Journal: Nature immunology

Article Title: Suppression by T FR cells leads to durable and selective inhibition of B cell effector functions

doi: 10.1038/ni.3578

Figure Lengend Snippet: (a) Volcano plot showing RNA-seq data from Figure 2 with all genes (gray) or Myc pathway (Hallmark_MYC_TARGETS_V1) genes (red) in activated versus suppressed B cells. (b) Quantification of IgG1+GL7+ B cells from suppression assays performed as in Figure 1 with the addition of the Myc inhibitor 10058-F4 (F4). (c) IgG antibody concentration in culture supernatants from suppression assays in (b). (d) IgG1+GL7+ (left) or GL7 expression (right) in B cells from suppression assays in which control (WT) or Myc overexpressing (Myc) B cells were cultured with TFH alone or with TFR cells. (e) IgG concentration in culture supernatants from assays as in (c). (f) Volcano plot showing RNA-seq data from Figure 2 with all genes (gray) or mTOR signaling (Hallmark_MTORC1_SIGNALING) genes (red) in activated versus suppressed B cells. (g) Quantification of IgG1+GL7+ B cells from suppression assays performed as in Figure 1 with the addition of the mTOR inhibitor rapamycin (Rapa). (h) IgG concentration in culture supernatants from suppression assays in (b). (i) Quantification of IgG1+GL7+ B cells from suppression assays performed as in (b) with the addition of the mTOR inhibitor PP242. (j) IgG concentration in culture supernatants from suppression assays in (i). *p<0.05, **p<0.01, ***p<0.001 (using 1-way ANOVA with Tukey’s correction (b,c,d,e,h,i,j) or Chi2 test (a,f). Data are from one experiment (technical replicate suppression assays utilizing cells from 20 pooled mice) and are representative of 3 independent experiments with similar results (b–e, g–j mean±s.e.m.).

Article Snippet: Analysis was performed using the CLC Genomics Workbench version 8.0.1 RNA-seq analysis software package (Qiagen).

Techniques: RNA Sequencing Assay, Concentration Assay, Expressing, Cell Culture

Journal: Nature immunology

Article Title: Suppression by T FR cells leads to durable and selective inhibition of B cell effector functions

doi: 10.1038/ni.3578

Figure Lengend Snippet: (a) Heat map of metabolic pathways using RNA-seq data from Fig. 2 (normalized to naive B cells) (Supplementary Figure 4). (b) Glut1 expression in B cells from activated (B + TFH), TFR-suppressed (B + TFH +TFR), or Treg-suppressed (B + TFH + Treg) cultures. (c) Glut1 expression in B cells, gated to indicate CTV division peaks. (d) Glut1 expression in B cells added to activated or suppressed day 3 cultures and harvested 20 hours later. (e) Glut1 expression in TFH cells. (f) Glucose uptake in culture supernatants as in (b). (g) Lactate production in culture supernatants from cultures as in (b). (h–i) IgG1+GL7+ B cells (left) and IgG concentration (right) in supernatants from cultures as in (b) to which 2DG was added. (j) Volcano plot of activated versus suppressed B cells showing all genes (gray) or genes encoding enzymes in one-carbon/serine/purine metabolism (red) using RNA-seq data from Fig. 2. (k) Shmt1 and Shmt2 expression in B cells added to activated or suppressed day 3 cultures and harvested 20 hours later. (l) IgG concentration in supernatants as in (c) with the addition of MTX. (m) IgG concentration in supernatants as in (c) with the addition of AZA. *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test (d), 1-way ANOVA with Tukey’s correction (b, e–i, l–m), or Chi2 test (j). Data are from one experiment (technical replicate suppression assays utilizing cells from 20 pooled mice) and are representative of 3 independent experiments with similar results (b–i, k–m mean±s.e.m.).

Article Snippet: Analysis was performed using the CLC Genomics Workbench version 8.0.1 RNA-seq analysis software package (Qiagen).

