Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The APC function of B cells is closely associated with their FA metabolism in metastatic OvCa. A GSVA pathway enrichment analysis of APC function low and APC function high B cells in patients with metastatic OvCa ( GSE235951 , GSE147082 and GSE154600 dataset in the GEO database, n = 13). B Representative images of the IHC staining of TLS structure (composed of CD3 + T cells, CD19 + B cells, and CD21 + FDC), CD80, and CD86, respectively, in the area adjacent to or away from tumor or adipose tissues in clinical HGSOC specimens (n = 20 for TLS and n = 5 for CD80 or CD86). Red dashed line area: TLS structure. Magnification × 200. C Representative image of lymphoid aggregates (white dashed line area) by immunofluorescent staining in ascites of OvCa mice with 3 w and 6 w in tumor-bearing mice. D Mean fluorescence intensity of CD80, CD86, CD83, and MHC class II molecules in ascitic CD19 + B cells of OvCa mice were detected by flow cytometry. E Comparison of mRNA levels of FA metabolic genes in ascitic B cells in OvCa mice. The relative expression of each gene was calculated using β -actin as the internal reference. F Protein expression of β-actin and FA metabolic proteins in ascitic B cells in OvCa mice was assessed by WB. β -Actin was used as the internal control to calculate the relative expression level of FA metabolic proteins. G Mean fluorescence intensity of Bodipy C16 in ascitic B cells of OvCa mice detected by flow cytometry. H Expression of A-CoA in ascitic B cells of OvCa mice detected by ELISA. I Expression of ATP in ascitic B cells of OvCa mice detected by ELISA. J Mean fluorescence intensity of intracellular oxidized lipid in ascitic B cells of OvCa mice detected by flow cytometry. I Comparison of mRNA levels of FA metabolic genes in ascitic B cells in OvCa mice. The relative expression of each gene was calculated using β -actin as the internal reference. Data are presented as the mean ± SD of three independent experiments. FA, fatty acid; TLS, tertiary lymphoid structure. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: Immunohistochemistry, Staining, Fluorescence, Flow Cytometry, Comparison, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The APC function and resulting anticancer immunity of B cells can be enhanced by oleic acid (OA) via reprogramming FA metabolism in vitro. A Mean fluorescence intensity of CD80 and MHC Class II molecules in CD19 + B cells from peripheral blood of healthy volunteers (n = 3) treated with OA and PA (both 150 μM), respectively. B Mean fluorescence intensity of CD80, CD86, CD83, MHC Class II molecules, and Ki67 in splenic CD19 + B cells of WT mice treated with 150 μM OA. C Mean fluorescence intensity of CD80, CD86, CD83, and MHC Class II molecules in ascitic CD19 + B cells from 3 w OvCa-bearing mice when treated with 150 μM OA. D Analysis of FA metabolism-related signaling pathways based on RNA-seq results. GSEA was used to analysised the FA metabolic pathways. E Comparison of mRNA levels of main FA metabolic genes in ascitic B cells from 3 w tumor-bearing mice when treated with 150 μM OA. The relative expression of each gene was calculated using β -actin as the internal reference. F Experimental scheme to detect the influence of inhibiting OA uptake on ascitic B cells. G Comparison of protein expressions of main FA metabolic molecules in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA. β -Actin was used as the internal control to calculate the relative expression level of the main FA metabolic molecules. H Mean fluorescence intensity of Bodipy C16 in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA was detected by flow cytometry. I The expression of A-CoA, ATP and the FAO activity in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS treated with OA was detected by ELISA. J Mean fluorescence intensity of intracellular oxidized lipid in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA was detected by flow cytometry. K Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS and treated with OA was detected by flow cytometry. L Mean fluorescence intensity of Bodipy C16, CD80, CD86 and CD83 in FABP4-knockdown CD19 + B cells, which are from the ascites of 3 w OvCa-bearing mice, treated with OA, was detected by flow cytometry. PBMC, peripheral blood mononuclear cell; SP, spleen; AS, Ascites; OA, oleic acid; PA, palmitic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Fluorescence, Protein-Protein interactions, RNA Sequencing, Comparison, Expressing, Control, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: Reprogramming of the FA metabolism of B cells by OA can improve anticancer immunity in vitro. A Experimental scheme to detect the in vitro effects of ascitic B cells treated with OA on anticancer immunity. B Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells after pulsed with ID8-Luc-cell-prepared antigenic peptides and pretreated with BMS and treated with OA, T cells and ID8-Luc. C Levels of IFN- γ , GZMB, and TNF-α in the supernatant of the coculture system constructed by ID8-Luc cells and 3 w OvCa-bearing mouse ascitic B and splenic T cells were detected by ELISA. D Cytotoxicity of T cells in the coculture system mentioned above was detected by luciferase assay. AS, Ascites; Ag, Antigen; OA, oleic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Fluorescence, Construct, Enzyme-linked Immunosorbent Assay, Luciferase

