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Human germinal centres engage memory and naïve B cells after influenza vaccination

Summary Influenza viruses remain a major public health threat. Seasonal influenza vaccination in humans primarily stimulates pre-existing memory B cells, leading to a transient wave of circulating antibody-secreting plasmablasts1–3. This recall response contributes to “original antigenic sin,” the selective boosting of antibody specificities from prior exposures to influenza virus antigens4. It remains unclear whether such vaccination can also induce germinal centre (GC) reactions in the draining lymph node (LN) where diversification and maturation of recruited B cells can occur5. Here we used ultrasound-guided fine needle aspiration to serially sample the draining LNs and investigate the dynamics and specificity of GC B cell responses after influenza vaccination in humans. We show that influenza vaccine-binding GC B cells can be detected as early as 1 week after vaccination. In 3 out of 8 participants, we detected vaccine-binding GC B cells up to 9 weeks after vaccination. Between 12% and 88% of the responding GC B cell clones overlapped with those detected among early circulating plasmablasts. These shared B cell clones had high frequencies of somatic hypermutation (SHM) and encoded broadly cross-reactive monoclonal antibodies (mAbs). In contrast, vaccine-induced B cell clones detected only in the GC compartment exhibited significantly lower SHM frequencies and predominantly encoded strain-specific mAbs, suggesting a naïve B cell origin. Electron microscopy-based epitope mapping revealed that some of these strain-specific mAbs recognized epitopes that were not targeted by the early plasmablast response. Our results indicate that influenza virus vaccination of humans can elicit a GC reaction to which B cell clones targeting novel epitopes are more likely to be recruited, thereby broadening the spectrum of vaccine-induced protective antibodies against this rapidly mutating pathogen.


Introduction
Seasonal influenza viruses kill 290,000 to 650,000 people globally every year 6 . As the virus drifts, novel antigenic targets emerge, creating a pressing need for the annual vaccine to engage new B cell clones that recognize such targets. The germinal centre (GC) reaction is critical for generating high-affinity and durable B cell responses 5 . It is currently unknown whether seasonal influenza virus immunization of humans can elicit a GC response in the draining lymph nodes (LN) where diversification and maturation of recruited B cells can occur. Studies examining human B cell responses have traditionally focused on sampling the easily accessible blood compartment, but ultrasound-guided fine needle aspiration (FNA) has enabled sampling of LNs with good representation of cell populations recovered by excisional biopsy, including GC B cells [7][8][9] .