Techniques: RNA Sequencing Assay, Expressing, Concentration Assay

Journal: Nature immunology

Article Title: Suppression by T FR cells leads to durable and selective inhibition of B cell effector functions

doi: 10.1038/ni.3578

Figure Lengend Snippet: (a) Bcl6 and Ki67 expression in TFH cells from reactivation cultures. B and TFH cells were cultured alone or along with TFR cells. After 3 days, B cells from the activated culture (Act B) or suppressed culture (Supp B) were sorted and cultured with new T cells (2°). Primary activation cultures are also included (1°) (b) Relative percentage of total number of TFH cells in cultures as in (a). (c) IgG1+GL7+ B cells from cultures as in (a). (d) Glut1 expression in B cells from cultures as in (a). (e) Glucose uptake in supernatants from cultures as in (a). (f) Venn diagram showing genes with lower expression in B cells upon suppression from RNA-seq data (as in Fig. 2) and gene loci showing evidence of chromatin inaccessibility by ATAC-seq. (g) RNA-seq data for gene transcripts expressed less in suppressed B cells by RNA-seq and which also showed less accessibility by ATAC-seq analysis. (h–j) ATAC-seq peaks and ChIA-pet annotated B cell regulome gene track33 for Aicda, Myc and Pou2af1. Red boxes indicate statistical downregulation (P<0.05). (k) Distance of ATAC-seq peaks from gene transcriptional start sites (TSS) for all peaks or peaks less accessible in suppressed B cells. *p<0.05, **p<0.01, ***p<0.001 (1-way ANOVA with Tukey’s correction (a–e). Data are pooled data from 10 independent experiments, each of which included cells from 20 pooled mice (a–e mean±s.e.m.), or are representative of two independent experiments (f–k).

Article Snippet: Analysis was performed using the CLC Genomics Workbench version 8.0.1 RNA-seq analysis software package (Qiagen).

Techniques: Expressing, Cell Culture, Activation Assay, RNA Sequencing Assay, ChIA Pet Assay

Journal: Nature immunology

Article Title: Suppression by T FR cells leads to durable and selective inhibition of B cell effector functions

doi: 10.1038/ni.3578

Figure Lengend Snippet: (a) (Left) B cell proliferation in B cells from cultures of TFH cells alone, TFH and TFR cells, or TFH and TFR cells with IL-21. (Right) Quantification of proliferation index. (b) IgG1+GL7+ B cells from cultures as in (a). (c) IgG concentration in culture supernatants from cultures as in (a). IL-4 (left) or IL-6 (right) was added. (d) Glut1 expression in B cells from cultures as in (a). (e) Glucose uptake from culture supernatants as in (a). (f) Lactate production in culture supernatants as in (a). (g) IgG concentration in culture supernatants as in (a), to which 2DG was added. (h) Volcano plot showing all genes (gray) or B cell function genes (red) from sorted B cells in Suppressed + IL-21 versus Suppressed cultures. Cultures were performed as in (a) except NP-OVA was used instead of anti-CD3/IgM. (i) Venn diagram of all genes differentially expressed (FDR adjusted p<0.05) for RNA-seq analysis as in (h). (j) Collapsed ssGSEA correlation plots for Activated, Suppressed or Suppressed + IL-21 B cell RNA-seq data. *p<0.05, **p<0.01, ***p<0.001 using 1 way ANOVA with Tukey’s correction (a–g). Data are from one experiment (technical replicate suppression assays utilizing cells from 20 pooled mice) are representative of 3 independent experiments with similar results (a–g, mean±s.e.m.), or are combined 3 biological replicates (h–j).

Article Snippet: Analysis was performed using the CLC Genomics Workbench version 8.0.1 RNA-seq analysis software package (Qiagen).

Techniques: Concentration Assay, Expressing, Cell Function Assay, RNA Sequencing Assay