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The enhanced APC function of B cells by OA in vitro is achieved through H3K27ac-mediated upregulation of PPAR γ expression. A Protein expression of β -actin and H3K27ac in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS and treated with OA was assessed by WB. β -Actin was used as the internal control to calculate the relative expression level of H3K27ac. B The enrichment percentage of H3K27ac at the PPAR γ , CD80, CD86, and CD83 promoter regions was quantified using ChIP-seq analysis. C Correlation analysis of PPAR γ and APC function-related genes (CD80, CD86, CD83, MHC II) in B cells in OvCa patients in the TCGA database (n = 426). D Analysis of protein interaction among FABP4, PPAR γ , CD80, CD86, and CD83. E The enrichment percentage of PPAR γ at the CD80, CD86, and CD83 promoter regions was quantified using ChIP-seq analysis. F Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS, GW9662/Trog, and treated with OA was detected by flow cytometry. AS, Ascites; Ag, Antigen; OA, oleic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Expressing, Control, ChIP-sequencing, Fluorescence, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The combination of adoptive immunotherapy with APC function-enhanced B cells and LDC improved anticancer immunity in metastatic OvCa mice. A Experimental scheme to evaluate the effects of combining adoptive immunotherapy with APC function-enhanced B cells and LDC in metastatic OvCa mice. B Representative BLI images and comparison of OvCa progression in each group (n = 3). C Representative images ( a ) and the number of tumor nodules ( b ) in the abdominal wall of mice in each group (n = 3). Areas marked by yellow dashed lines: Representative tumor nodules in the abdominal wall. D (a) Representative images of lymphoid aggregates (composed of CD3 + T cells, CD19 + B cells, and CD21 + FDC) in the ascitic cells of mice in each group. (b) Comparison of the amount of CD3 + T, CD8 + T, Ki67 + CD3 + T, Ki67 + CD8 + T, CD19 + B, CD80 + B, CD86 + B, and lymphoid aggregates respectively in the ascitic cells of mice in each group (n = 3) in frozen sections detected by immunofluorescence. E Comparison of the proportion of CD3 + T, CD8 + T, CD19 + B, CD19 − CD138 + B cells, mean fluorescence intensity of Ki67 in CD3 + T and CD8 + T, and mean fluorescence intensity of CD80, CD86, CD83, MHC II in CD19 + B, respectively, in ascitic cells of each group of mice (n = 3) detected by flow cytometry. F Mean fluorescence intensity values of IL-2, IFN- γ , GZMB, and CTLA-4 in CD8 + T cells, respectively. G Kaplan Meier analysis of survival time of each group of mice (n = 7). LDC, low-dose chemotherapy; B OA , B cells that have been treated with OA for 24h; L-DDP, low-dose DDP (1 mg/kg); H-DDP, high-dose DDP (2 mg/kg); BLI, bioluminescence imaging. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: Comparison, Immunofluorescence, Fluorescence, Flow Cytometry, Imaging

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Structural models of receptor-binding-deficient PIRYV (ePIRYV RBD ) engineered with K182A and Y352A mutations to eliminate low-density lipoprotein receptor (LDL-R) binding while preserving fusogenic capacity. Receptor-interacting region is highlighted in magenta b, Close up view of the two LDL-R contact residues, glycoprotein (green sticks) form hydrogen bond (dashed lines) with LDL-R (cyan) whereas in the mutant complex, substitutions abolish these bonds. c, Complete structural model of the engineered PIRYV RBD glycoprotein in complex with LDL-R depicting ablation of LDL-R recognition, alongside the four immunogenicity-reducing residues. d , Schematic of lentiviral production and T cell isolation. e, Representative flow cytometry plots of transduction efficiency (%GFP + ) in CD3 + T cells at MOI 2.5 with vector control (VC). ePIRYV wt . ePIRYV RBD or ePIRYV RBD+nbC3/7 . f , Bar graph showing the mean fluorescence intensity (MFI) of GFP + cells. Representative of three independent experiments (n=6 biologically independent samples). g, Humanized scFv design for bispecific BCMA/CD19 CAR construct showing complementarity-determining regions (CDRs) in heavy and light chains. h, Representative contour plots showing comparative CAR expression (PE) in transduced CD4 + (APC, top) and CD8 + (APC, bottom) T cell subsets in activated primary CD3 + T cells from healthy donors (n = 6). Bar graph quantifies the percentage of CAR + cells (using an anti-G4S linker antibody) among CD4 + and CD8 + T cells. i, PBMCs were either left unstimulated (None) or activated with anti-CD3/CD28 (CD3/CD28) Dynabeads before transduction with PIRVYwt or ePIRYRBD +nbC3/7 , followed by analysis of activation. j, T cell activation markers (CD69) measured as MFI following transduction with PIRYV wt or ePIRYV RBD+nbC3/7 at MOI 1 and MOI2.5 after 24 h post-transduction. Each point represents one donor (n = 8). k, l, In vitro cytotoxicity of CAR-T cells generated with PIRY wt or ePIRY RBD+nbC3/7 against BCMA + multiple myeloma targets MM.1S (k) and MM.1R (l), expressed as percentage target-cell survival across the indicated effector-to-target (E:T) ratios after 24 h coculture. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis with n = 3 biologically independent samples). See Supplementary Figures 7-11 for extended nanobody characterization and CAR construct optimization .

