Journal: Biology Open

Article Title: Monitoring and evaluation of the immune status of female Kunming mice maintained in different biosafety level laboratories

doi: 10.1242/bio.035006

Figure Lengend Snippet: Constitution of the lymphocyte subtypes of mouse blood and spleen samples. Mice from the ABSL-2/3/4 labs were euthanized at days 7, 14 and 28 of housing, and blood and spleen samples of each mouse were harvested ( n =4, each time point and each group). Percentages of CD3+, CD4+, CD8+ and CD19+ lymphocytes from blood (A,B) and spleen (C,D) samples were determined by flow cytometry. (A,C) Sorting illustrations of blood sample and spleen sample. (B,D) Statistics of total percentages of CD3+, CD4+, CD8+ and CD19+ lymphocytes of blood samples and spleen samples in each mouse group. The error bars represent the standard deviation of each mouse group ( n =12). The P value was conducted based on Student's t -test.

Article Snippet: Cell surface staining was done using Mouse BD Fc Block and anti-mouse CD3-PerCP (Cat. No. 551163, Lot 7038886, clone 145-2C11), CD4-PE (Cat. No. 553652, Lot 7038886, clone H129.19), CD8-FITC (Cat. No. 553030, Lot 7170636, clone 53-6.7), and CD19-APC (Cat. No. 550992, Lot 7081501, clone 1D3) (BD Biosciences).

Techniques: Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A ) Scheme of procedure used for generation of resistant models (left panel). Lymphoma (Raji) and B-ALL (Nalm-6) models were established through 19 subsequent and parallel expositions to effectors (unmodified T-cells, CD19-4-1BB- or CD19-CD28 CAR-T cells), in the same conditions and E:T ratio for every passage. The structures of CAR-CD19 constructs bearing 4-1BB or CD28 costimulatory domain (right panel) were identical to clinically used product tisa-cel and axi-cel, respectively. ( B ) Efficacy of T-cells transduction with CAR-CD19-4-1BB and CAR-CD19-CD28 constructs. Detection of CAR-positive T-cells (a representative plot for one of the PBMCs’ donors) was performed by flow cytometry staining with RTX followed by secondary AlexaFluor 647-conjugated anti-Fc anibody (left) or anti-scFv (FMC63) antibody (right). ( C ) Flow cytometry analysis of CD19 surface expression in Raji cells (left panel) and Nalm-6 cells (right panel) during the process of long-term co-culture with CD19 CAR-T cells bearing either 4-1BB or CD28 costimulatory domain. The CD19 expression was detected by the HIB19 antibody clone after targets-effectors co-culture separation and targets recovery (up to 10 days). Raji or Nalm-6 cells co-cultured with unmodified T-cells are shown in black (con), co-cultured with CD19-4-1BB CAR-T cells in blue (Raji/Nalm-6 CAR-4-1BB) and with CD19-CD28 CAR-T cells in magenta (Raji/Nalm-6 CAR-CD28). The staining of anti-CD19 antibody (filled histograms) was compared to isotype controls (transparent histograms). p11-p19 reffers to the passage number. ( D ) Flow cytometry-based analysis of sensitivity of Raji (upper panel) and Nalm-6 (lower panel) cells to unmodified T-cells, CD19-4-1BB CAR-T cells or CD19-CD28 CAR-T cells. In the experiment, control (con) Raji and Nalm-6 target cells and their counterparts after long-term co-culture (19 passages) with CD19 CAR-T cells incorporating 4-1BB or CD28 domain were used. The plot shows the % cytotoxicity of effectors against targets. Data are presented as the mean ± SD from 5-7 donors analyzed in 2 technical repeats each. Statistical analysis was performed using two-way ANOVA with interaction and Tukey’s post-hoc test for multiple comparisons. Only selected significances were shown on the plots.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Construct, Transduction, Flow Cytometry, Staining, Expressing, Co-Culture Assay, Cell Culture, Control

