. Q2 and Q4 comprise, respectively, double positive (dual sWTA probe labeling) and double negative (no sWTA probe binding) beads. Signals within Q1 and Q3 represent aspecific binding of, respectively, AF647 and BB515 streptavidin to beads. Histograms are included on the sides of the dot plots to visualize relative amounts of different bead populations within a fluorescent channel.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Biomarker Discovery, Fluorescence, Labeling, Binding Assay

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: sWTA specificity screening of pilot-scale produced mAbs. Binding profiles of 15 B cell-derived mAbs, expressed by HEK293T cells (production levels in ) as human IgG1, to streptavidin-coated beads immobilized with biotinylated sWTA to determine clone reactivity as measured by flow cytometry. Fluorescent signals are depicted in this figure as gMFI fold changes (mean + s.d. of three independent experiments) relative to the condition without antibodies to compensate for technical variation. One-way ANOVA was performed to determine significant binding to glycan-coated beads compared to empty beads. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Produced, Binding Assay, Derivative Assay, Flow Cytometry, Glycoproteomics

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Specificity verification of sWTA-reactive mAbs at equimolar level. Clones that displayed sWTA reactivity in the pilot screening (main ) were selected for large-scale production in HEK293 freestyle cells and purified through protein G agarose. Selected clones were categorized based on their α-GlcNAc A) or β-GlcNAc B) reactivity and relative binding capacities to sWTA beads were determined at a concentration of 3 μg/ml. Beads coated with polyrhamnose (PR) + β-1,3-GlcNAc and empty beads were used as controls for cross-reactivity and background, respectively. IgG1 binding to sWTA beads was measured by flow cytometry and data represent the gMFI mean + s.d. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine significant binding of sWTA-reactive clones to glycan-coated beads compared to empty beads. ns not significant ** P < 0.01, **** P < 0.0001. Index sort data of all sorted clones can be found in .

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Clone Assay, Purification, Binding Assay, Concentration Assay, Flow Cytometry, Glycoproteomics

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Binding of sWTA-reactive mAbs to WTA on S. aureus surface. For each glycoform specificity, one mAb clone was selected to assess bacterial opsonization. A-D) binding of W1C11 (anti-α-GlcNAc), W1F10 (anti-β-GlcNAc, with preference for β-1,3-GlcNAc), W1G7 (anti-β-GlcNAc), and B12 (isotype control) to S. aureus strains N315 Δ spa A), N315 Δ spa Δ tarSP B), Newman Δ spa Δ sbi C), and Streptococcus pyogenes strain 5448 Δ gacH D). IgG1 binding to bacteria was measured using flow cytometry and data represent normalized mean gMFI + s.d. (isotype signals set to 1) of three independent experiments. N315 Δ spa Δ tarSP and S. pyogenes 5448 Δ gacH were included as controls for WTA GlcNAc (species) specificity.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Binding Assay, Control, Bacteria, Flow Cytometry

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Effector functions of anti-WTA clones towards S. aureus . A-C) C3b deposition by sWTA-reactive mAbs onto S. aureus strains N315 Δ spa A), N315 Δ spa Δ tarSP B), and Newman Δ spa Δ sbi C). Data represent C3b binding (normalized gMFI + s.d.) of three independent experiments as measured by flow cytometry. Fluorescent signals are depicted as a fold change relative to the condition without antibodies to compensate for variation in background signals between biological replicates. D) neutrophil-mediated phagocytosis of GFP-expressing S. aureus Newman Δspa Δsbi by anti-WTA mAbs. Displayed data represent percentages of GFP-positive neutrophils and are representative of three biological replicates (individual replicates can be found in ). Curves were generated using nonlinear dose–response fitting model. E) relative phagocytic capacities of the anti-WTA mAbs. Absolute IC 50 values were determined for each replicate individually using nonlinear dose–response fitting model. Black lines represent means of the IC 50 values which are depicted as data points. Assays (A-D) were performed in the presence of 1% IgG-/IgM-depleted human serum as complement source. Statistical differences compared to isotype were determined by one-way ANOVA. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Clone Assay, Binding Assay, Flow Cytometry, Expressing, Generated

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Discovery and characterization of GAC-specific mAbs. A) schematic representation of sGAC probe generation for two glycoforms, i.e. PR and PR + β-1,3-GlcNAc. All sGAC probes were made for detection in two different fluorescence channels using streptavidin conjugated to Pe-Cy7 or BV421. B) sGAC probe binding to protein beads immobilized with goat polyclonal anti-GAC GlcNAc (Ab9191). N.B. no bead coat option was available to test PR specificity. Data in dot plots represent fluorescence signals (fluorophores: PE-Cy7 and BV421) on the beads. Q2 and Q4 comprise, respectively, double positive (dual sGAC probe labeling) and double negative (no sGAC probe binding) beads. Signals within Q1 and Q3 represent aspecific binding of, respectively, Pe-Cy7 and BV421 streptavidin to beads. C) specificity verification of sGAC-reactive mAbs at equimolar level. Clones were produced in HEK293 freestyle cells and purified through protein G agarose. Relative binding capacities to sGAC beads were determined at a concentration of 3 μg/mL. Beads coated with RboP +β-1,3-GlcNAc and empty beads were used as controls for cross-reactivity and background, respectively. IgG1 binding to sGAC beads was measured by flow cytometry and data represent the mean gMFI ± s.d. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine significant binding of sGAC-reactive clones to glycan-coated beads compared to empty beads. **** P < 0.0001. Index sort data of all sorted clones can be found in . D) binding of sGAC-reactive mAbs to natural GAC on Streptococcus pyogenes . For each glycoform specificity, one mAb clone was selected that showed evident binding to sGAC beads (panel C) and tested for bacterial opsonization. Titration of G1E8 (anti-PR), G1C4 (anti-β-1,3-GlcNAc PR), and B12 (isotype control) to S. pyogenes 5448 Δ emm1 . Bacterial opsonization was determined by measuring IgG1 binding to bacteria using flow cytometry and data represent normalized mean gMFI + s.d. (isotype signals set to 1) of three independent experiments. E) effector functions of anti-GAC clones towards S. pyogenes. C3b deposition by sGAC-reactive mAbs onto S. pyogenes 5448 Δ emm1 . Data represent C3b binding (normalized gMFI + s.d.) of three independent experiments and was measured by flow cytometry. Fluorescent signals are depicted as a fold change relative to the condition without antibodies to compensate for variation in background signals between biological replicates.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Fluorescence, Binding Assay, Labeling, Clone Assay, Produced, Purification, Concentration Assay, Flow Cytometry, Glycoproteomics, Titration, Control, Bacteria