Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 1 Chimeric antigen receptor (CAR) detection methods. Binding mechanisms of the different CAR detection reagents. (1) The APC- conjugated AffiniPure F(ab')2 fragment goat antihuman IgG, Fcγ fragment specific, also called anti-Fc, binds the human IgG1 derived hinge domain. (2–4) These three methods use a recombinant human CD19 protein fragment able to bind the anti-CD19 single-chain fragment variable (scFv). The CD19his (2) is fused to a histidine tag that is recognized by an APC-conjugated antihistidine antibody. The CD19bio (3) is fused to a biotin tag that is recognized by an APC-conjugated antibiotin antibody. The CD19-FITC (4) is directly conjugated to the FITC fluorochrome. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Binding Assay, Derivative Assay, Recombinant

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 2 Comparison between different chimeric antigen receptor (CAR) staining methods. (A) Histograms of CARCIK-CD19 cells staining with the anti-Fc antibody in presence of complete medium (RPMI + 10% fetal bovine serum) or after 1 h incubation in human serum at 100%. Negative controls are shown in the first row and represent the unstained samples. (B) Dot plot of CARCIK-CD19 cells detection on the infusion bag product and in the peripheral blood (PB) patient sample with the three CAR detection methods. Negative controls are shown in the first row and represent the samples stained with only the secondary antibody for the indirect methods (CD19his and CD19bio), while the unstained sample for the direct method (CD19-FITC). (C) Stain index for six independent PB samples stained with the three CAR detection methods. Stain index = ((MFI CAR+ population) – (MFI CAR population))/(2 standard deviation of the CAR population). Bars indicate the mean of each group of samples. (D) Comparison of CD19his, CD19bio, CD19-FITC CAR staining methods in six different PB samples from patients treated with CARCIK-CD19 cells. Statistically significant differences are noted in each figure (*p < 0.05; two-tailed paired t-test). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Comparison, Staining, Incubation, Standard Deviation, Two Tailed Test

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 3 Specificity and sensitivity of the three chimeric antigen receptor (CAR) staining methods using a recombinant human CD19 protein fragment. (A) The specificity was evaluated by staining healthy donor peripheral blood mononuclear cells with the three CAR detection staining reagents to assess background staining. Data are representative of three different donors acquired in one experiment and bars indicate the mean. (B) The sensitivity was evaluated serially diluting CARCIK-CD19+ cells into untransduced CIK cells at six different dilutions (from 100% to 0%). The graph shows mean values and the standard deviation of three independent experiments for each staining method (CD19his, CD19bio, CD19-FITC). The dotted line represents, in both graphs, the lower limit of quantification (LLOQ) for CD19his, identified at 0.1% CAR+

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining, Recombinant, Standard Deviation

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 4 Comparison between chimeric antigen receptor (CAR) expression measured with CD19his flow cytometry and real-time polymerase chain reaction (PCR) data. (A) CAR detection was concurrently assessed in 56 postinfusion peripheral blood samples from 4 patients treated with CARCIK-CD19 cells by flow cytometry using the CD19his method (CD3+CAR+ cell/μL) and qPCR (VCN/μg of DNA). The results show clear inter-method concordance by Pearson correlation test (R = 0.51, p = 0.0008). Data are displayed in a logarithmic scale to better represent values <1 cell/μL. (B,C) Monitoring of CARCIK-CD19 expansion, using CD19his flow cytometry staining and qPCR, in two representative patients. (D–F) CARCIK-CD19 cells were expanded in vitro and purified on a magnetic column for CAR expression. Data are representative of five independent experiments. Untransduced CIK cells, CAR+ and CAR purified cells were then analyzed by qPCR and for their cytotoxic activity. In each histograms columns represent the mean and bars the standard deviation. (D) CAR gene integration measured with quantitative PCR and expressed as vector copy number/μg of DNA. (E) CAR mRNA expression measured with qPCR and expressed as CAR mRNA copies normalized on the reference gene GUS. (F) Cytotoxic activity against a CD19+ REH cell line at a 1:1 effector to target ratio, after 72 h coculture. Statistically significant differences are noted in each figure (*p < 0.05; **p < 0.01; two-tailed paired t-test). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Comparison, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Staining, In Vitro, Purification, Activity Assay, Standard Deviation, Plasmid Preparation, Two Tailed Test

