Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: In vitro culture conditions maintaining the naïve B cell state. (A) Schematic diagram of the different steps of the in vivo CRISPR/Cas9 screening system. Briefly, naïve B cells isolated from donor mice of the indicated genotype are transduced with LV particles expressing sgRNAs specific for the genes of interest and a neutral control gene. After infection, the cells are cultured in vitro for 3 days. Transduced mCherry + donor B cells are sorted by flow cytometry and transferred to recipient mice of the indicated genotype, which are immunized with the hapten NP to induce an immune response. The resulting mCherry + plasmablasts are isolated, and the abundance of sgRNAs in plasmablasts is determined and compared with the abundance of the sgRNAs in the donor B cells. (B) Flow cytometric analysis of wild-type naïve splenic B cells cultured in vitro for 3 days under different conditions. B cells were cultured either in the B cell medium alone, or additionally in the presence of LPS, sBAFF, OP9 cells, or OP9 cells plus sBAFF. Before starting the culture, B cells were labeled with the CellTrace Violet dye. After 3 days, the cell viability, CD69 and IgD cell surface protein expression, differentiation into plasmablasts (CD138 + CD22 – ), and cell proliferation were assessed. (C) Summary of all the data generated with the different culture conditions indicated. The statistical data are shown as mean values with SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each dot corresponds to one mouse. Independent experiments were performed from two (IgD) to 10 (viability) times.

Article Snippet: Additionally, B cells were cultured with 10 ng/ml human recombinant sBAFF (60-mer, AG-40B-0112; AdipoGen Life Sciences) or on stromal OP9 cells in combination with or without sBAFF.

Techniques: In Vitro, In Vivo, CRISPR, Isolation, Transduction, Expressing, Control, Infection, Cell Culture, Flow Cytometry, Labeling, Generated

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Characterization of the Rosa26 LSL-EcoR allele. (A) Schematic diagram and expression of the Rosa26 LSL-EcoR allele. SLC7A1 (EcoR) protein expression was analyzed by flow cytometry in mature B cells from Cd23 -Cre Rosa26 LSL-EcoR/+ and control Cd23 -Cre Rosa26 +/+ mice and is displayed as a histogram or quantification of the geometric mean fluorescence intensity (gMFI). Statistical data are shown as mean values with SEM and were analyzed by the unpaired t test; ***P < 0.001. Each dot represents one mouse. (B) Flow cytometric analysis of B cells from Cd23 -Cre Rosa26 LSL-EcoR/+ and control Cd23 -Cre Rosa26 +/+ mice, which were transduced with ecotropic LV particles expressing the mCherry fluorescent reporter protein (mCherry-LV) and were subsequently cultured in vitro for 3 days in the presence of OP9 cells with sBAFF. The percentage of transduced mCherry + B cells is shown. The bar graph indicates the transduction efficiency relative to all B cells. Statistical data are shown as mean values with SEM and were analyzed by the multiple unpaired t test with Holm–Šídák’s correction; ****P < 0.0001. Two experiments were performed, and each dot corresponds to one mouse. (C) Flow cytometric analysis of splenic B cells from Cd23 -Cre Rosa26 LSL-EcoR/+ and control Cd23 -Cre Rosa26 +/+ mice. The frequencies are shown for total B cells (CD19 + B220 + ), immature B cells (CD19 + CD93 + ), T1 B cells (CD19 + CD93 + IgM hi CD23 − ), T2 B cells (CD19 + CD93 + IgM hi CD23 + ), mature B cells (CD19 + CD93 − ), MZ B cells (CD19 + CD93 − CD21 hi Cd23 −/lo ), FO B cells (CD19 + CD93 − CD21 lo CD23 hi ), GC B cells (CD19 + B220 + GL7 + CD95 + ), and plasma cells (CD138 + TACI + ). (D) Flow cytometric analysis of bone marrow plasma cells from Cd23 -Cre Rosa26 LSL-EcoR/+ and control Cd23 -Cre Rosa26 +/+ mice. (E) Quantification of the number of total B, immature B, mature B, GC B, and plasma cells in the spleen, and plasma cells in the bone marrow from Cd23 -Cre Rosa26 LSL-EcoR/+ and control ( Cd23 -Cre Rosa26 +/+ and Rosa26 +/+ ) mice. Statistical data are shown as mean values with SEM. All pairwise comparisons are nonsignificant, as analyzed by the multiple unpaired t test with Holm–Šídák’s correction test. Two (A and B) and three (C–E) independent experiments were performed. Each dot represents one mouse.