Vaccine-induced B cell responses in blood and lymph nodes
Eight healthy young adults were enrolled in a seasonal influenza vaccination study. Blood and FNA specimens were collected prior to vaccination and at 1, 2, approximately 4, and 9 weeks after vaccination with the 2018/2019 quadrivalent inactivated influenza virus vaccine (QIV) (Fig. 1a). QIV-binding antibody-secreting PBs were measured in blood by enzymelinked immune absorbent spot (ELISpot). PBs peaked in blood during the first week after vaccination in all participants, with the frequency varying from 160 to 3,400 IgG-secreting QIV-binding PBs per mL (Fig. 1b, Extended Data Fig. 1a). Haemagglutinin (HA)-binding PB were also measured by flow cytometry and peaked 1-week post-vaccination (CD20 lo HA + ) and activated B cells (ABC, CD20 hi HA + ) peaked during the second week before declining (Fig. 1d, Extended Data Fig. 1b,f) 10 . Four weeks after vaccination, anti-QIV IgG plasma antibody titers were elevated compared to those at baseline as measured by enzymelinked immunosorbent assay (ELISA), along with haemagglutination-inhibiting antibody titers against the four constituent viruses of the vaccine as measured by the haemagglutination inhibition (HAI) assay (Extended Data Fig. 1g, h).
In humans, QIV is injected into the deltoid muscle of the upper arm. This area primarily drains into the lateral axillary LNs 11,12 . Both axilla of each participant were examined by ultrasound before vaccination to identify an accessible LN. Once identified, the hypoechoic LN cortex, which contains B cell follicles, was sampled by FNA (Fig. 2a), and QIV was administered in the ipsilateral muscle. The same LN was serially sampled, and its dimensions and cortical thickness were measured. LN cortical thickening following vaccination was observed in participants 04, 05, 09, and 11 ( Fig. 2b, Extended Data Fig. 2a). The median number of live cells recovered per FNA was 8.9×10 5 (range, 2.3×10 4 to 6.9×10 6 ; Extended Data Fig. 2b). Flow cytometric analysis of FNA detected significantly higher frequencies of B cells and CD4 + T cells and lower frequencies of CD14 + monocytes or granulocytes than those in peripheral blood mononuclear cells (PBMCs) (Fig. 2c, Extended Data Fig. 1b). These trends were observed in FNA specimens from all participants except for participant 02 where the frequencies of CD14 + cells were consistently high, suggesting significant blood contamination (Extended Data Fig. 2c). FNA data from specimens in which fewer than 4×10 4 live cells were recovered or the CD14 + percentage exceeded 10% of CD45 + were excluded from further analyses, including all FNA specimens from participant 02.
Single cell RNA sequencing (scRNAseq) was performed on whole PBMCs and FNA samples from all timepoints for participant 05 to comprehensively examine their cellular compositions. Compared to PBMCs, B cells and CD4 + T cells comprised substantially larger populations in FNA (38.5% vs 5.8% for B cells and 42.3% vs 34.9% for CD4 + T cells), whereas monocytes and platelets were substantially lower (Fig. 2d, Extended Data Fig. 2d-h, Extended Data Table 1). Surface staining revealed a population of CD20 hi CD38 int IgD lo B cells that increased in frequency after vaccination in 5 of the 7 participants Turner et al. Page 3 Nature. Author manuscript; available in PMC 2021 February 28. analysed (participants 03, 04, 05, 08, and potentially 11, whose baseline FNA specimen was among those excluded) (Fig. 2e). Intranuclear staining confirmed that this CD20 hi CD38 int IgD lo B cell population expressed Bcl6, indicating that it was comprised of GC B cells, whereas equivalently gated PBMCs did not (Extended Data Fig. 2i). The expression of additional markers on the CD20 hi CD38 int IgD lo population was consistent with a GC phenotype, including CD24 lo , CD27 int , CD71 hi , CXCR5 int , and Ki-67 + (Extended Data Fig.  2j). HA + GC B cells were detected and increased in frequency after vaccination in participants 04, 05, and 11, along with HA + IgD lo CD20 − CD38 + PBs and IgD lo CD20 + CD38 − ABCs, which also were detected in participants 07 and 09 (Fig. 2f, Extended Data Figs. 1c, 2k-m, and Extended Data Table 2).

Clonal overlap between PB and GC B cells
We clonally analysed PBs and GC B cells from participants 04, 05, and 11. We sorted single  Fig.  4i). Sequences clonally related to QIV-binding mAbs were identified in PB and ABC compartments in 1-week post-vaccination PBMCs; thereafter, QIV-binding PBs declined rapidly and ABCs predominated. In FNA samples, QIV-binding GC B cells and PBs were observed after week 2, and QIV-binding RMB appeared at week 2-4 (Fig. 3e). The SHM frequency of clones recruited to the GC but not early PB response remained lower at all timepoints than that of clones found in both compartments (Extended Data Fig. 3h). We next constructed dendrograms of some responding B cell clonal lineages that were either highly mutated and extensively expanded (1A06, Fig. 3f) or minimally mutated and less expanded (1C10, Fig. 3g).

