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. 2024 May 14;57(5):1037-1055.e6.
doi: 10.1016/j.immuni.2024.03.016. Epub 2024 Apr 8.

Type I interferons induce an epigenetically distinct memory B cell subset in chronic viral infection

Affiliations

Type I interferons induce an epigenetically distinct memory B cell subset in chronic viral infection

Lucy Cooper et al. Immunity. .

Abstract

Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.

Keywords: B cells; IFN; LCMV; atypical; chronic viral infection; epigenetics; long COVID; memory B cells; scATAC-seq; scRNA-seq.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. MBCs in LCMV-Docile infection adopt phenotypic changes.
(A) Schematic of acute (LCMV-WE) or chronic (LCMV-Docile) LCMV infection in C57BL/6 mice. Antigen-specific B cells assessed at various timepoints post-infection. (B) Flow cytometric analyses of splenic MBCs (CD38+ % of B220+IgDTetramer+Decoy) in mice at day 14 (n = 6 per strain), day 28 (WE: n = 13, Docile: n = 15), or day 56 (WE: n = 10, Docile: n = 9) post-LCMV-WE or LCMV-Docile infection. Combined data from 7 independent experiments. Data represent mean ± SEM. (C) Flow cytometric analyses of splenic GCB (CD38CD95hi % of B220+IgDTetramer+Decoy) at day 7 (n = 7 per condition), day 14 (n = 6 per condition), day 28 (n = 10 per condition), or day 56 (WE: n = 10, Docile: n = 9) post-LCMV-WE or LCMV-Docile infection. Combined data from 8 independent experiments. Data represent mean ± SEM. (D and E) Flow cytometric analyses of (D) splenic PD-L2CD80 and (E) PD-L2+CD80+ within the MBC population (B220+ IgD Tetramer+ Decoy CD38+) at day 7 (n = 4 per condition), day 14 (WE: n = 11, Docile: n = 9), day 28 (WE: n = 9, Docile: n = 8), or day 56 (WE: n = 10, Docile: n = 8) post-infection. Combined data from 7 independent experiments. Data represent mean ± SEM. (F and G) Flow cytometric analyses of (F) splenic CD21+ and (G) CD11c+ within the MBC population at day 28 (n = 10 per condition), or day 56 (WE: n = 10, Docile: n = 9) post-LCMV-WE or LCMV-Docile infection. Combined data from 4 independent experiments. Data represents mean ± SEM. Related to Figure S1.
Figure 2.
Figure 2.. Acute and chronic LCMV infection drive (epi)genetically distinct MBC subsets.
(A) Schematic of scRNA-seq and scATAC-seq set-ups. (B) Unsupervised clustering of splenic antigen-specific B cells from 2 mice per condition visualized using UMAP, split by infection type. Each cell is represented by a point and colored by cluster. Graphical representation of percentage of total cells per cluster, split by infection type. (C) Unsupervised clustering of nuclei from splenic antigen-specific B cells from 2 mice per group visualized using UMAP. Each nuclei is represented by a point and colored by cluster. Graphical representation of percentage of total barcodes per cluster, split by infection type. (D) UMAPs of scRNA-seq (left) and scATAC-seq (right) data, with GCB (pink) and non-GCB (aqua). (E) Coverage plot showing chromatin accessibility at the Aicda gene region, split by cluster. (F) UMAP of scRNA-seq data, with Cd38+Ighd non-GC cells split by condition shown as purple dots. (G and H) Violin plots representing the log2 fold-change expression of a panel of genes associated with (G) classical murine MBCs (MBC: Pdcd1lg2, Cd80, Cr2, Sell, Cxcr5, Ccr7 and Cxcr4) or (H) PD-L2CD80 murine MBCs, in Cd38+Ighd non-GC antigen-specific MBCs isolated from LCMV-WE vs. LCMV-Docile-infected mice. Data represent mean ± SEM. (I) Heatmap of the top 25 DEGs in cluster R0 compared to all other clusters (excluding Ig genes). Also shown in Figure S2. (J) Dendrogram showing the relationships in chromatin accessibility between the scATAC-seq clusters. (K) UMAP of Tbx21 motif enrichment, with Z-score representing the sums of cut sites per cell which fall within all the peaks associated with the Tbx21 motif, split by condition. (L) UMAP plot with cluster A6 highlighted. Heatmap of the DARs between acute and chronic condition in cluster A6. Related to Figure S2, Figure S3 and Figure S4.