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A-B) Flow cytometry analysis of CD19 surface level with different anti-CD19 antibodies clones – HIB19 (black), FMC63 (orange), SJ25C1 (turquoise) and J3-119 (red) in Raji and Nalm-6 control (con, black) and after long-term co-culture (19 passages) with effectors - CD19-4-1BB CAR-T ( A , blue) or CD19-CD28 CAR-T cells ( B , magenta). The staining of anti-CD19 antibody (filled histograms) was compared to isotype controls (transparent histograms). ( C) The stability of resistance phenotype on Raji and Nalm-6 CAR-4-1BB and CAR-CD28 models was evaluated by flow cytometry CD19 surface level analysis with HIB19 clone (left panel) and FMC63 clone (right panel) after a culture of targets without effectors (8, 15, 22 and 29 days starting from day 0 – establishment of the models. ( D, F) Flow cytometry-based analysis of CD19 intracellular epitope (clone D4V4B recognizing residues surrounding Leu427) level in Raji ( D ) and Nalm-6 ( F ) WT, control, CAR-4-1BB and CAR-CD28 tumor cells. Data show the representative experiment from 2 biological replicates. ( E, G) Western blotting analysis of total CD19 protein level in Raji ( E ) and Nalm-6 ( G ) WT, control, CAR-4-1BB, and CAR-CD28 tumor cells evaluated by two antibodies recognizing different CD19 intracellular epitopes – residues surrounding Leu427 (clone D4V4B) or C-terminus residues (polyclonal antibody). Tubulin (TUB) was used as a loading control.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Flow Cytometry, Clone Assay, Control, Co-Culture Assay, Staining, Western Blot

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A) Scheme of CD19 gene fragment (exon 1 to exon 7; grey blocks) together with the location of the primers used for RT-qPCR experiments (ex1-2, ex3-4, ex4-5, black arrows) and codons coding amino acid residues (W159, R163, P222, K220) essential for FMC63 epitope recognition. ( B-C) RT-qPCR experiments showing relative CD19 mRNA level in Raji ( B ) and Nalm-6 ( C ) control (con) cells and after 19 passages of effectors’ exposures to CD19-4-1BB-CAR-T (4-1BB, blue) or CD19-CD28-CAR-T-cells (CD28, magenta). The relative mRNA expression was assessed by 3 different pairs of CD19 -specific primers covering exon 1-2, exon 3-4 or exon 4-5 junctions. The plot shows the CD19 mRNA level calculated with ΔCt method relative to the mean of TBP and GUSB as housekeeping genes. Data are presented as the mean ± SD from 3-4 biological repeats performed in technical triplicates. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. ( D) RNAseq analysis of CD19 transcript level in Raji and Nalm-6 control (con) and after exposition (19 passages) to CD19-4-1BB CAR-T cells (4-1BB) or CD19-CD28 CAR-T cells (CD28). The plot (left panel) shows raw expression (transcript per million, TPM) of CD19 transcript. Data is presented as mean ± SD from 3 biological replicates. Right panel shows the results of differential gene expression analysis (DESeq2) – the log 2 FC (fold change) and the p-value between control and 4-1BB or CD28 variants in Raji and Nalm-6 cell lines. ( E-F) CD19 sequence in Raji ( E ) and Nalm-6 ( F ) after exposition (19 passages) to CD19-4-1BB CAR-T cells (4-1BB) or CD19-CD28 CAR-T cells (CD28), as detected in RNAseq data and compared with CD19 RefSeq. The scheme shows the fragments of nucleotide sequences (blue – C, cytosine; orange – G, guanine; red – T, tymine; green – A, adenine) together with protein sequences, amino acid positions and exon numbers across the reference CD19 gene and those detected in Raji and Nalm-6 CAR-4-1BB/CD28 variants. The results of mutations detected in 4-1BB cell lines (amino acids and together with variant allele frequency, VAF [%]) are marked in blue. ( G-H) Intron 2 ( G ) or intron 6 ( H ) retention analysis in Raji and Nalm-6 control cells and after exposition (19 passages) to CD19-4-1BB CAR-T cells (4-1BB) or CD19-CD28 CAR-T cells (CD28) based on RNAseq data. Left panels show sashimi plots with coverage and the number of junction reads in Raji (upper panel) and Nalm-6 (lower panel) from pooled data of 3 biological replicates of each variant. The plots (right panels) show the frequency (% of all isoforms) of intron 2 ( F ) or intron 6 ( G ) retention calculated using the R/Bioconductor package ASpli. The data are presented as mean ± SD from 3 biological replicates. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. Only selected significances were shown on the plots.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Quantitative RT-PCR, Control, Expressing, Gene Expression, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A-B ) Venn diagrams showing the comparison of significantly upregulated or downregulated genes between Raji CAR-4-1BB and CAR-CD28 ( A, upper panel ), and Nalm-6 CAR-4-1BB and CAR-CD28 ( B, upper panel ). The significantly changed genes (p-value > 0.05, log 2 FC > 1 (up) or log 2 FC < −1 (down)) were extracted based on differential gene expression analysis (DESeq2). Volcano plots showing the results of differential gene expression analysis (DESeq2) between Raji control and CAR-4-1BB ( A, middle panel ), Raji control and CAR-CD28 ( A, lower panel ), Nalm-6 control and CAR-4-1BB ( B, middle panel ) and Nalm-6 control and CAR-CD28 ( B, lower panel ). The cut-off point for p-value was set as 0.05 and for log 2 FC as 1 or −1. ( C-D) Venn diagrams showing the comparison of significantly upregulated ( C ) or downregulated ( D ) genes between B-ALL patients treated with tisa-cel (re-analysis of RNAseq data from Orlando et al. (2018) and, Raji and Nalm-6 after long-term co-culture with CD19-4-1BB CAR-T cells (CAR-4-1BB). The significantly changed genes (p-value > 0.05; log 2 FC > 1 (up) or log 2 FC < −1 (down)) were extracted based on differential gene expression analysis (DESeq2) between relapsed samples vs. screening samples from Orlando’s data set and CAR-4-1BB vs. control samples in Raji and Nalm-6 models.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Comparison, Gene Expression, Control, Co-Culture Assay

