Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) IGHM (left) and IGHG1 (right) expression as transcripts per million (TPM) in IgM high ( n = 111) or IgG1 high ( n = 63) samples. ( B ) Kaplan-Meir curves displaying progression-free survival (probability) in IgM and IgG1 high samples. ( C ) Distribution of distinct DLBCL subtypes in IgM and IgG1 high groups. ( D to F ) Kaplan-Meir curves for progression-free survival (probability) in IgM high and IgG1 high in BN2 (D), EZB (E), and Other (F) genetic subtypes. sgRNA, single guide RNA. ( G ) Engineering of switched isogenic human lymphoma cell lines using CRISPR-Cas9. ( H ) IgM and IgG1 expression in BJAB, OCI-Ly7. and HBL-1 cells before and after CRISPR-Cas9 editing. ( I ) Tumor growth of IgM or IgG1 cells (1 million per mouse) subcutaneously injected into NSG mice ( n = 5 mice). ( J ) Tumor growth curves of IgM or IgG1 expressing BJAB subcutaneously injected into NSG mice. ( K ) Surface expression of Ig-kappa light chain (Igκ) in IgM BJAB cells (gray), IgM tumors (gray dashed), and IgG1 BJAB cells (red), tumors which grew from IgG1 BJAB cells (red dashed) and BJAB IgM cells stained with secondary Ab (no Ab control, black). ( L ) Cotransplantation of IgG1 and IgM lymphoma cells mixed at 1:1. Bottom; Representative plots of IgM and IgG1 at days 0 and 21 posttransplant. ( M ) Quantification of IgM and IgG1 cell frequency in tumors after 21 days ( n = 4 for Mino IgG1, Mino IgM, and BJAB IgG1; n = 5 for BJAB IgM). ( N and O ) Bar graphs quantifying percent IgM and IgG1 BJAB (N) and Mino (O) cells after mixing (day 0) and upon coculture for 5 or 8 days. Statistical significance calculated by log-rank test [(B) and (D) to (F)], t test (I), and two-way ANOVA [(M) to (O)]. Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. [(G) and (L)] Created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S1.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Expressing, CRISPR, Injection, Staining, Control

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Schematic of the experimental design. Cd19 cre Tet2 / Tet3- deficient DKO B cells (CD45.2+) were isolated from the spleen and cultured for 4 days using an iGB in vitro culture system. TET-deficient B cells (2 million) were transplanted into sublethally irradiated CD45.1+ recipient mice. ( B ) Representative flow cytometry plot showing the frequency of IgM and IgG1 before transplantation in TET-deficient YFP+, CD45.1− B cells 4 days after iGB culture. ( C ) Relative frequency of IgM and IgG1 expressing YFP+, CD45.2+ B cells in blood over time. IgM and IgG1 frequencies are normalized to their respective pretransplantation frequency ( n = 9 from two independent experiments). ( D ) Representative flow cytometry plot of YFP+, CD45.1− splenic B cells at week 10 posttransplantation (left). Quantification of IgM and IgG1 frequency (right) ( n = 9 from two independent experiments). ( E ) Quantification of the absolute number of IgM and IgG1 splenic B cells gated from YFP+, CD45.1− B cells. Cell numbers are normalized to IgM ( n = 9 from two independent experiments). ( F ) Representative flow cytometry plots of proliferating IgM and IgG1 cells with Ki67 staining from YFP+, CD45.1− B cells (left). Quantification of Ki67+ from YFP+, CD45.1− splenic B cells (right) ( n = 6 from two independent experiments). ( G ) Representative flow cytometry plots of apoptotic IgM and IgG1 cells measured by cleaved caspase-3 staining from YFP+, CD45.1− cells (left). Quantification of cleaved caspase-3 from YFP+, CD45.1− splenic B cells (right) ( n = 6 from two independent experiments). Statistical significance is calculated by ordinary two-way ANOVA test with Šídák multiple comparisons test (C) and paired t test [(D) to (G)]. Error bars represent means ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Schematic in (A) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S2.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Isolation, Cell Culture, In Vitro, Irradiation, Flow Cytometry, Transplantation Assay, Expressing, Staining

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Schematic for SRBC immunization. ( B ) Representative plots for gating GC B cells (left). Quantification of cleaved caspase-3 from IgM and IgG1 GC B cells (right). Connecting lines show data points within each mouse. ( C ) Schematic of iGB cocultures. ( D ) Representative plots for IgM and IgG1 frequency from CD19+CD138−, 12 hours poststimulation (BCR) with anti-kappa antibody (1 μg/ml). ( E ) Quantification of IgG1 and IgM B cell frequency with or without BCR stimulation. IgM and IgG1 are normalized to respective frequencies under untreated conditions ( n = 7, three independent experiments). ( F ) Quantification of IgM and IgG1 B cell numbers ( n = 7, three independent experiments). ( G ) Representative flow plots (left) and quantification of cleaved caspase-3 (right). Cleaved caspase-3 is normalized to IgM cells in the untreated group ( n = 17 from seven independent experiments). ( H ) Genome browser tracks of RNA-seq data in the IgH locus ( Ighg1 and Ighm genes are highlighted). Black and red tracks are from IgM and IgG1 cells from untreated and BCR-stimulated groups. Tracks are averaged from three replicates. ( I ) Volcano plot displaying DEGs in IgG1- versus IgM expressing cells from untreated (left) and BCR-stimulated conditions (right). ( J ) Up-regulated (red) and down-regulated (gray) pathways identified from DEGs in IgG1 compared with IgM using Metascape. The x axis represents the z score. ( K ) Gene set enrichment analysis (GSEA) of apoptosis and abnormal mitochondrial morphology gene sets. The y axis denotes enrichment score. NES, normalized enrichment score; P nominal P value. Statistical significance is calculated by the paired t test (B) and ordinary two-way ANOVA with Šídák multiple comparisons test [(E) and (F)]. Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Schematics in (A) and (C) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S3.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: RNA Sequencing, Expressing

