Clonal replacement sustains long-lived germinal centers primed by respiratory viruses

Abstract

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.

Keywords: B cells; Immunology; SARS-CoV-2; clonal dynamics; germinal centers; influenza; viruses.

Figure 1.. Kinetics and mutation profile of influenza-primed LLGCs up to 6 months after infection.

(A,B) Representative flow cytometry plos showing frequency of GC B cells (FAS⁺CD38^low) among total B cells (B220⁺) and frequency of HA-binding B cells among all GC B cells in the mLN of wild-type C57BL/6 mice at different time points after influenza infection. Graphs on the right show pooled data from 3–4 independent experiments. Non-infected (NI) age-matched control mice assayed at the 24 wpi time point are shown for comparison. Each symbol is one mouse, the line represents the mean. (C,D) Somatic mutations in the Igh variable gene (Ighv) in single-sorted GC B cells at various time points post-infection, as in (A). In (C), each symbol represents one sequenced B cell (n = 55–74 cells/mouse), at least 2 mice per time point are shown (C). The same data is binned into cells with low, medium, and high SHM in (D). See also Figure S1.

Figure 2.. Replacement of founder clones over time in influenza-primed LLGCs.

(A) Experimental setup. (B,C) Representative flow cytometry plots showing frequency of GC B cells (FAS⁺CD38^low) among total B cells (B220⁺) (B) and frequency of fate-mapped B cells among all GC B cells (C) in the mLN of tamoxifen-pulsed S1pr2-Tomato mice at different time points after influenza infection. Graphs on the right show pooled data from 2–5 independent experiments. Each symbol is one mouse, the line represents the mean. (D) Representative charts showing distribution of tdTomato⁺ founder (red) and tdTomato⁻ invader (grey) B cells sorted from GCs at different time points after influenza infection. Each slice represents one clone (defined as B cells with the same Igh VDJ rearrangement). Grey and red numbers in the center of the pie charts denote how many invader and founder GC B cells were sequenced, respectively, when GC B cell samples were sorted in an unbiased manner (thus the zero fate-mapped cells in the 24 wpi pie chart). Each chart is from one mouse; additional charts shown in Fig. S2. (E) Numbers of nucleotide mutations in Ighv among the cells shown in (D). Each symbol represents one sequenced B cell (n = 55–74 cells/mouse). Additional plates of rare fate-mapped GC B cells were sorted for the late time points (16 and 24 wpi). P-values are for Mann-Whitney U test. (F) Median SHM per mouse for combined data from (E) and from additional mice shown in Fig. S2A. (G) Same data as in (F), binned into cells with low, medium, and high SHM. (H) Experimental setup. A C57BL/6 (CD45.2) mouse was infected with influenza and joined parabiotically to a naïve B6.SJL mouse (CD45.1) at 4 wpi. (I) Representative flow cytometry plots showing the participation of CD45.2 founder and CD45.1 invader cells in mLN GCs of the infected CD45.2 parabiont at 8 wpi. Non-GC B cells are shown for comparison. Graph on the right summarizes data for 4 parabiosis pairs. Either the CD45.2 or CD45.1 partners were infected in different experiments. J) Experimental setup as in (A). S1pr2-Tomato mice are infected with mouse-adapted SARS-CoV-2, GC B cells are fate-mapped at 2 wpi, and GC composition is followed by flow cytometry at different time points. (K,L) Graphical representation of the kinetics of total GC B cells (K) and fate-mapped GC B cells over time (4–16 weeks), after infection with SARS-CoV-2. Results are pooled from 2 independent experiments. Each symbol is one mouse, the line represents the mean. (M) Pie charts showing distribution of tdTomato⁺ founder (red) and tdTomato⁻ invader (grey) B cells sorted from GCs at 16 weeks after SARS-CoV-2 infection. Each slice represents one clone (defined as B cells with the same Igh VDJ rearrangement). Grey and Red numbers in the center of the pie charts denote how many invader and founder cells were sequenced, respectively. Each plot is from one mouse. (N) Number of nucleotide mutations in Ighv among the cells shown in (M). Each symbol represents one sequenced B cell. Median SHM per mouse is shown on the right. P-values are for Mann-Whitney U test. See also Figure S2 and S3.

