Suppression by TFR cells leads to durable and selective inhibition of B cell effector function

Abstract

Follicular regulatory T cells (T_FR cells) inhibit follicular helper T cell (T_FH cell)-mediated antibody production. The mechanisms by which T_FR cells exert their key immunoregulatory functions are largely unknown. Here we found that T_FR cells induced a distinct suppressive state in T_FH cells and B cells, in which effector transcriptional signatures were maintained but key effector molecules and metabolic pathways were suppressed. The suppression of B cell antibody production and metabolism by T_FR cells was durable and persisted even in the absence of T_FR cells. This durable suppression was due in part to epigenetic changes. The cytokine IL-21 was able to overcome T_FR cell-mediated suppression and inhibited T_FR cells and stimulated B cells. By determining mechanisms of T_FR cell-mediated suppression, we have identified methods for modulating the function of T_FR cells and antibody production.

Figure 1. Suppressed B cells undergo early activation

(a) Quantification of IgG1⁺GL7⁺ B cells (left) and secreted antibody (right) from cultures of B and T_FH cells with or without T_FR cells in the presence of anti-CD3/IgM (Supplementary Figure 1a–b). (b) Quantification of secreted antibody as in (a) with the addition of a culture with CD4⁺ICOS⁻CXCR5⁻FoxP3⁺ T_reg cells. (c) Quantification of IgG1⁺GL7⁺ B cells from assays performed as in (a) with addition of supernatant from suppressed cultures (T_FR sup’). (d) Micrograph of culture containing B, T_FH and T_FR cells after 4 days. Scale bar = 5μm. (e) Proliferation of B cells measured by (cell trace violet) CTV dilution at day 4. B cells are pregated on CD19⁺IA⁺CD4⁻. (f) Proliferation of B cells measured in which B cells were cultured with LPS and IL-4 and T_FR cells for 4 days. (g) CD69 expression on B cells from suppression assays as in (a). (h) Cell death in B cells measured by zVAD staining. (i) Somatic hypermutation in B cells during suppression assays as in (a) with the addition of NP-OVA. (j) Expression of Bcl6 and Ki67 in T_FH cells from suppression assays as in (a). Cells are pregated on CD4⁺FoxP3⁻CD19⁻IA⁻. *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test (a,e,h) or 1-way ANOVA with Tukey’s correction (b,c,j)). Data are from one experiment (technical replicate suppression assays utilizing cells from 20 pooled mice) and are representative of 5 independent experiments with similar results (a–b, h, j) or 3 independent experiments (c–g; mean±s.e.m.).

Figure 2. Suppressed T_FH and B cells retain transcriptional programs except for inhibition of specific effector genes

(a) Principal component analysis of activated and suppressed B and T_FH cells. B and T_FH cells, from immunized FoxP3^GFP mice, were cultured alone (“Activated”) or with T_FR cells (“Suppressed”) from FoxP3^GFPActin^CFP mice, in the presence of NP-OVA. After 4 days B and T_FH cells were sorted. (b) Venn diagram of differentially expressed (FDR adjusted p<0.05) genes in activated versus suppressed B and T_FH cells. (c) (Left) Volcano plot of all genes (gray) or “T_FH genes” (red) in T_FH cells from cultures. (Right) Heat map showing T_FH genes. (d) Single sample GSEA showing correlation of activated B, activated T_FH and suppressed T_FH cells to ImmSig gene sets (GSE11924, GSE16697, GSE21380, GSE24574). (e) GSEA enrichment plot for effector T cells, senescent cells, exhaustion^, or anergy (GSE2323) signatures in activated versus suppressed T_FH cells. (f) Heat map of B cell genes in activated or suppressed B cells (only most upregulated or downregulated are shown). (g) Volcano plot showing data from all genes (gray) or B cell function genes (red) in activated versus suppressed B cells. (h) Single sample GSEA showing correlation of activated or suppressed B cells to ImmSig gene sets (GSE12366, GSE12845). (i) GSEA enrichment plot for gene sets for effector T cells, senescent T cells, exhaustion or anergy in activated versus suppressed B cells. Data are from 4 biological replicates on different experimental days, each containing cells from 20 pooled mice.

Figure 3. Inhibition of MYC and mTOR pathways suppress B cell effector functions

(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 T_FH alone or with T_FR 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.).

Figure 4. T_FR cells inhibit multiple metabolic pathways in B cells

(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 + T_FH), T_FR-suppressed (B + T_FH +T_FR), or T_reg-suppressed (B + T_FH + T_reg) 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 T_FH 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.).

Figure 5. T_FR cell suppression results in sustained inhibition and epigenetic changes in B cells

(a) Bcl6 and Ki67 expression in T_FH cells from reactivation cultures. B and T_FH cells were cultured alone or along with T_FR 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 T_FH 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 track 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).

Figure 6. IL-21 can overcome T_FR-mediated suppression of B cell metabolism and antibody production

(a) (Left) B cell proliferation in B cells from cultures of T_FH cells alone, T_FH and T_FR cells, or T_FH and T_FR 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).

Figure 7. IL-21 directly stimulates B cells and inhibits T_FR cells

(a) Quantitation of IgG1⁺GL7⁺ B cells from cultures of WT or Il21r^−/− B cells cultured with T_FH cells alone, T_FH and T_FR cells or T_FH, T_FR cells and IL-21. Cells are pregated on CD19⁺IA⁺CD4⁻. (b–c) GL7 expression in B cells from cultures as in (a). (d) Analysis of Ki67 in T_FR cells from cultures as in (a). Cells are pregated on CD4⁺FoxP3⁺ cells. (e) Numbers of T_FR cells remaining cultures as in (a). (f) Glut1 expression in T_FR cells from cultures as in (a) *p<0.05, **p<0.01, ***p<0.001 (1-way ANOVA with Tukey’s correction (a–c)) or (Student’s t-test (e,f)). 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–f, mean±s.e.m.), or 2 independent experiments (a–c).