Type III interferon drives thymic B cell activation and regulatory T cell generation

Abstract

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer's patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (T_reg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the T_reg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells.

Keywords: Treg cell selection; central tolerance; thymic B cells; type III IFN.

Fig. 1.

Class-switched thymic B cells exhibit a unique phenotype. (A) Flow cytometry of B cells from Peyer’s patch (Left), spleen (Middle), and thymus (Right) showing IgM and IgD expression, with number adjacent to outlined areas identifying proportion of isotype class-switched (IgM⁻IgD⁻) and activated IgM⁺IgD^lo B cells. Graph on right shows quantification of isotype class-switched B cells (IgM⁻IgD⁻). (B) Flow cytometry of total B cells from the Peyer’s patch (Left), spleen (Middle), and thymus (Right) showing CD38 and GL7 expression with number adjacent to outlined areas identifying proportion of activated memory B cell precursors. Graph on Right shows quantification of activated memory B cell precursors (CD38⁺GL7⁺). (C) Volcano plot representation of RNA-Seq data comparing isotype class-switched thymic and PP B cells showing Log₂ fold-change by -Log₁₀ FDR. Significantly up-regulated genes (FDR <= 0.05) are identified in the thymus (blue, FC < −2) and PP (black, FC > 2). (D) Gene set enrichment analysis normalized enhancement score (NES) for ranked RNA-Seq data. Negative NES are pathways overrepresented in thymic B cells; positive NES pathways overrepresented in PP B cells. (E) Flow cytometry histograms for CD69, I-A/I-E, H2-K^b, CXCR3, and Ly6C expression of B cells in the PP (black, Top), spleen (pink, Middle), and thymus (blue, Bottom). (F) Uniform Manifold Approximation and Projection (UMAP) plot for thymic B cells. (G) Heatmap representation of top 10 differentially expressed genes in subsampled thymic B cells from cluster 0 and 1. (H) Heatmap representation of log2-transformed counts per-million (CPM) of individual ISG mRNA in CD21⁻CD35⁻ and CD21⁺CD35⁺ thymic B cells compared with cord blood B cells. Each symbol (A and B) represents an individual mouse; small horizontal lines indicate the group mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 (A and B One-way ANOVA with Tukey’s multiple comparisons test).

Fig. 2.

Increased CD40L expression drives a burst of B cell licensing in early life. (A) Volcano plot representation of RNA-Seq data comparing isotype class-switched thymic and PP B cells (gray) with overlaid “Hallmark_G2M_Checkpoint” gene set (pink). (B) Flow cytometry histogram measuring BrdU incorporation after 24-h labeling (2-mg intraperitoneal injection) of B cells in the PP (black, Top), spleen (pink, Middle), and thymus (blue, Bottom). Graph of right shows quantification of BrdU-positive B cell after 24 h of labeling. (C) Flow cytometry histogram measuring BrdU incorporation after 6 d of labeling (0.8 mg/mL in drinking water) of B cells in the PP (black, Top), spleen (pink, Middle) and thymus (blue, Bottom). Graph on right shows quantification of BrdU-positive B cell after 6 d of labeling. (D) Time course of thymic B cell IgA isotype CSR (black, left axis) and CD38⁺GL7⁺ (pink, right axis) activation. Points represent mean (±SEM). (E) Flow cytometry histogram measuring DAPI in thymic B cells from neonatal (Left) and adult (Right). Graph on right shows quantification of DAPI-positive B cells. (F) Diagram of intrathymic transfer experiments into adult or neonatal hosts. (G) Flow cytometry of splenic B cells following intrathymic transfer showing CD38 by GL7 expression in neonatal (5 to 7 d old) (Left) and adult (6 to 8 wk old) (Right) host mice. Graph on right shows quantification of transferred CD38⁺GL7⁺ B cells. (H) Flow cytometry of splenic B cells following intrathymic transfer showing IgD and Cell Trace Violet (CTV) expression in neonatal (5 to 7 d old) (Left) and adult (6 to 8 wk old) (Right) host mice. Frequency of divided intrathymic transferred donor B cells normalized to the frequency of divided B cells in adult hosts. (I) Flow cytometry of CD4SP thymocytes (CD4+ CD8α- CD25-GITR-CD44-) showing CD40L by CD69 with number adjacent to outlined areas identifying proportion of CD40L+. The graph on right shows the normalized frequency of CD40L+ CD4SP thymocytes to adult CD40L frequency on the right. Each symbol (B, C, E, and G–I) represents an individual mouse; small horizontal lines indicate the group mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 (B, C, H One-way ANOVA with Tukey’s multiple comparisons test, E, G, I Two-tailed unpaired Student’s t test).