Figure 3.
Figure 3.. Chronic infectionexpands an ISG-associated subset.
(A) UMAP plot with cluster A1 highlighted (excluding cluster A5). (B) Heatmap of the DARs in cluster A1 compared to all other clusters (excluding cluster A5). (C) Representative selection of transcription factor binding motifs enriched in cluster A1 vs. all other clusters (excluding A5), generated using HOMER. (D and E) Heatmaps of the top 25 DEGs in (D) cluster R7 or (E) cluster R2 (excluding Ig genes), compared to all other clusters. Also shown in Figure S2. (F and G) Coverage plots showing chromatin accessibility at the Rtp4 gene region, split by (F) condition, and (G) split by cluster. (H) UMAPs of gene expression of Rtp4, Ifi44 and Oasl2 split by condition, with positive expression represented by pink dots. Related to Figure S5.
Figure 4.
Figure 4.. Chronic-emergent MBC have a reduced capacity to respond in vitro.
(A) IPA analysis of differentially expressed genes within the IFN pathway upregulated in MBCs (Cd38+Ighd non-GC) from LCMV-Docile-infected mice. (B) GSEA of hallmark IFNα response (left) and IFNγ response (right) gene sets in cluster R7 compared to other clusters. (C) UMAP of combined gene expression of Ly6c2, Irf7 and Lgals9, split by condition, with positive expression represented by pink dots. (D) Flow cytometric assessment of Ly6c and Galectin-9 in mice infected with LCMV-WE or LCMV-Docile (n = 4 per condition) 15 days prior. (E) Schematic of setup: Sort-purified Ly6cpos and Ly6cneg MBCs were stimulated in vitro with CD40L, anti-Ig and IL-21. (F) Number of plasmablasts formed 4 days post-stimulation of Ly6cpos and Ly6cneg MBCs (n = 5 per subset). Combined from 2 independent experiments. Related to Figure S5.
Figure 5.
Figure 5.. Type I IFN is a key driver of phenotypic and epigenetic changes to MBCs during chronic viral infection.
(A) Schematic of early type I or II IFN blocking treatment cohort and timepoint. (B, C and D) Flow cytometric analyses at day 15 post-infection of (B) antigen-specific MBCs (B220+IgDTetramer+DecoyCD38+), (C) CD11c+ frequency within MBCs, and (D) PD-L2 and CD80 MBC subsets in mice treated with anti-IFNAR1 (MAR1–5A3) (n = 8), anti-IFNγ (XMG1.2) (n = 5) or IgG control (n = 8) at day 15 post-infection in mice infected with LCMV-Docile. Combined data from 2 independent experiments. Data represents mean ± SEM. (E) Expression of Ly6c in antigen-specific MBCs in mice infected with LCMV-WE and treated with an IgG control, LCMV-Docile mice treated with anti-IFNAR1 or IgG control (n = 6 per condition) at day 15 post-infection. (F) Volcano plot of DARs in MBCs in IgG control (red) vs. anti-IFNAR1 blocking treatment (blue) in LCMV-Docile infection at day 15 post-infection. (G) Heatmap showing DARs in MBCs in LCMV-WE and LCMV-Docile infection, with IgG control, anti-IFNAR1 or anti-IFNγ blocking treatment at day 15 post-infection. (H) Representative selection of transcription factor binding motifs with increased (left) or decreased (right) chromatin accessibility in MBCs following early IFNAR1-blocking treatment generated using HOMER. Related to Figure S6.