Article Snippet: Additionally, B cells were cultured with 10 ng/ml human recombinant sBAFF (60-mer, AG-40B-0112; AdipoGen Life Sciences) or on stromal OP9 cells in combination with or without sBAFF.

Techniques: Expressing, Flow Cytometry, Control, Fluorescence, Transduction, Cell Culture, In Vitro, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Description and characterization of different aspects of the sgRNA screening system. (A) Flow cytometric analysis of mCherry expression in B cells at 2 and 3 days after infection with a control mCherry-LV. The cells were cultured on OP9 cells with sBAFF (right). The frequencies of mCherry + B cells are shown as mean values with SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001; ****P < 0.0001. (B) Flow cytometric analysis of dividing and nondividing mCherry + B cells after 3 days in culture on OP9 cells with sBAFF. Mature CD43 – B cells were incubated with CellTrace Violet prior to infection with a control mCherry-LV and were subsequently cultured for 3 days. The frequencies of mCherry + B cells among the dividing and nondividing B cell population are shown as mean values with SEM and were analyzed by the unpaired t test; ***P < 0.001. (C) Immunofluorescence staining of spleen sections from recipient mice at 5 and 7 days after immunization with NP-KLH/alum. LV infection and B cell transfer were performed as described in D. Spleen sections were stained with antibodies against IgD (green), mCherry (red), TCRβ (blue), and IRF4 (white). A nonimmunized mouse, which did not receive transduced mCherry + B cells, was used as a control. The scale bar represents 100 μm. (D) Time course of plasmablast formation upon immunization with NP-Ficoll. Naïve B cells isolated from the donor mice were transduced with LV particles expressing a neutral control sgRNA (sg. Chr1 ) and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of stromal OP9 cells and sBAFF. Transduced mCherry + B cells were isolated and transferred to recipient mice, which were immunized 16 h later with NP-Ficoll in PBS. Flow cytometric analysis of splenocytes from recipient mice at day 3, 5, and 7 after immunization (left) revealed the percentages of mCherry + B cells in total splenocytes and mCherry + plasmablasts (TACI + CD138 + ) within the mCherry + cells, as shown in the bar graphs (right). Absolute numbers of the mCherry + B cells and mCherry + plasmablasts in the spleen are also indicated (far right). Statistical data are shown as mean values with SEM and were analyzed by the Welch ANOVA with the Brown–Forsythe test; *P < 0.05; **P < 0.01; ****P < 0.0001. Each dot represents one mouse. AF, autofluorescence measured in the BV605 channel. (E) Scheme describing the steps taken for the selection of the 379 genes to be studied in the in vivo CRISPR/Cas9 screening experiments. The data shown in A, B, and D are based on two independent experiments. Each dot corresponds to one mouse.

Article Snippet: Additionally, B cells were cultured with 10 ng/ml human recombinant sBAFF (60-mer, AG-40B-0112; AdipoGen Life Sciences) or on stromal OP9 cells in combination with or without sBAFF.

Techniques: Expressing, Infection, Control, Cell Culture, Incubation, Immunofluorescence, Staining, Isolation, Transduction, In Vitro, Selection, In Vivo, CRISPR

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Testing of the in vivo model system used for CRISPR/Cas9 screening experiments. (A) Schematic diagram of the setup used for testing the in vivo CRISPR/Cas9 screening system. Naïve B cells isolated from donor mice were transduced with LV particles expressing a neutral control sgRNA and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mCherry + B cells were sorted by flow cytometry and transferred to recipient mice, which were immunized 16 h later with NP-KLH in alum. mCherry + plasma cells and mCherry + GC B cells were analyzed by flow cytometry at different time points after immunization. (B) Naïve B cells of the donor genotype were infected with a mCherry-LV expressing a neutral control sgRNA and sorted followed by transfer to mice of the recipient genotype and NP-KLH immunization, as described in A. Flow cytometric analysis of splenocytes from recipient mice was performed 3, 5, 7, and 10 days after immunization. The percentages of total mCherry + B cells in total splenocytes, mCherry + plasmablasts (TACI + CD138 + ), and mCherry + GC B cells (GL7 + CD95 + ) within the mCherry + B cell population (left), and the absolute numbers of these cells (right) are indicated. AF, autofluorescence measured in the BV605 channel. (C) Naïve B cells isolated from donor mice were transduced with LV particles expressing a control sgRNA (sg. Chr1 ) or sgRNAs targeting Prdm1 or Irf4 . After infection, B cells were cultured in vitro for 3 days and transduced mCherry + B cells were transferred to recipient mice followed by immunization with NP-KLH, as described in A. 6 days after immunization, the splenocytes were analyzed by flow cytometry. The percentages of total mCherry + B cells, mCherry + plasmablasts, and mCherry + GC B cells among total B cells are indicated. Statistical data (B and C) are shown as mean values with SEM and were analyzed by the multiple unpaired t test with Holm–Šídák’s correction test; *P < 0.05; **P < 0.01; ****P < 0.0001. Two independent experiments (B and C) were performed. Each dot represents one mouse.