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Representative plots of IgM and IgG1 GC B cells (Fas+, CD38low) (left) 14 days postimmunization with SRBCs. Histograms show MitoTracker Green staining in IgM and IgG1 cells (middle). Median fluorescence intensity of MitoTracker Green in IgM and IgG1 cells (right) ( n = 8). ( B ) Representative ImageStream analysis of IgM and IgG1 GC B cells stained with DAPI (nucleus), Tomm20 (PE: mitochondria), IgG1 (FITC), and IgM (PE-Cy7). ( C ) Histograms showing MitoTracker Green in IgM and IgG1 cells from iGB cultures stimulated with anti-kappa (1 μg/ml) for 12 hours (left). Quantification of relative MitoTracker Green mean fluorescence intensity (MFI) (middle). Values are normalized to untreated IgM from respective experiments. Relative MitoTracker Green MFI also normalized to cell size (right) ( n = 6 from two independent experiments). ( D ) Flow cytometry histograms showing MitoTracker Green staining (top) and quantification of MitoTracker Green MFI (bottom) in IgM and IgG1 splenic B cells from heterozygous IgH γ1μ /+ mice ( n = 3). ( E ) Representative confocal microscopy images of splenic B cells from heterozygous IgH γ1μ /+ mice (left) and quantified as mean fluorescence intensity (right). ( F ) Experimental design with IgG1 cells isolated from expressing homozygous IgH γ1μ/γ1μ mice and treated with TAT-Cre for 2 hours and stimulated with anti-kappa antibody. ( G ) Representative flow plots of IgM and IgG1 staining of IgH γ1μ/γ1μ splenic B cells before TAT-Cre treatment and 24 and 48 hours after TAT-Cre treatment. ( H ) Histograms showing MitoTracker Green staining (left) and quantification of MitoTracker Green MFI (right) in IgM and IgG1 B cells from IgH γ1μ /γ1μ mice 48 hours post-BCR anti-kappa stimulation (2 μg/ml) ( n = 5). Statistical significance is calculated by the paired t test (A), ordinary two-way ANOVA with Šídák multiple comparisons test [(C), (D), and (H)], and unpaired t test (E). Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. See also fig. S4.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Staining, Fluorescence, Flow Cytometry, Confocal Microscopy, Isolation, Expressing

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Changes in OCR measured over time in IgM (black) and IgG1 (red) IgH γ1μ /+ B cells stimulated with anti-kappa antibody, treated with oligomycin, phenylhydrazone (FCCP), and antimycin A and rotenone. ( B ) Changes in OCR measured over time in IgM and IgG1 BJAB lymphoma cells treated with anti-Igκ antibody, oligomycin, FCCP, antimycin A, and rotenone. ( C ) Representative flow cytometry plots of IgM and IgG1 splenic GC B cells (Fas+, CD38 low) stained with MitoTracker Green ( x axis) and TMRM ( y axis). Gated population indicates dysfunctional mitochondria (left), identified as lower TMRM and positive MitoTracker Green staining. Quantification of the frequency of dysfunctional mitochondria in IgM and IgG1 GC B cells (right) ( n = 3). ( D ) Representative flow cytometry plots of IgM and IgG1 B cells from in vitro iGB cultures stained with MitoTracker Green ( x axis) and TMRM ( y axis) (left). Quantification of the frequency of dysfunctional mitochondria measured by identifying as lower TMRM and positive MitoTracker Green staining (right) ( n = 5 from two independent experiments). ( E ) Frequency of cleaved caspase-3–positive cells in IgM and IgG1 B cells from in vitro iGB cultures ( n = 5 from two independent experiments). ( F and G ) Representative flow cytometry plots of OCI-Ly7 (F) and BJAB (G) IgM and IgG1 lymphoma cells stained with MitoTracker Green and TMRM after BCR treatment for 24 hours (left). Quantification of the frequency of dysfunctional mitochondria (right) ( n = 6 from two independent experiments (F) and n = 3 (G) (right). Statistical significance is calculated using a paired t test (C) and ordinary two-way ANOVA with Šídák multiple comparisons test [(D) to (G)]. Error bars represent means ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Schematic in (F) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S4.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Flow Cytometry, Staining, In Vitro