Journal: bioRxiv

Article Title: The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies

doi: 10.1101/2025.02.28.640707

Figure Lengend Snippet: ( A ) General simulation algorithm presented on activity diagram with the six subsequent experimentation stages. ( B) Graphical representation on the histogram of the distinct subpopulations (resistant, low-antigen and high-antigen cells with defined antigen levels. ( C) Main parameters set for simulations. ( D) Histograms showing changes in CD19 MFI in subsequent passages (iterations) mimicking contact with CD19-4-1BB CAR-T cells (left) or CD19-CD28 CAR-T cells (right). The graphs show selected iteration from 19 that were performed.

Article Snippet: Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-CD19 recognizing epitope surrounding Leu427 (Clone 1; 1:1000 dilution, Cell Signaling Technology, Cat #90176, RRID: AB_2800152), anti-CD19 recognizing C-terminus residues (Clone 2; 1:1000 dilution, Cell Signaling Technology, Cat #3574, RRID: AB_2275523) or anti-tubulin (1:1000, Sigma-Aldrich, Cat #CP06, RRID: AB_2617116).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity

doi: 10.1101/2023.09.23.559132

Figure Lengend Snippet: (A) Diagrammatic representation of the experimental set-up. WT, Gla -KO, or A4galt -KO mice were immunized with NP-OVA adsorbed on alum. (B) FACS plots and percentages of centroblasts (CB) and centrocytes (CC) in the spleen on day 10 post immunization. Each dot represents one mouse (7 or 8 mice per group), and the experiment was repeated three times. (C) Immunoblot showing BCR and CD19 downstream signaling molecules and transcription factors. Anti-BCR antibodies (F(ab) 2 ) were used to stimulate FACS-sorted GC B cells for 2 or 5 minutes. U = unstimulated. (D) Proximity ligation assay (PLA) performed on GC B cells to probe for vicinity between CD81 and CD19 (top panel; blue = DAPI, red = CD19:CD81 PLA signal), and BCR and CD19 (bottom panel; blue = DAPI, red = CD19:BCR PLA signal). In all panels, experiments were repeated at least three times, and PLA signal on more than 30 cells in different fields was calculated for statistical analysis. (E) PLA performed on FACS-sorted GC B cells to probe for proximity between CD19 and Gb3 (blue = DAPI, red = CD19:Gb3 PLA signal). PLA signal was captured by confocal microscopy, and images were processed and analyzed by Image J software. Experiment was repeated at least three times, and PLA signal on more than 30 cells in different fields was calculated for statistical analysis. (F) Structures of different lipids and Gb3 analog used in the study. (G) Phospho-flow to examine the effect of lipid reconstitution on Akt phosphorylation in GC B cells from A4galt -KO mice. Histogram overlay and mean fluorescence intensity (MFI) of pAkt staining in GC B cells cultured with different lipids (bottom panel). FACS-sorted GC B cells from A4galt -KO mice were seeded with a complex of lipid and BSA for 2h at 37°C (see methods), and Akt phosphorylation was quantified after stimulation of GC B cells with F(ab) 2 . (H) Isothermal titration calorimetry (ITC) to measure the binding between Gb3 and CD19. CD19 and the Gb3 analog were dissolved in PBS, and thermodynamic analysis of their binding was carried out at 25°C on a MicroCal ITC 200 instrument. Top panel: x-axis depicts time, and y-axis represents rate of heat release (μcal/sec). Bottom panel: x-axis represents molar ratio between CD19 and Gb3-analog, and y-axis depicts change in enthalpy. (I) A mechanistic scheme of the effect of Gb3 on the CD19 translocation and BCR downstream signaling pathway. The p -values in all graphs were calculated by Kruskal-Wallis H test with Dunn’s multiple comparison test.