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 5 Inclusion of CD19his chimeric antigen receptor (CAR) staining method in a 12 colors flow cytometry panel. Representation of CD3 cells staining in a single patient treated with CARCIK-CD19 cells at different time point after infusion, using viSNE map visualization tool. To interpret high-dimensional single-cell data that were produced by multicolor flow cytometry panel, we used a tool based on the viSNE algorithm [32], which allows visualization of high-dimensional cytometry data on a two-dimensional map at single-cell resolution and preserve the nonlinearity [33]. In the viSNE map, cell position reflects their proximity in high-dimensional space based on the similarity of marker expression. The first column shows the distribution of the four major T-cell populations: CD4+ CAR+ cells (blue), CD4+ CAR cells (orange), CD8+ CAR+ cells (green), and CD8+ CAR cells (red). All the other columns show the expression level of each marker included in the panel, excluding CD45 and CD3 used for the identification of the T cells. D, day; M, month after CARCIK infusion. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining, Flow Cytometry, Produced, Cytometry, Marker, Expressing

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

doi: 10.1002/cyto.a.24796

Figure Lengend Snippet: FIGURE 6 Application of the CD19his staining method on different commercial chimeric antigen receptor (CAR)-T products and within different sample sources. (A) Representative dot plots of three peripheral blood (PB) samples from patients previously infused with tisagenlecleucel (Kymriah®), axicabtagene ciloleucel (Yescarta®),brexucabtagene autoleucel (Tecartus®) and lisocabtagene maraleucel (Breyanzi®). (B) Representative dot plots of CAR detection on different sample sources: bone marrow (BM), pleural effusion and cerebrospinal fluid (CSF) in patients infused with CARCIK-CD19 cells. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense together with APC-conjugated antihistidine antibody (Miltenyi Biotec, Bergisch, Gladbach, Germany); (2) the CD19 CAR detection reagent, a biotinylated human CD19 protein, together with APC-conjugated antibiotin antibody (Miltenyi Biotec); (3) the FITC-labeled human CD19 protein (Acro biosystems).

Techniques: Staining

Journal: Nature Communications

Article Title: Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy

doi: 10.1038/ncomms15507

Figure Lengend Snippet: ( a ) Schematic representation of PEG engager constructs, which code for a murine immunoglobulin kappa chain leader sequence (Igκ), a humanized 6.3 light chain (6.3 VL–C κ ), an internal ribosome entry site (IRES) sequence, a humanized 6.3 heavy chain fragment (6.3 VH–CH 1 ), a glycine-serine peptide linker (GGGGS), an antitumour single chain disulfide-stabilized variable fragments (dsFv, anti-CD19 or anti-EGFR), and a polyhistidine-tag (His tag). ( b ) Reducing (i) and non-reducing (ii) sodium dodecyl sulfate polyacrylamide gel electrophoresis showing Coomassie blue staining of Erbitux, PEG engager EGFR and PEG engager CD19 . M, PageRuler prestained protein ladder (Fermentas). H (heavy chain), L (light chain). ( c ) Precise molecular weight of PEG engager EGFR (i) and PEG engager CD19 (ii) analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Mass-to-charge ratio ( m/z ). ( d ) Binding affinity of PEG engager EGFR (i) and PEG engager CD19 (ii) to PEG 5k analysed by microscale thermophoresis ( n =3). ( e ) Binding affinity of PEG engager EGFR to recombinant EGFR protein (i) and PEG engager CD19 to recombinant CD19 protein (ii) analysed by microscale thermophoresis ( n =3). Equilibrium dissociation constant ( K D ). Data are shown as mean±s.d. Representative microscale thermophoresis data from three independent experiments are shown.

Article Snippet: To analyse the tumour antigen-binding affinity of PEG engagers, 2 nM of Alexa Fluor 647-conjugated PEG engager CD19 or PEG engager EGFR were mixed at a 1:2 volume ratio with graded concentrations (0.027–180 nM) of recombinant CD19 or EGFR proteins (Sino Biological Inc.).

Techniques: Construct, Sequencing, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Mass Spectrometry, Binding Assay, Microscale Thermophoresis, Recombinant

Journal: Nature Communications

Article Title: Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy

doi: 10.1038/ncomms15507

Figure Lengend Snippet: PEG engager EGFR (i–iii) and PEG engager CD19 (iv–vi) supplemented with Hoechst 33342 (blue) were incubated with MDA-MB-468 ( a ), A431 ( b ) or MCF7 ( c ) cells followed by PEG-Qdot655 (red) and observed in real time with a digital confocal microscope. Scale bar, 10 μm. Representative confocal images from three independent experiments are shown.