Figure 3.. Clonal replacement results from invasion of ongoing GCs by naïve-derived B cells.

(A) Experimental setup. (B) Cellular composition of invaded GCs. (C) Multiphoton imaging of an infected mLN (collapsed 3-slice, 40 μm Z-stack), highlighting GCs with different degrees of invasion by parabiont-derived B cells (i., ii., iii. and iv.). GCs are defined based on follicular dendritic cell networks, stained in vivo by administration of a monoclonal antibody to CD35 (leftmost panel). (D) Representative flow cytometry plot showing the identification of parabiont-derived invader cells (AID-GFP) in whole mLN. (E) Frequency of AID-GFP⁺ invaders among individual GCs, determined by imaging. Each symbol represents the ratio of GFP⁺ over tdTomato⁺ cells in one individual GC. Each column shows all of GCs analyzed in 4 individual mice from 4 independent experiments.

Figure 4.. Clonal and transcriptional characteristics of founder and invader B cells.

(A,B) Experimental setup. (C) Representative images of founder- and invader-dominated GCs, estimated from frequency of fate-mapped CD45.2⁺tdTomato⁺ B cells (red). (D) Clonal distribution of founder (orange) and invader (green) clones in sorted GCs. CD45.2⁺tdTomato⁻ cells (hatched) were not sequenced. The size of the orange, green, and hatched sectors of the chart was determined by flow cytometry. Internal slices are based on Igh sequencing. 6 GCs from 2 parabiosis pairs were analyzed. (E) Clonal diversity (D50, or the fraction of clones accounting for 50% of cells sequenced) plotted against the degree of invasion for founder (left) and invader (right) clones. Each symbol represents one GC. P-values are for Spearman correlation. (F) D50 and fraction of small clones (accounting for <10% of sequenced cells in that population) among founder and invader cells. Each symbol represents one GC. P-values are for Mann-Whitney U test. (G) Number of nucleotide mutations in Ighv among founder and invader B cells in each GC. Each symbol represents one sequenced cell. P_Mann-Whitney < 0.0001 for all comparisons of invaders vs. founders within the same GC. (H) Clonal phylogenies of expanded clones from one founder and 3 invader-dominated GCs. Colors and roman numerals correspond to those in panel (D). (I-M) scRNA-seq of GC B cells derived from infected mLNs of S1pr2-Tomato mice treated as in Fig. 2A, sorted at 3 (tdTomato⁺ cells only) or 8 (tdTomato⁺ and tdTomato⁻) wpi. UMAP plots showing distribution of sequenced B cells colored by transcriptionally-defined cluster (I) or by timepoint/origin (J). (K) Distribution of clones according to size and timepoint/origin (L) Percentage of tdTomato⁺ founder B cells in each transcriptional cluster (as defined in (I)). Includes only cells 8 wpi. (M) Genes differentially expressed between tdTomato⁺ founder and tdTomato⁻ invader B cells (left) or between 3 wpi and 8 wpi timepoints (right). The number of differentially expressed genes is indicated above each plot.

