Journal: bioRxiv

Article Title: An IRF4-MYC-mTORC1 integrated pathway controls cell growth and the proliferative capacity of activated B cells during B cell differentiation in vivo

doi: 10.1101/2021.06.20.449155

Figure Lengend Snippet: (A ) Schematic of experimental design. Ctrl (CD45.2 + Cd19 +/+ Irf4 fl/fl ) or IRF4cKO (CD45.2 + Cd19 Cre/+ Irf4 fl/fl ) splenic B cells were CTV-labeled and adoptively transferred into µMT (CD45.1+) mice, as described in the methods. At 24 h post transfer, mice were inoculated with LPS i.v. At the indicated time points, spleens were harvested and analyzed. ( B ) Flow cytometry histograms displaying cell division and ASC differentiation (CD138 + ). The frequency of CD138+ cells are shown. (C) Frequency of transferred (CD45.2 + ) cells at discrete divisions for 48, 60, and 72 h. (D) Mean division number of all responding cells at each time point. ( E) Ctrl (top) and IRF4cKO (bottom) representative flow cytometry plots of CD45.1 versus CD45.2 with gates drawn and frequencies shown for the transferred population. (F) Quantification of the frequency of CD45.2 transferred cells from E . All data are representative of at least two independent experiments using at least 3 mice per group. Data in C, D , and F represent mean ± SD. Statistical significance in C was determined by a two-way ANOVA with Sidak’s multiple comparisons test. Statistical significance in D was determined by a paired two-tailed Student’s t test, while statistical significance in F was determined by determined by a two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p <0.001.

Article Snippet: For adoptive transfers, naïve splenic CD43 − B cells were magnetically isolated using the B cell isolation kit (Miltenyi Biotec, Inc.; 130-090-862) and LS columns (Miltenyi Biotec, Inc.; 130-042-40).

Techniques: Labeling, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: An IRF4-MYC-mTORC1 integrated pathway controls cell growth and the proliferative capacity of activated B cells during B cell differentiation in vivo

doi: 10.1101/2021.06.20.449155

Figure Lengend Snippet: ( A ) Schematic of experimental design. Ctrl and IRF4cKO B cells were prepared and adoptively transferred as in and the methods section. Here, animals were stimulated with either NP-Ficoll or infected with influenza strain X31 as described in the methods. Spleens from NP-Ficoll inoculated animals were harvested at d5; and for influenza, both spleens and the draining mediastinal lymph nodes were isolated at d6 post-challenge. (B) Representative flow cytometry plots of CD138 versus CTV or CTV histograms for Ctrl and IRF4cKO. The frequency of CD138 + (top) and division 8 (bottom) cells are shown. Frequency of division 8 cells for Ctrl and IRF4cKO from B following NP-Ficoll ( C ) or influenza X31 ( D ) challenge. All data are representative of two independent experiments using at least 3 mice per group. Data in C and D represent mean ± SD with statistical significance determined by a two-tailed Student’s t test.

Article Snippet: For adoptive transfers, naïve splenic CD43 − B cells were magnetically isolated using the B cell isolation kit (Miltenyi Biotec, Inc.; 130-090-862) and LS columns (Miltenyi Biotec, Inc.; 130-042-40).

Techniques: Infection, Isolation, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: An IRF4-MYC-mTORC1 integrated pathway controls cell growth and the proliferative capacity of activated B cells during B cell differentiation in vivo