Article Snippet: To analyse the tumour antigen-binding affinity of PEG engagers, 2 nM of Alexa Fluor 647-conjugated PEG engager CD19 or PEG engager EGFR were mixed at a 1:2 volume ratio with graded concentrations (0.027–180 nM) of recombinant CD19 or EGFR proteins (Sino Biological Inc.).

Techniques: Incubation, Microscopy

Journal: Nature Communications

Article Title: Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy

doi: 10.1038/ncomms15507

Figure Lengend Snippet: BT-20 ( a ), MDA-MB-468 ( b ) and MDA-MB-231 cells ( c ) were incubated with PEG engager EGFR for 30 min followed by addition of serial dilutions of Doxisome (white circles) or empty liposomes (white squares) in triplicate for 4 h. The cells were also incubated with culture medium or PEG engager CD19 for 30 min followed by addition of serial dilutions of Doxisome (black circles and red squares, respectively). Serial dilutions of free doxorubicin (white triangles) or PEG engager EGFR alone (white diamonds) were also added to cells for 4 h. The incorporation of 3 H-thymidine into cellular DNA was measured 72 h later. The data are representative of three independent experiments. ( d ) The half maximal effective concentration (EC 50 ) values of BT-20, MDA-MB-468 and MDA-MB-231 cells treated with PEG engager CD19 plus Doxisome or PEG engager EGFR plus Doxisome were analysed ( n =3). Data are shown as mean±s.d. Significant differences in mean EC 50 values are indicated: ** P ≤0.001, *** P ≤0.0001 (two-way analysis of variance).

Article Snippet: To analyse the tumour antigen-binding affinity of PEG engagers, 2 nM of Alexa Fluor 647-conjugated PEG engager CD19 or PEG engager EGFR were mixed at a 1:2 volume ratio with graded concentrations (0.027–180 nM) of recombinant CD19 or EGFR proteins (Sino Biological Inc.).

Techniques: Incubation, Concentration Assay

Journal: Nature Communications

Article Title: Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy

doi: 10.1038/ncomms15507

Figure Lengend Snippet: ( a ) NSG mice were intravenously injected with 6 mg kg −1 PEG engager EGFR (white circles) or PEG engager CD19 (red squares). Mean plasma concentrations of the PEG engagers were measured by sandwich ELISA ( n =3 mice). ( b ) Five hours before intravenous administration of 4armPEG 10k -NIR-797 probe (5 mg kg −1 ), NSG mice bearing subcutaneous MDA-MB-468 tumours were intravenously injected with 6 mg kg −1 PEG engager EGFR or PEG engager CD19 and the whole-body imaging were sequentially imaged at 24, 48 and 72 h with an IVIS Spectrum imaging system. ( c ) The uptake of PEG-NIR797 in MDA-MB-468 tumours was determined by measuring fluorescence intensities ( n =3). Data are shown as mean±s.d. Significant differences in mean fluorescent intensity between PEG engager EGFR and PEG engager CD19 groups are indicated: * P ≤0.01, ** P ≤0.001 (two-way analysis of variance). NS, not significant.

Article Snippet: To analyse the tumour antigen-binding affinity of PEG engagers, 2 nM of Alexa Fluor 647-conjugated PEG engager CD19 or PEG engager EGFR were mixed at a 1:2 volume ratio with graded concentrations (0.027–180 nM) of recombinant CD19 or EGFR proteins (Sino Biological Inc.).