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Binding Assay, Preserving, Mutagenesis, Immunopeptidomics, Cell Isolation, Flow Cytometry, Transduction, Plasmid Preparation, Control, Fluorescence, Construct, Expressing, Activation Assay, In Vitro, Generated

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Experimental timeline of in vivo CAR-T delivery in humanized xenograft model. NSG mice received intravenous engraftment of human hematopoietic stem cells (HSCs) on days −28 with prior busulfan treatment. viroVbot1 (ePIRYV RBD+nbC3/7 encoding bi-CAR transgene) was administered intravenously at doses ranging from 0.25 × 10 6 to 1 × 10 6 TU/mouse on day 0. Blood, was collected at days −4, 7, 14, 21, and 28 post-vector administration and bone marrow (BM), and spleen at the end of the experiment. b , Kinetics of CAR-T cell expansion in peripheral blood. Percentage of BCMA-CAR + CD3 + T cells over time (Day −4 to 28). c , d, CAR-T cell frequency in (c) bone marrow and (d) spleen at Day 28. Each point represents pooled samples from 15 animals (n = 5 from 3 pooled in each group). e, f, CAR-T cell transduction efficiency in peripheral blood cells determined by flow cytometry by collecting the samples at day 7 and day 28. g , Flow cytometry analysis of CD19 + cells among hCD45 + cells showing rapid B cell depletion over time. h, i, Similarly in bone marrow and spleen. j, Representative IHC images of major organs (spleen, liver, lungs, and kidneys) showing CAR expression as indicated by red arrowheads with quantitative analysis (n=6 images); Bar graph shows CAR+ cells per 1×10 3 cells by tissue in visceral tissues. k, Schematic of vector production of the two lentiviral vectors used: (left) ePIRY wt with MHC-I −/− modification and bispecific BCMA/CD19 CAR; (right) viroVbot1 with TcrBM-detargeted envelope (ePIRY RBD+nbC3/7 ), with the same bispecific CAR. Both vectors were used to transduce a panel of 65 distinct human-derived cell lines to assess CAR expression at MOI 2.5. l, CAR expression profile across 65 cell lines (ex vivo transduction assay). Dot plot showing CAR fluorescence (n=3 biologically independent samples). Data represents mean ± SEM. **p < 0.01; *p < 0.05. A non-parametric t-test was used for statistical analysis between groups. Scale bar; d: 200 μm.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: In Vivo, Plasmid Preparation, Transduction, Flow Cytometry, Expressing, Modification, Derivative Assay, Ex Vivo, Fluorescence

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic showing receptor-mediated CAR uptake by B cells with wild-type (CD19 wt Raji, BCMA wt multiple myeloma (MM) phenotype and CRISPR-Cas9 knockout (BCMA/CD19 −/− MM, CD19 −/− Raji) phenotype. Cell lines were transduced with viroVbot1 or wild type PIRYV b, Flow cytometry showing ex vivo CAR transduction the wild type and knockout cells (n=6). c, Schematic of receptor competition assay showing that pre-incubation of viroVbot1 with recombinant BCMA (BCMAR) and CD19 (CD19R) extracellular domain proteins before transduction of wild-type Raji and MM.1S cells. d, Transduction efficiency (% CAR + cells) in Raji and MM.1S cells, with viroVbot1 incubated with vehicle (PBS) or viroVbot1 incubated with CD19R or BCMAR (n=8 biologically independent samples). e, Schematic showing CAR glycoprotein displayed on wild-type producer cell line (left) is incorporated onto the lentiviral envelope in contrast to CAR-TRAP producer cell (right) where CD19-ECD-KDEL retains the CAR in ER lumen rather than plasma membrane. f , Representative Immunofluorescence image of HEK293T control cells (left), show robust CD19-CAR (green) throughout the cell surface; CAR-TRAP-expressing cells (right) show minimal surface CD19-CAR fluorescence. Bottom panel is quantification of CD19-CAR intensity density (integrated intensity per cell area) in HEK293T versus CAR-TRAP (n=9 cells). g, Contour plots of CAR (FITC-CD19) surface expression under non-permeabilising conditions with quantitative analysis shown as MFI (n=6), h, Representative Immunofluorescence image showing intracellular ER-localized CAR (anti-CD19-CAR, green) colocalized with calnexin (ER marker, red) in CAR-TRAP cells, along with the lines scans (LS) showing co-localisation of ER signal with CD19 CAR. i, Illustration of viroVbot1 particle produced from wild type producer cell and viroVbot1.1 particle produced from CAR-TRAP cells with bi-CAR as transgene. j, Dot plots showing % BCMA or % CD19 CAR expression in Raji wt or MM.1S wt cells with LVV obtained from HEK293T or CAR-TRAP cells. Quantification bar graphs (right) under same conditions (n= 8). k, BCMA CAR expression in patient multiple myeloma samples (PMM-1 to PMM-12) showing % of BCMA/CAR + cells after transduction with viroVbot1 versus viroVbot1.1. l-m, Similarly, BCMA CAR expression in patient-derived leukemia (PLK-1 to PLK-10) and lymphoma (PLM-1 to PLM-15) samples. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups. Scale bar; f: 50 μm, n: 10 μm.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: CRISPR, Knock-Out, Transduction, Flow Cytometry, Ex Vivo, Competitive Binding Assay, Incubation, Recombinant, Clinical Proteomics, Membrane, Immunofluorescence, Control, Expressing, Fluorescence, Marker, Produced, Derivative Assay