doi: 10.1101/2021.06.20.449155

Figure Lengend Snippet: (A) Ctrl (black) and IRF4cKO (red) B cells were prepared, adoptively transferred, and inoculated with LPS as in . At 72 h, mice were sacrificed and the spleens harvested. Cells were stained with Ki67 and 7AAD and representative flow cytometry plots at the indicated divisions are shown. Flow cytometry gates indicating G0/G1, S, and G2/M phase of the cell cycle are shown with the frequency of cells for each. (B) Quantification of the data from A displaying the frequency of cells found in each phase of the cell cycle at each division. (C) Following the above adoptive transfer scheme described in A , mice were injected with BrdU 1 h prior to sacrifice to assess active S phase of the cell cycle. Representative flow cytometry plot of BrdU versus 7AAD (left) and CTV histograms (right) of the total transferred population (grey) overlaid with the BrdU + cells to visualize the distribution of actively proliferating cells. (D) Quantification of the data from C displaying the frequency of BrdU + cells. All data are representative of at least two independent experiments using at least 3 mice per genotype. Data in B and D represent mean ± SD. Statistical significance in D was determined by a two-tailed Student’s t test. Statistical significance in B was determined by a two-way ANOVA with Sidak’s multiple comparisons test. P-values are shown at points of significance.

Article Snippet: For adoptive transfers, naïve splenic CD43 − B cells were magnetically isolated using the B cell isolation kit (Miltenyi Biotec, Inc.; 130-090-862) and LS columns (Miltenyi Biotec, Inc.; 130-042-40).

Techniques: Staining, Flow Cytometry, Adoptive Transfer Assay, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: An IRF4-MYC-mTORC1 integrated pathway controls cell growth and the proliferative capacity of activated B cells during B cell differentiation in vivo

doi: 10.1101/2021.06.20.449155

Figure Lengend Snippet: (A) Ctrl and IRF4cKO B cells were prepared, adoptively transferred, and inoculated with LPS as in and harvested at 72 h. Cells at the indicated divisions were sorted and subjected to RNA-seq as described in methods. Representative flow cytometry plots of B220 and CTV histograms and projections of the sorted populations are shown and labeled by division number. (B) Bar plot quantifying the number of differentially expressed genes (DEG) at each division that increase (top) or decrease (bottom) expression in IRF4cKO cells compared to Ctrl. Solid bars indicate the proportion of genes that represent a new DEG appearing in that division while striped bars indicate the proportion of genes that were a DEG in an earlier division. (C) Hierarchical clustering of the expression of 10,404 genes detected from A. (D) t-SNE projections of RNA-seq data from control samples (highlighted in grey) and IRF4cKO samples (highlighted in red). (E) Heat map of normalized enrichment scores (NES) calculated by gene set enrichment analysis (GSEA) for pathways upregulated and downregulated in IRF4cKO. (F) GSEA examples for the indicated gene sets for IRF4cKO up and down DEG from divisions 4, 5, and 6. NES values are indicated for each division. (G) Bar plot displaying reads per kilobase million (RPKM) values for the indicated genes at all sequenced divisions for Ctrl and IRF4cKO cells. Asterisks above IRF4cKO division data indicate significance (FDR < 0.001) when compared to the corresponding Ctrl division. Data were derived from 3 independent adoptive transfers for Ctrl and IRF4cKO. One division 0 IRF4cKO sample was excluded due to a high frequency of duplicate reads.

Article Snippet: For adoptive transfers, naïve splenic CD43 − B cells were magnetically isolated using the B cell isolation kit (Miltenyi Biotec, Inc.; 130-090-862) and LS columns (Miltenyi Biotec, Inc.; 130-042-40).

Techniques: RNA Sequencing, Flow Cytometry, Labeling, Expressing, Control, Derivative Assay

Journal: bioRxiv

Article Title: An IRF4-MYC-mTORC1 integrated pathway controls cell growth and the proliferative capacity of activated B cells during B cell differentiation in vivo