Article Snippet: Additionally, B cells were cultured with 10 ng/ml human recombinant sBAFF (60-mer, AG-40B-0112; AdipoGen Life Sciences) or on stromal OP9 cells in combination with or without sBAFF.

Techniques: In Vivo, CRISPR, Isolation, Transduction, Expressing, Control, Infection, Cell Culture, In Vitro, Flow Cytometry, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Indel sequencing data, identification of GC B cell– and PB-specific sgRNA hits, and sgRNA representation at different stages of the screening experiments. (A and B) Indel sequencing. Mature CD43 – B cells were infected with the indicated sgRNA mCherry-LVs, cultured for 3 days on OP9 cells with sBAFF, and then stimulated with CpG, IL-4, and IL-5 for another 3 days, followed by flow cytometric sorting of the mCherry + B cells and DNA preparation. Indel sequencing was performed by PCR amplification and sequencing of a DNA fragment spanning the sgRNA break site, followed by TIDE analysis . (A) Percentages of indels are indicated relative to the break site of sg. Cd44 (position 0, black), demonstrating that the percentage of indels is 88% for the sg. Cd44 . (B) Percentages of indels are shown for the indicated sgRNAs, as determined by the indel sequencing and TIDE analysis. (C) Identification of GC B cell–specific and PB-specific positive and negative regulators, as determined by CRISPR/Cas9 screening experiments at day 7 after NP-KLH immunization. The log 2 FC plot (left) indicates the sgRNA hits that were determined by a more than twofold change in sgRNA abundance in GC B cells and PBs (corresponding to ). The genes corresponding to the GC B cell–specific and PB-specific sgRNA hits are shown (right), and their fold changes and P values are indicated in . The common positive regulators are shown in , as being significant in both cell types, while the PB-specific positive regulators are indicated as being nonsignificant in the GC B cell analysis . (D) sgRNA representation at different stages of the TD and TI screening experiments. Flow cytometric analysis was used to determine the number of sorted mCherry + B cells at the start, the splenic mCherry + B cells at day 3 after B cell transfer ( [TD] and S1D [TI]), and the splenic mCherry + PBs, GC B cells, and memory B cells at day 7 ( [TD] and S4D [TI]). As the screening library contained 882 sgRNAs, the number of identified B cells was divided by 882 to determine how many cells contained one specific sgRNA (cells/sgRNA) under the assumption that each cell was only infected by one sgRNA virus (MOI = 1). (E) Schematic representation of the LV vectors used in this study. LVs containing the PGK-mCherry or EF1a-mCherry gene were used for establishing and testing of the in vivo CRISPR/Cas9 screening system. The sgRNA library was cloned in a LV vector containing the EF1as-mCherry gene. The validation experiments were performed with LVs containing the EF1as-mCherry or EF1as-mAmetrine gene. 5′LTR, 5′ long terminal repeat; Ψ, psi packaging signal; RRE, Rev response element; cPPT, central polypurine tract; PBS, primer binding site for DNA sequencing library preparation; hU6, human U6 promoter; hPGK, human phosphoglycerate kinase promoter; hEF1a, human elongation factor 1a promoter; hEF1as, human elongation factor 1a short promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; 3′LTR (SIN), 3′ long terminal repeat (self-inactivating); FC, fold change; PBs, plasmablasts.

Article Snippet: Additionally, B cells were cultured with 10 ng/ml human recombinant sBAFF (60-mer, AG-40B-0112; AdipoGen Life Sciences) or on stromal OP9 cells in combination with or without sBAFF.