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic of miR-122-mediated hepatocyte-specific transgene silencing strategy incorporating five tandem miR-122 target sequences (5×miR-122T) in the 3’ untranslated region (UTR) downstream of CD3ζ costimulatory domain of bi-CAR transgene (viroVbot2). b, Flow cytometry analysis of % CAR expression (BCMA CAR-PE) in Huh-7 hepatoma cells transduced with LVV containing miR-122 (viroVbot2) or non-targeting control shRNA (NTC) with bi-CAR as transgene. c, Schematics of CD47 overexpression strategy in CAR-TRAP producer cells for macrophage evasion (viroVbot2.1). d, Flow cytometry analysis of % CAR expression in THP1 cells transduced with either viroVbot2 or viroVbot2.1. e, Schematic flow diagram of PromoterForge pipeline (details of the pipeline are provided in methods) . f, Candidate synthetic promoters fused upstream with GFP reporter cassette in reporter-plasmid format. Each synthetic promoter represents a unique combination of core promoter elements, TFBS motifs and enhancer arrangements. g, Flow cytometric analysis of GFP expression as MFI in primary human T cells transduced with constructs in which GFP is driven by the EF-1α promoter or the indicated synthetic promoters (Syn-T3, Syn-T8, Syn-T15) (n=5 biologically independent samples). h, Schematic of the dual-luciferase reporter assay used to validate promoter activity. i, Relative luciferase activity in T cells transfected with reporter constructs containing EF-1α or the indicated synthetic promoters (Syn-T3, Syn-T8, Syn-T15), normalized to the CMV control (n=6 biologically independent samples). j, Schematic of the viroVbot2.2 construct and LVV design. The transfer vector encodes bi-CAR under the Syn-T8 promoter with mir-122. Producer cells co-express the CAR-TRAP system and CD47, generating viroVbot2.2 particles displaying CD47 on the envelope. k, Flow cytometry analysis of CAR expression on CD3 + T cells in PBMCs from six healthy donors (HD-1 to HD-6) following ex vivo transduction with viroVbot2.1 (top row) and viroVbot2.2 (bottom row). Numbers in gates indicate the percentage of CAR-FITC + cells. The bar graph shows the %CAR + of CD3 + cells per donor. l, Scheme of the humanized mouse model. NCG mice were engrafted with human PBMCs (day −5), inoculated with 1×10 6 MM.1S multiple myeloma cells (day −4), and treated with a single dose of viroVbots (day 0). Peripheral blood was collected and BLI performed on days 7, 14, 21, 28, and 56; all organs were harvested at endpoint (day 56). m Representative BLI images of MM.1S tumor burden in mice treated with PBS control (MM.1S), viroVbot2.1, or viroVbot2.2 at the indicated time points. Red “X” denotes deceased animals. n, Quantification of whole-body BLI radiance (p/sec/cm 2 /sr) over time for each treatment group. Each line represents an individual mouse (n=5 per group). o, Percentage of circulating MM.1S tumor cells per 100 µl of blood over time in mice treated with viroVbot2.1 (orange) or viroVbot2.2 (blue). Data shown as mean ± SEM. p, Percentage of CAR + T cells in peripheral blood over time in the same treatment groups, demonstrating in vivo expansion kinetics and contraction of CAR-T cells. q, Kaplan-Meier survival curves of mice bearing MM.1S tumors and treated with PBS (red), viroVbot2.1 (orange), or viroVbot2.2 (blue); n = mice per group, log-rank test. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Flow Cytometry, Expressing, Transduction, Control, shRNA, Over Expression, Plasmid Preparation, Construct, Luciferase, Reporter Assay, Activity Assay, Transfection, Ex Vivo, In Vivo

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic of the screening strategy for selecting a synthetic IL-7R agonist. Three synthetic binding proteins (sBC-P1, sBC-P2, sBC-P3) were paired with full-length IL-7R or minimized IL-7R variants (min1-, min2-, min3-IL7R), expressed in T cells, and assessed for downstream STAT5 phosphorylation using the PathScan Phospho-STAT5 assay. b, Representative flow cytometry histograms of phospho-STAT5 (p-STAT5) in T cells expressing the indicated constructs, compared with isotype and non-transduced (NTP) controls. Anti-p-STAT5 antibody was used followed by Alexafluor 488 and acquired in FITC, channel. c, Quantification of pSTAT5 MFI across conditions, showing comparable STAT5 activation by sBCP3-IL7R and the minimized sBCP3_min3-IL7R receptor relative to NTP-IL7R control (n=8 biologically independent samples). d, Dose-response curves of ligand-induced receptor activation measured by ELISA across a concentration range (mM) for NTP-IL7R, sBCP3-IL7R, and sBCP3_min3-IL7R (n=5 biologically independent samples). e, f Schematic of the viroVbot3 transfer vector and producer cell design. The bi-CAR (BCMA/CD19) cassette is driven by the Syn-Tp promoter, linked via T2A, and detargeted from hepatocytes by 5× miR-122 target sites in the 3′ UTR to co-express the synthetic sBCP3-min3-IL7R (sBCP-mIL7R) cytokine receptor module. g, In vitro cytotoxicity assay showing % MM.1S tumor cell survival at increasing effector-to-target (E:T) ratios following co-culture with untransduced T cells (UT), viroVbot2.2-, or viroVbot3-generated CAR-T cells (n=5 biologically independent samples) after 24 h. h, IL-2 secretion (pg/mL) by CAR-T cells co-cultured with MM.1S target cells at the indicated E:T ratios (n=5 biologically independent samples). i, Similarly, intracellular Granzyme B expression (MFI) in CAR-T cells across the same E:T ratios (n=5). j, Schematic of the serial tumor-rechallenge assay. k, Percentage of Patient MM (PMM) cell survival over time during serial rechallenge in co-cultures with UT, viroVbot2.2, or viroVbot3 CAR-T cells. l, Absolute CAR-T cell counts (5×10 4 ) during serial rechallenge, demonstrating superior expansion and persistence of viroVbot3-generated CAR-T cells. m, Bar graph of frequency of PD1 low (gray) versus PD1 high (orange) populations within CD8 + effector memory (T EM ) cells at days 14, 21, and 28 of co-culture for the three groups. n, Similarly, CD8 + central memory (T cm ) cells at the same time points (n=5 biologically independent samples). o, Memory subset distribution (% of CD8 + T cells); naive/stem-cell memory (T n /T scm ), effector (T eff ), effector memory (T em ), and central memory (T cm ), across UT, viroVbot2.2, and viroVbot3 groups. p, Pie charts showing the relative proportions of T n (gray) and T scm (blue) compartments within CD8 + T cells (n=5 biologically independent samples). Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Binding Assay, Phospho-proteomics, Flow Cytometry, Expressing, Construct, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Plasmid Preparation, In Vitro, Cytotoxicity Assay, Co-Culture Assay, Generated, Cell Culture