Mapping vaccine-induced PB and GC B cells
Influenza A viruses are categorized into two phylogenetic groups based on the HA sequence 17 . The QIV contains antigens that are derived from circulating H1N1 (group 1 HA) and H3N2 (group 2 HA) strains, in addition to strains from the two antigenically distinct influenza B lineages. Antibodies are considered broadly cross-reactive if they recoginze HAs from H1N1 or N3N2 strains that no longer circulate in humans. We analyzed the binding breadth of mAbs derived from PB and GC B cell using influenza virus protein microarrays (IVPMs) 18,19 and found that both broadly cross-reactive and strain-specific clones were recruited to the GC and PB responses (Fig. 4a, Extended Data Fig. 5a-c). However, analysis of all QIV binding mAbs combined from participants 04, 05, and 11 indicated that the proportion of strain-specific mAbs among H1 HA and influenza B HA binders was significantly higher in clones recruited to the GC but not early PB response. Similar trends were observed for the individual participants (Fig. 4b, Extended Data Fig.  5d).
We next examined the epitopes targeted by plasma anti-HA antibodies in participants 04, 05, and 11. We used electron microscopy polyclonal epitope mapping (EMPEM) 20,21 of antigen binding fragments (Fabs) purified from the plasma of these participants to identify the binding landscape to the H1 and H3 HAs included in QIV. The polyclonal antibody (pAb) response diversified over time in participants 04, 05, and 11; new pAbs that appeared in the first four weeks after vaccination likely represented contributions from the early PB response, whereas later additions were likely also contributed by GC-derived longer-lived plasma cells (Fig. 4c, Extended Data Fig. 5e-g). We then mapped the epitopes targeted by some GC clones that did not participate in the early PB response by negative stain electron microscopy of Fab/HA complexes and found that the binding footprint of some Fabs overlapped with those found in plasma, whereas others targeted unique epitopes (Fig. 4d, Extended Data Fig. 5h-k). We identified two mAbs that targeted unique H1 HA epitopes and were protective in mice against lethal influenza challenge: 1B05 and 2C09 from participants 04 and 05, respectively (Fig. 4e). Notably, these and another mAb not detected in plasma, 1B08 from participant 05, were strain-specific and had low Vh mutation frequencies of 0.033, 0.026, and 0.026, respectively.

Discussion
This study sought to define the role of the GC reaction in human B cell responses to influenza virus vaccination. We found that a substantial proportion of vaccine-binding GC B cell clones were part of the early circulating PB response. These shared clones exhibited the two cardinal features of a recall B cell response to influenza: they possessed high levels of SHM, and they were broadly cross-reactive. By contrast, a subset of vaccine-induced GC B cells that were not detected among early circulating PBs had low SHM levels and were more likely to be strain-specific, indicating a probable naïve B cell origin. Studies of secondary GC responses in mice have found that naïve B cells and less somatically mutated MBCs are more efficiently recruited to GCs than MBCs that have undergone extensive GC selection 22-25 . One key difference is that in the current study, the vaccine antigens were likely different from the influenza antigens the participants had previously encountered, as none had been vaccinated for three years, whereas in the mouse studies, animals were boosted with the same antigen. Compared to homologous reimmunization, heterologous boosting may recruit MBCs to GC responses more efficiently due to decreased competition from preexisting antibodies.
To our knowledge, the current study provides the first direct evidence of vaccine-induced GC responses in humans, but it does have some limitations. We detected HA-binding GC B cells in only three participants despite readily detectable peripheral B cell responses in all eight participants. It is possible that the LNs sampled in the other five participants were not the primary draining LNs. It is also possible that vaccination did not elicit a GC response in these participants. A second limitation pertains to our clonal analyses of vaccine-responding B cells, in which we excluded those encoding mAbs that did not show detectable binding by QIV ELISA. Studies in mice 26,27 and humans 28 indicate that low-affinity B cells can be recruited to GCs despite encoding BCRs that, when expressed as mAbs, are below the ELISA detection threshold. This suggests that some very low-affinity clones that are in fact specific for QIV could have been excluded from our analyses, resulting in overestimation of the proportion of higher-affinity clones in the response. In a similar vein, the epitope analysis by EMPEM may have failed to detect some low-affinity and/or -frequency circulating pAbs, overestimating the contribution from more abundant, higher-affinity PBand plasma cell-derived antibodies.
A significant fraction of vaccine-induced GC-and PB-derived mAbs were cross-reactive with previously circulating influenza virus strains. This response is reminiscent of the original antigenic sin (OAS) phenomenon, where antibody responses are preferentially directed against previously encountered influenza strains when a person is exposed to a contemporary strain 4 . We propose that suboptimal GC B cell responses after influenza vaccination result in a more pronounced OAS effect due to inefficient engagement of naïve B cells targeting novel epitopes. High levels of baseline antibodies against conserved influenza epitopes may interfere with the formation or continued maintenance of vaccination-induced GC responses in humans. If so, new vaccine formulations that promote robust GC reactions are more likely to induce a more diverse antibody response against circulating and emerging influenza virus strains.