Fig. 3.

Thymic B cell class switching does not require T_FH. (A) Immunofluorescence of the thymus showing UAE-1 (green), β5T (red), and CD19 (cyan). Medullary regions are outlined by a white dotted line. (B) Volcano plot representation of RNA-Seq data comparing isotype class-switched thymic and PP B cells (gray) with overlaid “GSE12366_GC_VS_MEMORY_BCELL_UP” gene set (pink). (C) Flow cytometry overlays of thymic B cells showing IgM by IgD from WT (black) and respective knockout mice (TCRα^−/−, Aicda^−/−, Cd40l^−/−, Icos^−/−, and CD4^Cre/+ Bcl6^fl/fl). (D) Quantification of activated B cells (CD69⁺), isotype class-switched (IgM⁻IgD⁻) and activated IgM⁺IgD^lo thymic B cells. (E) Quantification of isotype class-switched (IgM⁻IgD⁻) B cells in Peyer’s patch of WT and Cd4^Cre/+ Bcl6^fl/fl mice. Each symbol (D and E) represents an individual mouse; small horizontal lines indicate the group mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 [(D) Two-way ANOVA with Sidak’s multiple comparisons test, (E) Two-tailed unpaired Student’s t test].

Fig. 4.

Type III IFN drives thymic B cell licensing. (A) Volcano plot representation of RNA-Seq data comparing isotype class-switched thymic and PP B cells (gray) with overlaid “Hallmark_Interferon_Alpha_Response” gene set (pink). (B) Flow cytometry of thymic B cells showing CD19 and Mx1^gfpin WT and Mx1^gfp/gfp mice with number adjacent to outlined areas identifying proportion of Mx1^gfp+ thymic B cells. Graphs on right showing quantification of Mx1^gfp+ PP, splenic and thymic B cells in adult mice, and thymic B cells from 10-d-old mice (C) Flow cytometry histogram overlays of Mx1^gfp+ (pink) and Mx1^gfp− (black) thymic B cells showing expression of CD69, GL7, Ly6C, CXCR3, IgD, and IgA. (D) UMAP plot of thymic B cells with module score identifying cells expressing genes identified in the “Hallmark_Interferon_Alpha_Response” gene set. (E) UMAP plots of thymic B cells derived from WT and Infar1^−/−/Inflr1^−/− hosts identified by HTO. (F) Flow cytometry histograms showing Ly6C expression (Top) or isotype class-switched (IgM⁻IgD⁻) thymic B cells derived from WT, Ifnar1^−/−, Ifngr1^−/−, Ifnlr1^−/−, Ifnar1^−/−/Ifnlr1^−/−, and Stat1^−/− mice. Numbers adjacent to outlined areas identifying the proportion of cells within the gate. (G) Quantification of Ly6C⁺, CXCR3⁺, and isotype class-switched (IgM⁻IgD⁻) thymic B cells from WT, Ifnar1^−/−, Ifngr1^−/−, Ifnlr1^−/−, Ifnar1^−/−/Ifnlr1^−/,⁻ and Stat1^−/− mice. Each symbol (B and G) represents an individual mouse; small horizontal lines indicate the group mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 [(G) Two-way ANOVA with Sidak’s multiple comparisons test].