Article Snippet: Anti-mouse p-STAT-2 (polyclonal), STAT-1 (STAT1-79), and p-CD19 (polyclonal) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), while anti-mouse CD77 (BGR23) was purchased from Creative Biolabs.

Techniques: Western Blot, Proximity Ligation Assay, Confocal Microscopy, Software, Fluorescence, Staining, Cell Culture, Isothermal Titration Calorimetry, Binding Assay, Translocation Assay, Comparison

Journal: bioRxiv

Article Title: The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity

doi: 10.1101/2023.09.23.559132

Figure Lengend Snippet: (A) Proximity ligation assay (PLA) performed on GC B cells to quantify the recruitment of PI3K to CD19 (blue = DAPI, red = CD19:PI3K PLA signal). FACS-sorted GC B cells were stimulated with anti-BCR antibodies (F(ab) 2 ) and were probed with specific antibodies. Experiment was repeated three times, and PLA signal on more than 30 cells in different fields was calculated for statistical analysis. U = unstimulated. (B) Protein lipid overlay assay to assess the CD19 binding capability of different lipids. Lipids were deposited on a PVDF membrane and their binding to CD19 (containing histidine) was tested by using anti-His-HRP secondary antibody using chemiluminescence. The p -values in graph were calculated by Kruskal-Wallis H test with Dunn’s multiple comparison test.

Article Snippet: Anti-mouse p-STAT-2 (polyclonal), STAT-1 (STAT1-79), and p-CD19 (polyclonal) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), while anti-mouse CD77 (BGR23) was purchased from Creative Biolabs.

Techniques: Proximity Ligation Assay, Protein-lipid Overlay Assay (PLOA), Binding Assay, Membrane, Comparison

Journal: bioRxiv

Article Title: The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity

doi: 10.1101/2023.09.23.559132

Figure Lengend Snippet: (A) Representative FACS plots showing gating strategy to identify B cell subsets among human tonsil cells. GC B cells (CD38 + ); plasma cells (CD27 + , IgD - ). CD77-positive and CD77-negative GC B cells were sorted from human tonsils, and CD77-negative GC B cells were subsequently cultured with Gb3. FACS analysis shows Gb3-positivity after culture (bottom panel). CD77 = Gb3. (B) Proximity ligation assay (PLA) performed on GC B cells to probe for vicinity between Gb3 and CD19 (blue = DAPI, red = CD19:Gb3 PLA signal). CD77-negative, CD77-positive, and CD77-negative B cells reconstituted with Gb3 were used for PLA. (C) Immunoblot showing CD19 downstream signaling molecules (Akt) and transcription factors (Foxo1). CD77-negative, CD77-positive, and CD77-negative B cells reconstituted with Gb3 were used for immunoblot analysis. Anti-BCR antibodies (F(ab) 2 ) were used to stimulate B cells. U = unstimulated. (D) Workflow for stimulation of human tonsil organoids with antigens and adjuvants. (E) Graphs depicting GC B cell and plasma cell percentages from unstimulated or antigen/adjuvant-stimulated tonsil organoids on day 10 post treatment measured by flow cytometry. Each dot in bar graph represents a separate human tonsil donor (n=7). (F) IgG1 secretion from unstimulated or antigen/adjuvant-stimulated tonsil organoids on day 21 post treatment. Each dot in graph represents a different human tonsil donor (n=7). In all graphs, data were analyzed using Kruskal-Wallis H test with Dunn’s multiple comparison test. (G) Illustration depicts the regulation of the GC B cell response by Gb3 (left panel) and its translation as an adjuvant against influenza infection (right panel). Gb3 binds to CD19 to promote BCR downstream signaling and Foxo1 modulation, driving the cycling of GC B cells to the light zone. Moreover, Gb3 regulates MHC-II antigen presentation to Tfh cells to facilitate selection of antibodies against subdominant epitopes. Use of Gb3 as an adjuvant induces IgG2c class switch and selects antibodies recognizing the stalk region of hemagglutinin. Collectively, these mechanisms provide superior protection against heterologous influenza infection. GC = germinal center; Tfh = T follicular helper cell; TCR = T cell receptor; FDC = follicular dendritic cell; rHA = recombinant hemagglutinin; dLN = draining lymph node; H1N1/H3N2 = influenza virus strains.

Article Snippet: Anti-mouse p-STAT-2 (polyclonal), STAT-1 (STAT1-79), and p-CD19 (polyclonal) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), while anti-mouse CD77 (BGR23) was purchased from Creative Biolabs.

Techniques: Cell Culture, Proximity Ligation Assay, Western Blot, Adjuvant, Flow Cytometry, Comparison, Infection, Selection, Recombinant, Virus