Sample collection, preparation, and storage.
All studies were approved by the Institutional Review Board of Washington University in St.
Louis. Written consent was obtained from all participants. Eight participants who had not been vaccinated against influenza for at least three years were enrolled, including 1 female and 7 males, aged 26-40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated using Vacutainer CPT tubes (BD), the remaining RBCs were lysed with ammonium chloride lysis buffer (Lonza), and cells were immediately used or cryopreserved in 10% dimethylsulfoxide in FBS. Ultrasound guided fine-needle aspiration (FNA) of axillary lymph nodes was performed by a qualified physician's assistant under the supervision of a radiologist. Lymph node dimensions and cortical thickness were measured before each FNA. For each FNA sample, 6 passes were made using 25 -gauge needles, each of which was flushed with 3 mL of RPMI 1640 supplemented with 10% FBS and 100 U/mL penicillin/ streptomycin, followed by three 1 -mL rinses. Red blood cells were lysed with ammonium chloride buffer (Lonza), washed twice with PBS supplemented with 2% FBS and 2 mM EDTA, and immediately used or cryopreserved in 10% DMSO in FBS. Participants reported no adverse effects of phlebotomy, serial FNA, or vaccination.

ELISpot.
Direct ex-vivo ELISpot was performed to determine the number of total, vaccine-binding, or recombinant HA-binding IgM-secreting cells present in PBMC samples. ELISpot plates (Millipore) were coated overnight at 4°C with QIV (diluted 1:100), tetanus/diphtheria vaccine (diluted 1:50), and 10 µg/mL anti-human Ig (Jackson ImmunoResearch) and 3 µg/mL recombinant HA proteins. Plates were blocked the following morning for 90 min at 37°C with RPMI 1640 supplemented with 10% FBS. Dilutions of washed PBMCs were incubated for 18 h in RPMI supplemented with 10% FBS. After washing the plates, secreted antibodies were detected with anti-human IgM-biotin (Invitrogen, 1:2000) and streptavidin-HRP (Jackson ImmunoResearch), and plates were developed with AEC substrate (Sigma). Total, vaccine-binding, or recombinant HA-binding IgG and IgA-secreting cells were detected using IgG/IgA double -color ELISpot Kits (Cellular Technologies, Ltd.) according to the manufacturer's instructions. ELISpot plates were analyzed using an ELISpot counter (Cellular Technologies Ltd.).

ELISA.
Assays were performed in 96-well plates (MaxiSorp; Thermo). Each well was coated with 100 µL of QIV (diluted 1:100), tetanus/diphtheria vaccine (diluted 1:50), or with 0.5 μg/mL of the recombinant HA antigens in PBS, and plates were incubated at 4 °C overnight. Plates were then blocked with 0.05% Tween20 and 10% FBS in PBS. Plasma or mAbs were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween-20 in PBS. Goat antihuman IgG-HRP (Jackson ImmunoResearch, 1:2,500) was diluted in blocking buffer before adding to wells and incubating for 90 min at room temperature. Plates were washed 3 times with 0.05% Tween20 in PBS, and then washed 3 times with PBS before the addition of peroxidase substrate (SigmaFAST o-Phenylenediamine dihydrochloride, Sigma-Aldrich). Reactions were stopped by the addition of 1 M HCl. Optical density measurements were taken at 490 nm. The half-maximal binding dilution for plasma was calculated using nonlinear regression (Graphpad Prism v7). The minimum positive concentration for mAbs was defined as that with optical density at least 3-fold above background.

Hemagglutination inhibition (HAI).
Plasma and monoclonal antibody HAI titers were determined for the four constituent viruses of 18 μL of 0.5% turkey RBCs (Lampire) suspended in PBS was added to each well, and the plates were gently agitated. After an additional 30 min at room temperature, the titers were computed as the reciprocal of the final dilution for which non-agglutination was observed.