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, NCG mice received MM.1S cells (day −7), human PBMCs (day −5), and viroVbot3 (day 0). On day 45, mice were rechallenged with BCMA negative but expressing GPRC5D (GR) MM.1S (GR + /BCMA − /CD19 − ) and given a second PBMC infusion plus viroVbot-CO or viroVbot-VS on day 47. Blood and BLI were collected at indicated time points; organs were harvested at day 90. b, Representative BLI images of tumor burden over time (days 10-90) in five treatment groups as indicated (n=5 mice in each group). c, Quantification of whole-body BLI radiance (p/sec/cm 2 /sr) over time for each group; each line represents an individual mouse. d, Kaplan-Meier survival curves of the five groups (Group-1 to Group-5) across 12 weeks. Log-rank test (n=10 mice in each group). e, Longitudinal flow cytometric quantification of BCMA + CAR-T cells (% of T cells) in peripheral blood from days 0-90 across Groups 2-5, showing initial expansion and contraction kinetics of the first-line CAR-T population. f, Frequency of GPRC5D + CAR-T cells (% of T cells) in peripheral blood across Groups 2, 4, and 5, demonstrating expansion of the second-line viroVbot-CO/VS-derived CAR-T cells following antigen-loss rechallenge. g, Quantification of BCMA + (left axis) and GPRC5D + (right axis) CAR-T cells in bone marrow (BM) at endpoint (day 90) for Groups 2, 4, and 5. ND, not detected. h, Similarly in spleen. Data represents mean ± SEM. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Expressing, Derivative Assay