Figure 5.. Invader B cells are not specific for influenza antigens

(A) Experimental setup. (B) Representative flow cytometry plots showing the frequency of endogenous and parabiont-derived GC B cells that bind HA in mLNs of both mice at 8 (left) and 12 (right) wpi. (C) Quantification of data as in (B). (D) Binding of founder (tdTomato⁺) and invader (tdTomato⁻) GC B cells to influenza HA, NA, and NP antigens at 4 or 13 wpi. GC B cells were sorted from mLNs of S1pr2-Tomato mice into 96-well plates for single cell (Nojima) culture, and IgG⁺ supernatants were assayed against influenza antigens by ELISA. Each bar represents one IgG⁺ culture. Data are pooled from two mice. (E-H) Igs of expanded invader clones were sequenced and their encoded mAbs were produced for binding assays. (E) Phylogeny inferences based on Ighv sequences for 3 selected clones. The arrowhead indicates the produced mAb. (F) Binding to different influenza antigens was assayed for 9 such mAbs at 100 nM. (G,H) mAbs were titrated and incubated in the presence of PR8-infected cell lysate, uninfected cell lysate (negative control) (G), and panels of influenza (HA, NA, NP, M1 and NS1) and polyreactivity (dsDNA, ssDNA, insulin, KLH, and LPS) antigens (H). For polyreactivity assays, mAbs ED38 and MG053 were included as positive and negative controls, respectively. (I) Experimental setup. (J) Flow cytometry plots showing absence of invasion by mutant (CD45.2) B cells in the mLN of the infected CD45.1 mouse. Data from multiple mice are quantified to the right of each flow cytometry plot. Data representative of at least three independent experiments on biological replicates (at least 2 parabiotic pairs per experiment).

Figure 6.. Binding properties of long-lasting founder B cells.

(A) Phylogeny inferences based on Ighv sequences of 14 selected founder clones present in the mediastinal GC 24 weeks post infection. (B) mAbs derived from founder clones were assayed for binding to recombinant PR8 influenza proteins by ELISA. Shown are the Abs. 450 nm values for each mAb assayed at 100nM. (C) ELISA titration curves for mAb F.01 binding to recombinant influenza proteins. (D) ELISA titration curves for mAbs F.01, F.07, and F.09 binding to antigens of the polyreactivity panel. mAbs ED38 and MG053 were included as positive and negative controls, respectively. (E) Bio-layer interferometry for Fabs F.01, F.07, and F.09 binding to NP, assayed at concentrations of 20, 40, 80, and 160 nM of Fab. Affinities are average of four measurements fitted globally at 20–160 nM range. The off-rate of Fab F.09 was below the detection limit of the assay. Results are medians of technical triplicates and are representative of two independent experiments (B-D).

Figure 7.. Late founder-derived memory B cells from LLGCs contribute to recall responses.

(A) Experimental setup. (B) Expected abundance and sequence-level phenotypes of founder and invader GC B cells labeled by tamoxifen treatment at 12 wpi. MBCs, memory B cells. (C,D) Representative flow cytometry plots showing frequency of total PBs (CD138^hi, gated on total TCR-β⁻ LN cells) and fate-mapped PBs (tdTomato⁺, gated on CD138^hi cells) in pLNs boosted with NP (C) or HA (D) in PR8-infected mice pulsed with tamoxifen at early (W2FM) or late (W12FM) time points after infection, compared to age-matched non-infected controls fate mapped at the late time point. Graphs on the right show pooled data from 2 independent experiments. Each symbol is one mouse, line represents the median. (E) Mean Ighv SHM loads of tdTomato⁻ and tdTomato⁺ B cell clones sorted from pLN of NP or HA W12FM mice at 6 days post-boosting. Only pLN with substantial recruitment of fate-mapped B cells were analyzed. Data are from 4 mice from 2 independent experiments. Each symbol represents the mean SHM load of one clonal family. P-values are for student’s t test. (F) Pie charts showing clonal distribution of tdTomato⁺ PBs as in (E). Highly expanded clones (representing > 5% of sequenced cells in the compartment) are colored by whether or not sister cells bearing the same V(D)J rearrangement were identified in the non-fate-mapped PB compartment in the same node (not shown). Clones representing <5% of all cells are in dark grey. Numbers in the center of pies are no. clones /total cells sequenced. Roman numerals represent clones for which phylogenies are shown in (G). (G) Clonal phylogenies for clones indicated in (F). Grey and red circles indicate sequences obtained from tdTomato⁻ and tdTomato⁺ cells, respectively. Identical sequences found in both compartments are represented as pie charts. (H) Mean Ighv SHM load in tdTomato⁻ and tdTomato⁺ members of the same clone, obtained from clones with at least 5 cells from W12FM mice. P-value is for paired student’s T test.