Influenza virus protein microarray (IVPM).
IVPMs were generated as described previously 18,19 . Briefly, recombinant influenza virus HAs were spotted onto expoxysilane-coated glass sides (Schott) using a Versa 100 automated liquid handler (Aurora Biomed). Each array included 13 HAs (Extended Data Table 5) diluted in 0.1% milk in PBS, and spotted in triplicate. Each slide contained 24 identical arrays. HAs were printed at a volume of 30 nL per spot with a concentration of 100 µg/mL. Slides were vacuum-packed and stored at -80°C until use. At the start of each assay, slides were removed from the -80°C freezer and allowed to warm to room temperature, then incubated in a humidity chamber at 95-98% relative humidity for 2 h. The slides were then inserted into 96-well microarray gaskets (Arrayit), dividing the slide into 24 separate arrays, which were blocked with 220 µL 3% milk in PBS containing 0.1% Tween 20 (PBS-T) for 2 h. The blocking solution was then removed, and mAbs diluted in 1% milk in PBS-T were incubated with arrays at a volume of 100 µL and a concentration of 5 µg/mL for 1 h. The arrays were washed 3 times with 220 µL PBS-T, and secondary antibody solution containing Cy5-labeled anti-human IgG secondary antibody (Abcam) diluted 1:3,000 in 1% milk in PBS-T was added to each well at a volume of 50 µL and incubated for 1 h. After removing the secondary antibody solution, arrays were washed 3 times with PBS-T and removed from their gaskets. The slides were rinsed with PBS-T, followed by a rinse with deionized water, and then dried with an air compressor. Arrays were imaged and analyzed with a Vidia microarray scanner (Indevr) using an exposure time of 1000 ms to measure spot median fluorescence.
Cell sorting and flow cytometry.
Staining for analysis and sorting was performed using fresh or cryo-preserved PBMCs or Cells were washed twice with permeabilization/wash buffer and resuspended in P2 for acquisition on an Aurora using SpectroFlo v2.2 (Cytek). Flow cytometry data were analyzed using FlowJo v10 (Treestar).

Monoclonal antibody (mAb) generation.
Antibodies were cloned as described previously 13 . Briefly, VH, Vκ, and Vλ genes were amplified by reverse transcription-PCR and nested PCR reactions from singly sorted GC B cells and PBs using primer combinations specific for IgG, IgM/A, Igκ, and Igλ from previously described primer sets 30 and then sequenced. To generate recombinant antibodies, restriction sites were incorporated via PCR with primers to the corresponding heavy and light chain V and J genes. The amplified VH, Vκ, and Vλ genes were cloned into IgG1 and Igκ expression vectors, respectively, as described previously 1,30,31 . Heavy and light chain plasmids were co-transfected into Expi293F cells (Gibco) for expression, and antibody was purified with protein A agarose (Invitrogen).

Bulk B cell receptor sequencing.
RNA was purified from whole PBMCs and sorted PBs from participants 04, 05, and 11 (Extended Data  36 ]. Additional quality control of these sequences was performed with the following requirements: aligned exclusively to heavy chain V and J genes; had Ns at fewer than 10% of V segment positions; had a minimum V segment coverage from nucleotide position 1 to 310 under the IMGT unique numbering scheme 37 ; and had a junction length that was a multiple of 3, where junction was defined as from IMGT codon 104 encoding the conserved cysteine to codon 118 encoding phenylalanine or tryptophan.

Processing 10× Genomics single-cell B cell receptor reads.
Demultiplexed pair-end FASTQ reads from participant 05 were preprocessed using the "cellranger vdj" command from 10× Genomics' Cell Ranger v3.1.0 for alignment against the GRCh38 human reference v3.1.0 (refdata-cellranger-vdj-GRCh38-alts-ensembl-3.1.0). Initial germline V(D)J gene annotation was performed as described for bulk sequencing BCR reads. Additional quality control was performed, requiring sequences to be productively rearranged and have valid V and J gene annotations, consistent chain annotation (excluding sequences annotated with heavy chain V gene and light chain J gene), and a junction length that was a multiple of 3. The 429 heavy chain BCRs from the sample day 0 PBMC 2 were removed after they were found to have identical cell barcodes and sequences as BCRs from the sample day 5 PBMC 2. Only cells with exactly one heavy chain sequence paired with at least one light chain sequence were retained.