Fig. 5.

Requirement for type III IFN receptor is B cell intrinsic. (A) RT-qPCR of Ifnlr1 in sorted IgM⁻IgD⁻ B cells from the PP (black) and thymus (blue) normalized to Gapdh. (B) Proportion of thymic B cells from mixed bone marrow chimeras with the following phenotypes: Ly6C⁺, CXCR3⁺, and IgM⁻IgD⁻. (C) Flow cytometry of thymic B cells from Ifnlr1^fl/fl and Mb1^Cre/+Ifnrl1^fl/fl showing isotype class-switched (IgM⁻IgD⁻) (Left) and Ly6C expression (Right). Numbers adjacent to outlined areas identifying the proportion of cells within the gate. (D) Quantification of the proportion of CD38⁺GL7⁺, CXCR3⁺, Ly6C⁺, and isotype class-switched (IgM⁻IgD⁻) B cells from Ifnlr1^fl/fl and Mb1^Cre/+Ifnrl1^fl/fl mice located in the PP, spleen, or thymus. Each symbol (A, B, and D) represents an individual mouse; small horizontal lines indicate the group mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 [(A) Two-tailed unpaired Student’s t test, (B) One-way ANOVA with Tukey’s multiple comparisons test, (D) Two-way ANOVA with Sidak’s multiple comparisons test].

Fig. 6.

Type III IFN signaling in thymic B cells is required for Foxp3+ regulatory T cell selection. (A) Flow cytometry of live CD19⁻ CD11b⁻ CD11c⁻ F4/80⁻ TCRβ⁺ CD4⁺ T cells showing Cre:I-A^b-PE by Cre:I-A^b-APC to identify Cre_61–71:I-A^b-specific CD4 T cells. Numbers adjacent to outlined areas identify the average number of cells in the gate ± the SD of all samples. (B) Quantification of the absolute number (Left) and Foxp3+ regulatory T cell frequency (Right) of Cre_61–71:I-A^b-specific CD4 T cells 7 d following Cre_61–71/CFA subcutaneous immunization. (C) Flow cytometry of CD4SP thymocytes showing Foxp3 by CD4 in WT (C57BL/6) and Mb1^Cre/Cremice (Left). Numbers adjacent to outlined areas identify the proportion of cells within the gate. Quantification of the proportion of Foxp3+ regulatory T cells in the CD4SP thymocyte population (Right). (D) Flow cytometry of CD4SP thymocytes showing Foxp3 by CD4 in Ifnlr1^fl/fl and Mb1^Cre/+Ifnrl1^fl/fl mice (Left). Numbers adjacent to outlined areas identify the proportion of cells within the gate. Quantification of the proportion of Foxp3+ regulatory T cells in the CD4SP thymocyte population (Right). (E) Flow cytometry of CD4SP thymocytes showing Foxp3 by CD4 in Aicda^Cre/− and Aicda^Cre/Cremice (Left). Numbers adjacent to outlined areas identify the proportion of cells within the gate. Quantification of the proportion of Foxp3+ regulatory T cells in the CD4SP thymocyte population (Right). (F) Comparison of the mean CDR3 clonotype counts per million reads mapped in Tconv (Left) and Treg cells (Right) from WT or Aicda^Cre/Cre Tcra^+/− Tclib^Tg Foxp3^eGFP mice (n = 4 mice per genotype). The Log₁₀(FDR) of for each CDR3 peptide counts per million is shown by heatmap, and CDR3 clonotypes with an FDR ≤ 0.05 are represented by square points and CDR3 clonotypes with an FDR >0.05 are represented by circular points. CDR3 clonotypes identified in red dotted ellipses represent clonal deletion (Left) and regulatory T cell generation (Right) dependent upon class-switched B cells. Each symbol (B–E) represents an individual mouse; small horizontal lines indicate the group mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 [(C–E) Two-tailed unpaired Student’s t test, (A) Two-way ANOVA with Sidak’s multiple comparisons test].