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: (A) Representative flow cytometric histograms showing the specificity of dimeric CD20 aptamer WB1/1.CD20.1_3S across CD20-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (B) Bar graph quantifying the mean fluorescence intensities of WB1/1.CD20.1_3S binding, highlighting significant specificity (*p < 0.0001). (C; E) Fluorescence histograms for CD19 homodimeric aptamer WB17/17.CD19.1_3S and WB15/15.CD19.1_3S, respectively, demonstrating selective binding to CD19-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (D; F) Quantification of homodimeric aptamer WB17/17.CD19.1_3S and WB15/15.CD19.1_3S, respectively, binding specificity using mean fluorescence intensity. (G) Fluorescence histograms for CD19 heterodimeric aptamer WB15/17.CD19.1_3S, demonstrating selective binding to CD19-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (H) Quantification of heterodimeric aptamer WB15/17.CD19.1_3S binding specificity using mean fluorescence intensity. (I-L) Confocal microscopy images showing colocalization of bivalent WB1/1.CD20.1_3S (L1-L2: Cy3, RED) with a CD20-specific antibody (J1-J2: APC, GREEN) on Raji cells. DNA random aptamer controls and isotype antibody controls confirmed specificity. The aptamer binds to the cell surface membrane, as shown in zoomed-in views (L1-L2). Panel M-P: Confocal microscopy images of bivalent WB17/17.CD19.1_3S (M1-M2: Cy3, RED) colocalizing with a CD19-specific antibody (N1-N2: APC, GREEN) on Raji cells. The aptamer demonstrates specificity and surface binding with no significant off-target interactions. Zoomed-in views (P1-P2) confirmed aptamer binding to the cell membrane. Scale bars = 7 and 5 μm. Mean fluorescence intensity was calculated using the formula: Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence. The aptamer and random mean fluorescence values correspond to the mean fluorescence observed in their respective histograms. Bar graphs represent mean ± standard deviation from three independent experiments with statistical significance indicated (****p < 0.0001). Data represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Fluorescence, Binding Assay, Confocal Microscopy, Membrane, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Internalization studies of CD19 Antibody and Bivalent CD19 Aptamers in Ramos and Raji Cells. (A–H) Time-course analysis of CD19 antibody and bivalent CD19 aptamer internalization in Ramos cells (A, C, E, and G: CD21-negative) and Raji cells (B, D, F, and H: CD21-positive). (A-B) demonstrate internalization of APC-CD19 antibody in Ramos (A) and Raji (B) cells. In Ramos cells (A), CD19 antibody shows efficient internalization over 48 hours, while in Raji cells (B), CD21 expression inhibits internalization. (C-D) show internalization of bivalent CD19 aptamer WB17/17.CD19.1_3S in Ramos (C) and Raji (D) cells. WB17/17.CD19.1_3S shows robust internalization in Ramos cells (C), but reduced uptake in Raji cells owing to CD21-mediated blocking. (E-F) illustrate internalization of bivalent CD19 aptamer WB15/17.CD19.1_3S in Ramos (E) and Raji (F) cells. The aptamer demonstrates high internalization efficiency in Ramos cells (E), but not CD21-positive Raji cells (F). Panels G and H: Internalization of bivalent CD19 aptamer WB15/15.CD19.1_3S in Ramos (G) and Raji (H) cells. Similar to other bivalent aptamers, WB15/15.CD19.1_3S internalizes efficiently in CD21-negative Ramos cells (G), but CD21 expression in Raji cells (H) significantly blocks its internalization. Data are expressed as the percentage of internalization calculated as . Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Expressing, Blocking Assay, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Colocalization of CD19 and CD21 was confirmed by flow cytometry and confocal microscopy, whereas CD20 does not co-localize with CD21. (A) demonstrates flow cytometry histograms showing CD19 (A1: PE-CD19 mAb, Red) and CD21 (A2: APC-CD21 mAb, Light Blue) fluorescence intensity in Raji cells. (A3) Bi-parametric dot plot confirms colocalization of CD19 and CD21 on the same population of cells. (B) shows bar graph quantifying mean fluorescence intensity of CD19 and CD21, highlighting their robust expression on Raji cells. (C–E) Confocal microscopy images showing CD19 (C: PE-CD19, Red) colocalized with CD21 (D: APC-CD21, Light Blue) on the surface of Raji cells. Arrowheads indicate regions of colocalization (D). Panels F1–F3 illustrate flow cytometry analysis of bivalent CD19 aptamer WB17/17.CD19.1_3S (F1: Pink) binding to Raji cells and its colocalization with CD21 (F2: APC-CD21 mAb, Light Blue). (F3) Dot plot shows overlapping signals, confirming aptamer-CD21 interaction. (G) demonstrates the bar graph of mean fluorescence intensity of bivalent CD19 aptamer (WB17/17.CD19.1_3S) and CD21 antibody. (H–J) present confocal microscopy images showing WB17/17.CD19.1_3S (H: Red) colocalized with CD21 (I: Green) on Raji cells. Arrowheads highlight colocalized regions (J). (K1–K3) Flow cytometry analysis showing CD20 (K1: FITC-CD20 mAb, Green) and CD21 (K2: APC-CD21 mAb, Light Blue) fluorescence intensity in Raji cells. (K3) Dot plot shows no significant overlap between CD20 and CD21, indicating no colocalization. (L) Bar graph showing fluorescence intensity of CD20 and CD21. (M–O) Confocal microscopy images showing no colocalization between CD20 (M: Red) and CD21 (N: Green) on Raji cells (O). (P1–P3) Flow cytometry analysis of bivalent CD20 aptamer WB1/1.CD20.1_3S (P1: Blue) and CD21 (P2: APC-CD21 mAb, Light Blue). (P3) Dot plot confirms the absence of colocalization on the same population of cells. Panel Q: Bar graph of fluorescence intensity for CD20 aptamer and CD21. (R–T) Confocal microscopy images showing that WB1/1.CD20.1_3S (R: Red) and CD21 (S: Green) do not co-localize on Raji cells (T). Scale bars = 5 μm. Mean fluorescence intensity was calculated using the formula: Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence for aptamer. As for antibody it was calculated using the formula: Mean fluorescence intensity=Antibody Mean Fluorescence − Isotype Control Mean Fluorescence. The aptamer/random and antibody/isotype mean fluorescence values corresponds to the mean fluorescence observed in their respective histograms. Data represents mean ± standard deviation from three independent experiments. Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Flow Cytometry, Confocal Microscopy, Fluorescence, Expressing, Binding Assay, Control, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: The expression of CD19, CD20, and CD21, the binding activity of bivalent CD19 aptamers, and the internalization dynamics of CD19 antibody and bivalent aptamers in OCl-LY7 and HBL-1 (DLBCL) cells. Antibody staining for CD19, CD20, and CD21 expression. (C; G) demonstrate flow cytometry histograms showing the expression of CD19 (C2: Red), CD20 (C1: Green), and CD21 (C3: Light Blue) on OCI-Ly7 (C), whereas HBL-1 (G) cells express CD19 (G2: Red), CD20 (G1: Green), and CD21 (G3: Light Blue). Isotype controls (gray) confirm specific binding. (D; H) Bar graphs showing mean fluorescence intensities, confirming robust expression of CD19 and CD20, but the absence of CD21 expression in both cell lines. Binding assay with bivalent CD19 aptamers (A1–A4, E1–E4). Fluorescence intensity histograms of bivalent CD19 aptamers (WB17/17.CD19.1_3S, WB15/17.CD19.1_3S, and WB15/15.CD19.1_3S) compared to random DNA control in OCI-Ly7 (A1–A4) and HBL-1 (E1–E4) cells. (B; F) illustrate bar graphs of mean fluorescence intensities showing high binding specificity of bivalent aptamers compared to controls. Internalization of CD19 antibody with and without Proteinase K (I, K). Flow cytometry histograms (L1) and bar graphs (L2) showing internalization dynamics of CD19 antibody in OCI-Ly7 (I), whereas flow cytometry histograms (K1) and bar graphs (K2) show internalization dynamics of CD19 antibody in HBL-1 (K) cells. Internalization was analyzed at 0 hour and 24 hours in the presence and absence of Proteinase K. Data reveal a significant reduction in surface fluorescence intensity after Proteinase K treatment, confirming internalization. Internalization of bivalent CD19 aptamers under the same conditions (J, L). Fluorescence intensity histograms (J1-J3, L1-L3) and bar graphs (J4 and L4) show the internalization of bivalent CD19 aptamers (WB17/17.CD19.1_3S (J2, L2), WB15/17.CD19.1_3S (J1, L1), and WB15/15.CD19.1_3S (J3, L3)) in OCI-Ly7 (J) and HBL-1 (L) cells. Internalization is measured at 0 hour and 24 hours with and without Proteinase K, demonstrating the effective uptake of bivalent aptamers. Data are expressed as the percentage of internalization calculated as fluorescence intensity was calculated using the formula. Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence for aptamer. As for antibody it was calculated using the formula: Mean fluorescence intensity=Antibody Mean Fluorescence − Isotype Control Mean Fluorescence. The aptamer/random and antibody/isotype mean fluorescence values corresponds to the mean fluorescence observed in their respective histograms. Each bar represents mean ± standard deviation from three independent experiments. Data are presented as mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Expressing, Binding Assay, Activity Assay, Staining, Flow Cytometry, Fluorescence, Control, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Internalization Assay of CD19 Antibody and Bivalent CD19 Aptamer in Ramos and OCI-Ly7 Cells Visualized by Confocal Microscopy. Internalization of CD19 Antibody in Ramos Cells (A-D). Confocal images showing surface-bound APC-CD19 antibody (A1-A2: Red) at 0 hour with and without Hoechst nuclear staining (A2: Blue). (B1–B2) demonstrate the isotype control at 0 hour. Panels C1-C2 show the internalization of APC-CD19 after 24 hours (C1-C2: Red) with and without Hoechst nuclear staining (C2: Blue). (E) Bar graph quantifying the mean fluorescence intensity of APC-CD19 antibody with and without Proteinase K treatment at 0 and 24 hours. Internalization of Bivalent CD19 Aptamer WB17/17.CD19.1_3S in Ramos Cells (F-I). Initial binding of WB17/17.CD19.1_3S (F1-F2: Green) at 0 hours, as shown with Hoechst-stained nuclei (F2: Blue). (H1–H2) demonstrate internalization of WB17/17.CD19.1_3S at 24 hours with and without Hoechst nuclear staining (H2: Blue). (J) shows the bar graph quantifying the mean fluorescence intensity of WB17/17.CD19.1_3S with and without Proteinase K treatment at 0 and 24 hours. Internalization of CD19 Antibody in OCI-Ly7 Cells (K-N) Surface-bound APC-CD19 antibody at 0 hours (K1-K2). (M1–M2) illustrate reduced surface-bound fluorescence at 24 hours, consistent with internalization. After 24 hours, CD19 antibody is completely uptaken on OCl-Ly7 cells. (O) Bar graph quantifying the mean fluorescence intensity of APC-CD19 antibody with and without Proteinase K treatment at 0 and 24 hours. Internalization of Bivalent CD19 Aptamer WB17/17.CD19.1_3S in OCI-Ly7 Cells (P-S). Confocal images showing WB17/17.CD19.1_3S (P1: Green) binding at 0 hours with and without Hoechst nuclear staining (P2: Blue). (R1–R2) highlight internalized WB17/17.CD19.1_3S at 24 hours with Hoechst nuclear staining (R2). Panel T shows the bar graph quantifying the mean fluorescence intensity of WB17/17.CD19.1_3S with and without Proteinase K treatment at 0 and 24 hours. Scale Bars: 5 μm. Data represent mean ± standard deviation from three independent experiments. Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Confocal Microscopy, Staining, Control, Fluorescence, Binding Assay, Standard Deviation