B cell receptor genotyping.
Processed BCR sequences from bulk sequencing (Extended Data Table 3) and, if available, single-cell sequencing (Extended Data B cell clonal lineages were inferred based on productively rearranged heavy chain sequences using hierarchical clustering with single linkage 15 . First, sequences were partitioned based on common V and J gene annotations and junction region lengths. Within each partition, sequences whose junction regions were within 0.1 normalized Hamming distance from each other were clustered as clones. This distance threshold was determined by manual inspection in conjunction with kernel density estimates to identify the local minimum between the two modes of the within-participant bimodal distance-to-nearest distribution (Extended Data Fig.  3c). Following clonal clustering, full-length clonal consensus germline sequences were reconstructed for each clone with D-segment and N/P regions masked with N's, resolving any ambiguous gene assignments by majority rule. Within each clone, duplicate IMGTaligned V(D)J sequences from bulk sequencing were collapsed with the exception of duplicates derived from different B cell compartments or isotypes. Clonal rank abundance distributions for GC and early PB (Extended Data Fig. 3e) were produced using the "estimateAbundance" function from Alakazam v0.3.0 [ref 36 ].

Clonal overlap analysis.
Clonal overlap between week 1 PBs from blood and the GC B cells from later weeks was determined by the presence of sequences from both compartments in the same B cell clone. Visualization was achieved using the circlize package v0.4.8 [ref 39 ].

Calculation of SHM frequency.
Mutation frequency was calculated by counting the number of nucleotide mismatches from the germline sequence in the heavy chain variable segment leading up to the CDR3, while excluding the first 18 positions that could be error-prone due to the primers used for generating the mAb sequences. This calculation was performed using the calcObservedMutations function from SHazaM v0.1.11 [ref 36 ].

Construction of B cell lineage trees.
Phylogenetic trees for responding B cell clones were constructed using IgPhyML v1.1.0 [ref. 40 ] with the HLP19 model 41 .

Processing of 10× Genomics single-cell 5′ gene expression data.
Demultiplexed pair-end FASTQ reads from participant 05 were first preprocessed on a bysample basis using the "cellranger count" command from 10× Genomics' Cell Ranger v3.1.0 for alignment against the GRCh38 human reference v3.0.0 (refdata-cellranger-GRCh38-3.0.0). To avoid a batch effect introduced by sequencing depth, the "cellranger aggr" command was used to subsample from each sample so that all samples had the same effective sequencing depth, which was measured in terms of the number of reads confidently mapped to the transcriptome or assigned to the feature IDs per cell. The feature biotypes were retrieved using biomaRt v2.42.0 [ref 42 ] from Ensembl release 93 [ref 43 ]. Additional quality control was performed as follows. 1) To remove presumably lysed cells, cells with mitochondrial content greater than 12.5% of all transcripts were removed. 2) To remove likely doublets, cells with more than 7,000 features or 70,000 total UMIs were removed. 3) To remove cells with no detectable expression of common housekeeping genes, cells with no transcript for any of ACTB, GAPDH, B2M, HSP90AB1, GUSB, PPIH, PGK1, TBP, TFRC, SDHA, and LDHA were removed 44 . 4) The feature matrix was subset, based on their biotypes, to protein-coding, immunoglobulin, and T cell receptor genes that were expressed in at least 0.05% of the cells in any sample. The resultant feature matrix contained 15,479 genes. 5) Cells with detectable expression of fewer than 200 genes were removed. After quality control, there were a total of 130,757 cells from 21 samples (Extended Data Table 4).