Figure 6.
Figure 6.. Chronic LCMV infection induces delayed but sustained IFN exposure.
(A) Schematic of BrdU treatment groups and timepoints following LCMV-WE or LCMV-Docile infection. (B, C and D) Flow cytometric analyses of BrdU+ frequency within MBCs (B220+IgDTetramer+DecoyCD38+) at various timepoints post-infection in mice infected with LCMV-WE or LCMV-Docile and treated with BrdU at (B) days 5–7 post-infection, (C) days 12–14 post-infection or (D) days 25–27 post-infection. Combined data from 3 independent experiments. Data represents mean ± SEM. (E and F) LegendPlex assay results of sera (E) IFNα or (F) IFNγ at various timepoints post-infection with LCMV-WE or LCMV-Docile (n = 4–8 per condition). Combined data from 3 independent experiments. Data represents mean ± SEM. Related to Figure S6.
Figure 7.
Figure 7.. IFN blocking antibody administration late in the primary response to LCMV-Docile infection did not induce changes in the phenotype and epigenetic profile of MBCs.
(A) Schematic of late type I or II IFN blocking treatment cohort and timepoint. (B, C and D) Flow cytometric analyses of (B) antigen-specific MBCs (B220+IgDTetramer+DecoyCD38+), (C) CD11c+ frequency within MBCs, and (D) CD21+ frequency within MBCs in mice treated with anti-IFNAR1 (MAR1–5A3; n = 7), anti-IFNγ (XMG1.2; n = 6) or IgG control (n = 5) at day 41 post-infection. Combined data from 2 independent experiments. Data represents mean ± SEM. (E) Flow cytometric analyses of PD-L2 and CD80 MBC subsets in mice treated with anti-IFNAR1 (MAR1–5A3; n = 3), anti-IFNγ (XMG1.2; n = 3) or IgG control (n = 2) at day 41 post-infection. Combined data from 2 independent experiments. Data represents mean ± SEM. (F) Volcano plot of DARs in MBCs in IgG control (red) vs. anti-IFNAR1 blocking treatment (blue) in LCMV-Docile infection at day 41 post-infection. (G) Graph representing number of DARs in MBCs in treatment groups versus IgG control at day 15 and day 41 post-infection. (H) Heatmap showing DARs in MBCs in IgG control, anti-IFNAR1 or anti-IFNγ blocking treatment in LCMV-Docile infection at day 41 post-infection. (I) Coverage plots showing chromatin accessibility at the Cd28 (left), Adgrg5 (center) and Pou2af1 (right) gene regions, split by treatment group. Related to Figure S6 and Figure S7.

References

    1. Maruyama M, Lam KP, and Rajewsky K (2000). Memory B-cell persistence is independent of persisting immunizing antigen.[see comment][erratum appears in Nature 2001 Jan 18;409(6818):382]. Nature 407, 636–642. - PubMed
    1. Schittek B, and Rajewsky K (1990). Maintenance of B-cell memory by long-lived cells generated from proliferating precursors. Nature 346, 749–751. - PubMed
    1. Good KL, and Tangye SG (2007). Decreased expression of Kruppel-like factors in memory B cells induces the rapid response typical of secondary antibody responses. Proc Natl Acad Sci U S A 104, 13420–13425. - PMC - PubMed
    1. Di Pietro A, Polmear J, Cooper L, Damelang T, Hussain T, Hailes L, O’Donnell K, Udupa V, Mi T, Preston S, et al. (2022). Targeting BMI-1 in B cells restores effective humoral immune responses and controls chronic viral infection. Nat Immunol 23, 86–98. 10.1038/s41590-021-01077-y. - DOI - PubMed
    1. Cooper L, and Good-Jacobson KL (2020). Dysregulation of humoral immunity in chronic infection. Immunol Cell Biol 98, 456–466. 10.1111/imcb.12338. - DOI - PubMed

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