doi: 10.1101/2021.06.20.449155

Figure Lengend Snippet: (A) GSEA using the top differentially expressed genes dysregulated in MYC-deficient B cells stimulated with LPS and IL-4 for 72 h . FDR values are displayed for each division as indicated by color. Splenic B cells from Ctrl and IRF4cKO mice were isolated and treated with LPS, IL2, and IL5 ex vivo as described in methods. (B) Quantitative RT-PCR expression of Myc relative to 18S rRNA expression before (0 h) or 24 h after stimulation. (C) Representative intracellular staining of MYC for naïve untreated B cells (nB) and 24 h stimulated Ctrl and IRF4cKO cells (top). ( D ) Geometric mean fluorescence intensity (gMFI) quantified for the stimulated samples for C. (E) Representative CTV histograms of Ctrl (left) and IRF4cKO (right) transduced with empty-RFP retrovirus (black) or MYC-RFP expressing retrovirus (blue). (F) (Left) Quantification of the mean division number (MDN) for Ctrl and IRF4cKO cells transduced with empty-RFP retrovirus or MYC-RFP retrovirus from E . (Right) Quantification of the change in MDN after MYC overexpression in Ctrl and IRF4cKO cells from E . All data are representative of at least two independent experiments using at least 3 mice per genotype. Data in B, D , and F represent mean ± SD. Statistical significance in B and D was determined by a two-tailed Student’s t test. Statistical significance in F when comparing IRF4cKO samples was determined by a paired two-tailed Student’s t test, while significance between Ctrl and IRF4cKO samples was calculated by a two-tailed Student’s t test.

Article Snippet: For adoptive transfers, naïve splenic CD43 − B cells were magnetically isolated using the B cell isolation kit (Miltenyi Biotec, Inc.; 130-090-862) and LS columns (Miltenyi Biotec, Inc.; 130-042-40).

Techniques: Isolation, Ex Vivo, Quantitative RT-PCR, Expressing, Staining, Fluorescence, Transduction, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: An IRF4-MYC-mTORC1 integrated pathway controls cell growth and the proliferative capacity of activated B cells during B cell differentiation in vivo

doi: 10.1101/2021.06.20.449155

Figure Lengend Snippet: (A) RNA-seq (described in ) average RPKM of all detected genes (22/24) in the B cell-activation UPR gene set . (B) Heatmap of z score-normalized gene expression data for all detected genes from A for the indicated divisions. (C) Representative flow cytometry histograms displaying intracellular phosphorylated S6 (pS6) protein staining for Ctrl or IRF4cKO activated B cells cultured ex vivo with LPS, IL2, and IL5 for 48 h. Grey histogram is representative of Ctrl cultures treated with rapamycin to block mTORC1 activity 2 h before harvest. (D) Quantification of geometric mean fluorescence intensity (gMFI) for pS6 from C. (E) Histograms displaying cell size distribution via forward scatter area (FSC-A) at divisions 0 - 6 48 h post-LPS inoculation of adoptive transfer host mice, as described in . Grey histogram represents cell size at division 0, with the dashed line drawn from the summit to better visualize changes in cell size across the divisions. Cell divisions are indicated to the right of each trace. (F) Quantification of data from E indicating cell size at division 0 (bottom) and the average change in cell size among responding cells (top). (G) Quantification of cell size via forward scatter area (FSC-A) for all responding cells in Ctrl and IRF4cKO transduced with empty-RFP retrovirus or MYC-RFP expressing retrovirus. All data are representative of at least two independent experiments using at least 3 mice per genotype. Data in D, F , and G represent mean ± SD. Statistical significance in D and F was determined by a two-tailed Student’s t test. Statistical significance in G when comparing IRF4cKO samples was determined by a paired two-tailed Student’s t test, while significance between Ctrl and IRF4cKO samples was calculated by a two-tailed Student’s t test.

Article Snippet: For adoptive transfers, naïve splenic CD43 − B cells were magnetically isolated using the B cell isolation kit (Miltenyi Biotec, Inc.; 130-090-862) and LS columns (Miltenyi Biotec, Inc.; 130-042-40).

Techniques: RNA Sequencing, Activation Assay, Gene Expression, Flow Cytometry, Staining, Cell Culture, Ex Vivo, Blocking Assay, Activity Assay, Fluorescence, Adoptive Transfer Assay, Transduction, Expressing, Two Tailed Test