Journal: Immunity

Article Title: Memory B cells that cross-react with group 1 and group 2 influenza A viruses are abundant in adult human repertoires

doi: 10.1016/j.immuni.2017.12.009

Figure Lengend Snippet: Single B cells were sorted from PBMCs and cultured in the presence of MS40Llo feeder cells with exogenous recombinant human IL-2, IL-4, IL-21, and BAFF. (A) Representative flow diagrams from 4 or more independent experiments showing the gating strategy used to isolate human Bmem cells (CD19+CD27+CD24hiIgM−IgD−). (B and C) Kinetics of B cell numbers (B) and IgG concentrations in culture supernatants (C) during single-cell cultures of switched Bmem cells. We analyzed 22 individual cultures from a single experiment for each timepoint; data shown are values for samples that exceeded the background for cell counting and IgG determinations.

Article Snippet: The following human surface antigen specific mAbs were used: anti-human IgM-FITC (MHM-88), CD3-PE-Cy5 (UCHT1), CD14-Tri (TuK4), CD16-PE-Cy5 (3G8), CD19-PE-Cy7 (HIB19), IgG-APC (G18-145), IgD-APC-Cy7 (IA6-2), CD27-BV421 (M-T271), and CD24-BV510 (ML5), purchased from BD Biosciences or BioLegend or Thermo Scientific.

Techniques: Cell Culture, Recombinant, Cell Counting

Journal: Immunity

Article Title: Memory B cells that cross-react with group 1 and group 2 influenza A viruses are abundant in adult human repertoires

doi: 10.1016/j.immuni.2017.12.009

Figure Lengend Snippet: (A) Representative flow diagrams used to isolate rHA-specific IgG+ human Bmem cells (H3 Wisconsin+CD19+CD27+CD24hiIgM−IgD−IgG+) from PBMCs of KEL01 and KEL03. The donors received TIV 2 weeks before blood collection. (B) Representative Luminex diagram showing reactivity of culture supernatant IgGs from individual single B cell cultures (n = 231 for KEL01, n = 610 for KEL03) against 14 antigens including 4 positive and negative controls (anti-IgG, anti-Igκ, anti-Igλ, BSA) and a panel of rHAs (HA H3 WI-05 = H3 A/Wisconsin/67/2005; HA H3 X31 = H3 A/Aichi/2/1968 (X31); HA H1 SI-06 = H1 A/Solomon Islands/03/2006; HA H1 MA-90 = H1 A/Massachusetts/1/1990; HA H1 CA-09 = H1 A/California/04/2009; HA X181 = H1 A/reassortant/NYMC X-181 (California/07/2009 × NYMC X-157); HA H5 VN-04 = H5 A/Vietnam/1203/2004; and HA B Malaysia = B/Malaysia/2506/2004. We also included head-only HA constructs: HA H3 WI-05h = H3 A/Wisconsin/67/2005; and HA H3 Joburg-94h = H3 A/Johannesburg/33/1994. Each dot represents an individual test for each antigen. Bars in blue indicate the threshold median fluorescence intensities (MFIs) for each antigen (average + 6 SD of B cell negative, mock-treated samples). Above each column is the number of supernatants testing above this threshold. (C) Distributions of rHA AvIn for rHA (H3 WI-05)-reactive IgG+ human Bmem cells relative to Ab2210 monoclonal standard. Curves were created by binning with 3-fold intervals AvIn values for all samples. Data from one (for KEL01) and two individual experiments (for KEL03) are shown. See also Figures S1, S2, and S3 and Table S1.

Article Snippet: The following human surface antigen specific mAbs were used: anti-human IgM-FITC (MHM-88), CD3-PE-Cy5 (UCHT1), CD14-Tri (TuK4), CD16-PE-Cy5 (3G8), CD19-PE-Cy7 (HIB19), IgG-APC (G18-145), IgD-APC-Cy7 (IA6-2), CD27-BV421 (M-T271), and CD24-BV510 (ML5), purchased from BD Biosciences or BioLegend or Thermo Scientific.

Techniques: Luminex, Construct, Fluorescence

Journal:

Article Title: Human Endothelial Cells Enhance Human Immunodeficiency Virus Type 1 Replication in CD4 + T Cells in a Nef-Dependent Manner In Vitro and In Vivo

doi: 10.1128/JVI.79.1.264-276.2005

Figure Lengend Snippet: ECs enhance HIV-1 replication in purified CD4+ T cells. (A) Replication of CXCR4-utilizing strain NL4-3 in unstimulated, purified CD4+ T cells alone, IFN-γ-pretreated ECs alone, or T cells cocultured with untreated or IFN-γ-pretreated ECs as assessed by RNA quantification via real-time RT-PCR. The y axis represents the increase in viral RNA (n-fold) over that observed in cultures containing T cells alone. Experiments were performed in triplicate, and the values represent the peak viral RNA concentration. (B) FACS analyses of ECs transduced with a retrovirus containing a CIITA construct, stained for expression of MHC-II or ICAM-1, in untreated or IFN-γ-pretreated cultures. (C) Replication of HIV-1 in T cells cocultured with ECs that were either pretreated with IFN-γ or transduced with CIITA as assessed by HIV-1 p24Gag production in culture supernatants. The y axis represents the concentration of HIV-1 p24Gag (nanograms per milliliter). The data shown are representative of two independent experiments with similar results. In the above experiments, the error bars represent the standard error from an individual experiment performed in triplicate.

Article Snippet: For FACS analyses, all the fluorescently conjugated antibodies α-CD3, α-CD4, α-CD11c, α-CD14, α-CD19, α-CD83, α-HLA-DR, and α-ICAM-1 (BD Biosciences, Palo Alto, Calif.) were used at a 1:50 to 1:100 dilution.

Techniques: Purification, Quantitative RT-PCR, Concentration Assay, Transduction, Construct, Staining, Expressing