Techniques: Sequencing, Infection, Cell Culture, Amplification, CRISPR, Cell Analysis, Virus, In Vivo, Clone Assay, Plasmid Preparation, Biomarker Discovery, Binding Assay, DNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: CRISPR/Cas9 screen for identifying new regulators of the TD B cell responses. (A) Schematic diagram of the in vivo CRISPR/Cas9 screening experiments used to study the TD B cell responses. Naïve B cells isolated from donor mice were transduced with LV particles, each expressing one of the 882 sgRNAs and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mCherry + donor B cells were sorted by flow cytometry and transferred to recipient mice, which were immunized 16 h later with NP-KLH/alum. 7 days after immunization, splenic mCherry + CD45.2 + cells were enriched by anti-CD45.1 antibody–mediated MACS depletion of the CD45.1 + CD45.2 + recipient cells. Plasmablasts and GC B cells were isolated from the enriched fraction by flow cytometric sorting, followed by DNA extraction, library preparation, and DNA sequencing. DNA was also extracted from a fraction of the sorted transduced mCherry + donor B cells before immunization, followed by library preparation and DNA sequencing. (B) Flow cytometric analysis of the enriched mCherry + CD45.2 + B cells from recipient mice. The gates used for the sorting of plasmablasts (TACI + CD138 + ) and GC B cells (CD19 + GL7 + CD95 + ) are indicated. The percentages of mCherry + plasmablasts and mCherry + GC B cells within the sorted cell population are shown. (C) Numbers of sorted plasmablasts and GC B cells (left), the percentage of mCherry + plasmablasts and mCherry + GC B cells within the sorted cell population (middle), and the numbers of mCherry + plasmablasts and mCherry + GC B cells (right) were determined for individual recipient mice analyzed in two independent screening experiments. (D) Total sgRNA representation in each plasmablast and GC B cell replicate sample analyzed in two screening experiments. The sgRNA representation was estimated by dividing the number of mCherry + plasmablasts or GC B cells by the number of the 882 sgRNAs constituting the sgRNA library. Each plasmablast and GC B cell replicate sample contained cells that were obtained from five or six recipient mice after pooling. Each colored box represents the individual contribution of each mouse to the sgRNA representation of the entire replicate sample.

Article Snippet: Additionally, B cells were cultured with 10 ng/ml human recombinant sBAFF (60-mer, AG-40B-0112; AdipoGen Life Sciences) or on stromal OP9 cells in combination with or without sBAFF.

Techniques: CRISPR, In Vivo, Isolation, Transduction, Expressing, Infection, Cell Culture, In Vitro, Flow Cytometry, DNA Extraction, DNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Validation of gene hits of the TD B cell responses by sgRNA-mediated gene inactivation. (A) Schematic diagram of the experiments used for validating potential regulators identified by the in vivo CRISPR/Cas9 screens. Naïve splenic B cells of the donor genotype were transduced with LV particles expressing the mCherry reporter protein and a sgRNA targeting the gene of interest. In parallel, donor B cells were transduced with LV particles expressing the mAmetrine reporter protein and the control sgRNA (sg. Chr1 ). After infection, the cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mAmetrine + and mCherry + B cells were sorted by flow cytometry, mixed, and transferred to recipient mice, which were then immunized with NP-KLH/alum 16 h later. The frequency of mAmetrine + and mCherry + PBs in the spleen was analyzed by flow cytometry 7 days after immunization. (B) mCherry + and mAmetrine + sgRNA-expressing B cells were mixed at a 2.5:1 ratio and transferred into recipient mice, followed by immunization. Flow cytometric analysis of splenocytes from recipient mice was performed 7 days after immunization. mCherry + B cells expressed either the control sgRNA (sg. Chr1 ) or sgRNAs targeting Prdm1 (sg. Prdm1 ), positive regulator genes (sg. Amigo2 , sg. Slc22a17 , and sg. Qpctl ), and a negative regulator gene (sg. Cd44 ). The percentage of mCherry + (red gate) and mAmetrine + (blue gate) B cells among the total B cells (CD19 + CD138 –/lo and CD19 lo CD138 + ; green gate) is shown. The percentages of mCherry + and mAmetrine + PBs (TACI + CD138 + ) and GC B cells (GL7 + CD95 + ) in total B cells (green numbers) and within the mCherry + B cells (red numbers) or mAmetrine + B cells (blue numbers) are indicated. PB, plasmablast.

Article Snippet: Additionally, B cells were cultured with 10 ng/ml human recombinant sBAFF (60-mer, AG-40B-0112; AdipoGen Life Sciences) or on stromal OP9 cells in combination with or without sBAFF.

Techniques: Biomarker Discovery, In Vivo, CRISPR, Transduction, Expressing, Control, Infection, Cell Culture, In Vitro, Flow Cytometry