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Calcium flux measured by fluorescence intensity of Fluo4 ( y axis) over time in IgM and IgG1 IgH γ1μ /+ splenic B cells in response to BCR (anti-Igκ) stimulation. ( B ) Calcium flux measured by fluorescence intensity of Fluo4 in IgM and IgG1 OCI-Ly7 lymphoma cells in response to BCR (anti-Igκ) stimulation. ( C ) Schematic of the iGB in vitro culture system with treatment groups. ( D ) Representative flow cytometry plots of IgM and IgG1 B cells derived from iGB in vitro cultures stained with MitoTracker Green ( x axis) and TMRM ( y axis) 12 hours posttreatment. ( E ) Quantification of dysfunctional mitochondria frequency in IgM and IgG1 B cells from iGB in vitro cultures ( n = 6 from two independent experiments). ( F ) Representative flow cytometry plots of cleaved caspase-3 from IgM and IgG1 B cells from iGB in vitro cultures 12 hours posttreatment. ( G ) Quantification of relative frequency of cleaved caspase 3 from IgM and IgG1 B cells from iGB in vitro cultures 12 hours poststimulation. Values are normalized to DMSO IgM and IgG1. ( H ) Proposed model showing that IgG1 B cell signaling triggers greater calcium flux, leading to increased mitochondrial dysfunction, which contributes to reduced cell survival. Dampening of calcium flux with BAPTA-AM rescues the survival of IgG1 expressing B cells. Statistical significance is calculated by repeated measures two-way ANOVA with Tukey’s multiple comparisons test [(E) and (G)]. Error bars represent means ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Schematics in (C) and (H) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S5.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Fluorescence, In Vitro, Flow Cytometry, Derivative Assay, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Natural Killer Cells from Patients with Recombinase-Activating Gene and Non-Homologous End Joining Gene Defects Comprise a Higher Frequency of CD56 bright NKG2A +++ Cells, and Yet Display Increased Degranulation and Higher Perforin Content

doi: 10.3389/fimmu.2017.00798

Figure Lengend Snippet: Combination of antibodies used to characterize natural killer cell phenotype.

Article Snippet: , PE/Cy7-conjugated goat anti-mouse IgG2b , Southern Biotech.

Techniques: Control

Journal: bioRxiv

Article Title: Development of attenuated coxsackievirus B3 vectored intranasal pre-emptive pan-coronavirus vaccine

doi: 10.1101/2023.11.22.568225

Figure Lengend Snippet: (A) Genome Schematic diagram of attenuated rCVB3 delivery vector based intranasal pre-emptive pan-coronavirus vaccines construction. (B) Cartoon models of rCVB3-EPI and rCVB3-RBD-trimer. (C) Western Blot analysis of the intranasal pre-emptive pan-coronavirus vaccines. Collection and analysis the protein in serial passages of the recombinant viruses (1-6 passages) from Vero culture cells. (D) Plaque morphology of viruses on Vero cells. For each recombinant virus, we randomly measured 10 Plaque diameter for statistical analysis. (E) Experimental schema of vaccination and sampling timeline. Nasal washes and serum were collected on day 28, n=6 /group. (F) SARS-CoV-2 S1 specific serum IgG antibodies levels on day 28, group rCVB3-RBD-trimer and rCVB3-EPI SARS-CoV-2 S1 specific serum IgG antibodies levels on day 28 compare to control group. IgG levels measured by ELISA and presented as OD450 nm values, n=4 / group. (G) The neutralizing activity of serum samples isolated from vaccinated mice (n =6 per group) against SARS-CoV-2 (Wuhan-Hu-1) pseudovirus. The vertical axis represents the pseudovirus inhibition rate of each immune serum at a serum dilution of 1:45. (H) SARS-CoV-2 S1-specific secretory IgA (sIgA) response in nasal lavage fluid of immunized mice was assessed on day 14 post booster immunization. n=6/group. (L-J) Percentages of IFN-γ + CD3 + and CD4 + splenic T cells by FCM analysis on day 14 after the last immunization. The percentage of IFN-γ + CD4 + Th cells among from splenocytes was measured under non-stimulation (NON), PMA stimulation (PMA), and SARS-CoV-2 peptide pool (PP) stimulation, respectively, for both experimental group and vaccine group (n=3/ group). (K-L) The percentage of IL-4 + Th cells and IFN-γ + Th cells among CD4 + T lymphocytes was determined under SARS-CoV-2 PP stimulation, respectively. Significance analysis was performed using a two-tailed t-test for unpaired samples. One-way analysis of variance (ANOVA) was also employed for significance analysis, data are represented as mean ± standard error of the mean (SEM) *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: Next, cells were fixed and permeabilized using the Fixation/Permeabilization Kit (554714, BD Pharmingen, USA) to enable intracellular staining with PE Rat Anti-Mouse IFN-γ (554412, BD Pharmingen, USA) and PE-Cy™7 Rat Anti-Mouse IL-4 (560699, BD Pharmingen, USA).

Techniques: Plasmid Preparation, Vaccines, Western Blot, Recombinant, Virus, Sampling, Enzyme-linked Immunosorbent Assay, Activity Assay, Isolation, Inhibition, Two Tailed Test