Techniques: Injection, Sandwich ELISA, Imaging, Fluorescence

Journal: Nature Communications

Article Title: Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy

doi: 10.1038/ncomms15507

Figure Lengend Snippet: ( a ) Groups of eight SCID mice bearing MDA-MB-468 tumours were intravenously injected with 6 mg kg −1 PEG engager CD19 (red squares), 6 mg kg −1 PEG engager EGFR (white circles) or 18 mg kg −1 PEG engager EGFR (white squares) 5 h before intravenous injection of 1 mg kg −1 Doxisome. Groups of eight mice were also intravenously injected with 6 mg kg −1 free doxorubicin (white triangles), 3 mg kg −1 Doxisome (black diamonds), PBS (black circles) or 18 mg kg −1 PEG engager EGFR (white diamonds). Treatment was performed once a week for 4 weeks (arrows). Results show mean tumour sizes±s.d. ( b ) Mean body weights of treated MDA-MB-468 mice ( n =8). ( c ) Groups of six SCID mice bearing MDA-MB-231 tumours were intravenously injected with 6 mg kg −1 PEG engager EGFR (white circles) or 6 mg kg −1 PEG engager CD19 (red squares) 5 h before intravenous injection of 1 mg kg −1 Doxisome. Groups of six mice were also intravenously injected with PBS (black circles), PEG engager EGFR alone (white diamonds) or 1 mg kg −1 Doxisome (black triangles). Treatment was performed once a week for 4 weeks. Results show mean tumour sizes at 43 days post first treatment). Statistical analysis of the differences in tumour volumes between treatment and control groups was performed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons. * P ≤0.05, ** P ≤0.005.

Article Snippet: To analyse the tumour antigen-binding affinity of PEG engagers, 2 nM of Alexa Fluor 647-conjugated PEG engager CD19 or PEG engager EGFR were mixed at a 1:2 volume ratio with graded concentrations (0.027–180 nM) of recombinant CD19 or EGFR proteins (Sino Biological Inc.).

Techniques: Injection

Journal: Cellular and Molecular Immunology

Article Title: Oncolytic virus M1 reinvigorates CD8 + T-cell immunity against glioblastoma through B-cell-dependent antigen cross-presentation in the spleen

doi: 10.1038/s41423-026-01396-w

Figure Lengend Snippet: Splenic B cells are indispensable for the antiglioma activity of OVM. A Uniform manifold approximation and projection (UMAP) plot of splenic T-cell scRNA-seq data, showing six distinct subsets, with each dot representing a single cell colored by cluster. B Frequencies of the six T-cell subsets. C CellChat analysis illustrating the interaction strengths among major immune cell populations, including neutrophils, myeloid cells, NK cells, B cells, plasma cells, and T cells, in the OVM-treated group. D Differential interaction strengths between the OVM- and vehicle-treated groups across the indicated immune cell populations. E Interaction numbers between B cells and the six T-cell subsets in the OVM-treated group. F Schematic of the B-cell–CD8 + T-cell coculture experiment. GL261 cells were intracranially implanted on Day 0. The mice received daily intravenous injections of OVM or vehicle from Days 5–9. Splenic B cells were isolated on Day 12 and cocultured with tumor-derived CD8 + T cells at a 5:1 (B:T) ratio. G Proliferation of CD8 + T cells was assessed by CFSE dilution, and the proportions of IFN-γ + and GZMB + CD8 + T cells were quantified by flow cytometry. H Kaplan‒Meier survival curves of GL261 tumor-bearing mice receiving intraperitoneal injections of an anti-CD19 antibody or isotype control combined with intravenous injections of OVM or vehicle. I , J Quantification of total, CD4 + , and CD8 + T cells in the TME and spleen of GL261 tumor-bearing mice across the four treatment groups. K Kaplan‒Meier survival curves of GL261 tumor-bearing C57BL/6J wild-type or Ighm-KO mice receiving vehicle or OVM treatment. Quantification of T-cell subsets in the TME ( L ), analysis of spleen morphology and weight ( M ), and the proportion of effector CD8 + T cells (CD44 + CD62L − ) within total CD8 + T cells in the PBMCs ( N ) of GL261 tumor-bearing C57BL/6J WT or Ighm-KO mice

Article Snippet: The antibodies and reagents used were as follows: CD21/CD35 recombinant antibody (Proteintech, 84565-2-RR), CD19 polyclonal antibody (Proteintech, 27949-1-AP), BST2 polyclonal antibody (Proteintech, 30118-1-AP), FlexAble 2.0 Cora Lite® Plus 488 Antibody Labeling Kit for Rabbit IgG (Proteintech, KFA501), FlexAble 2.0 Cora Lite® Plus 594 Antibody Labeling Kit for Rabbit IgG (Proteintech, KFA509), and FlexAble 2.0 Cora Lite® Plus 555 Antibody Labeling Kit for Rabbit IgG (Proteintech, KFA502).

Techniques: Activity Assay, Single Cell, Clinical Proteomics, Isolation, Derivative Assay, Flow Cytometry, Control