Single-cell gene expression analysis.
Single-cell gene expression analysis was performed using Seurat v3.1.1 [ref 45 ]. UMI counts measuring gene expression were log-normalized. The top 2,000 highly variable genes (HVGs) were identified using the "FindVariableFeatures" function with the "vst" method. A set of 317 immune-related, "immunoStates" marker genes 46 were added to the HVG list, whereas immunoglobulin and T cell receptor genes were removed. The data were scaled and centered, and principal component analysis (PCA) was performed based on HVG expression. The PCA-guided t-distributed stochastic neighbor embedding (tSNE) plot was generated using the top 20 principal components (PCs).
Overall clusters (Extended Data Table 1) were identified using the "FindClusters" function with resolution 0.07 (Extended Data Fig. 2b). Cluster identities were assigned by examining the expression of a set of marker genes for different cell types (Extended Data Fig. 2c- Fig. 4c) and high expression levels of CD2 and CD3E (Extended Data Fig. 4e), whereas cluster 9 was labelled "Unassigned" as its cells tended to have no BCR (Extended Data Fig. 4c) and high expression levels of CD2 and CD3E (Extended Data Fig. 4e). Clusters 5 and 9 were excluded from the final B cell clustering (Extended Data Fig. 4f). Cells that were found in the GC cluster but came from the blood samples were labelled "PB-like" (Extended Data Fig. 4f-g). To further confirm the identities of these B cell subsets, we assessed their corresponding heavy chain BCRs for SHM frequency (Extended Data Fig. 4h) and isotype usage (Extended Data Fig. 4i), which were consistent with expected values.

EMPEM sample preparation and complexing.
Polyclonal antibodies were prepared similarly to Bianchi

Negative stain electron microscopy.
For negative stain analysis, 400 mesh copper grids (Electron Microscopy Sciences, EMS) were coated with collodion 2% in amyl acetate (EMS), deposited with carbon using a Cressington carbon evaporator, and plasma cleaned (EMS). Immune complexes were diluted to 30 µg/mL, loaded onto prepared grids, and stained with 2% w/v uranyl formate. Samples were imaged on three Tecnai transmission electron microscopes (FEI): Spirit T12 with a CMOS 4k camera (TVIPS), T20 with an Eagle CCD 4k camera (FEI), and Talos 200C with a Falcon II direct electron detector and a CETA 4k camera (FEI). Micrographs were obtained using Leginon 47 , 100,000-300,000 particles were picked and stacked using Appion 48 , and data were processed using 2D and 3D classification and reconstruction in Relion 49,50 . Fab densities were segmented and resampled onto reference trimers (PDB IDs 4M4Y for group 1 HA and 3M5G for group 2 HA), and final images were made using UCSF Chimera 51 and Photoshop (Adobe). Polyclonal Fab specificities were determined using 3D reconstructions and 2D class averages. Some specificities did not reconstruct fully due to limited angular sampling caused by orientation bias, sub-stoichiometric binding of Fabs, and/or low abundance of these immune complexes even within the large datasets collected. However, in addition to suggestive 3D density, our large internal database of images of HA-Fab complexes provide references to extrapolate the 2D images onto 3D models of HA using the same procedure we recently reported for interpreting neuraminidase-Fab complexes 52 .

In vivo challenge.
Animal experiments were approved by approved by the Washington University IACUC. Mice were housed under specific-pathogen free conditions at 55±10% relative humidity with a 12h day/night cycle. Female 6-8-week-old C57BL/6 mice (5-7 mice/group) received 5 mg/kg mAb 321-04 1B05, 321-05 2C09, the broadly protective mAb 1G01 [ref 14 ] as a positive control, or irrelevant human IgG control mAb in 100 μL PBS via intraperitoneal administration. One day later, the mice were anesthetized with isofluorane and challenged intranasally with 10 4 TCID 50 A/California/04/2009 E3 (H1N1). Weight was measured daily for 14 days; mice were euthanized if weight loss exceeded 20% of original weight.  CD19 + CD4 − live singlet lymphocytes as in (b). Symbols at each timepoint represent one sample (n=8). g, h) IgG plasma antibody titers against QIV and Tetanus/Diptheria vaccine measured by ELISA (g) and hemagglutination inhibition titers against QIV constituent viruses (h) pre-and 4-weeks post-vaccination. Symbols at each timepoint represent one sample (n = 8). Horizontal lines in a, g, and h represent means. Dotted lines represent limit of detection. P-values from paired two-sided Student's t -tests. a) Schematic of single cell mAb cloning and expression. Paired heavy and light chain genes were amplified from singly sorted PBs or GC B cells. Variable portions of heavy chains were cloned into a Cγ1 expression vector and variable portions of κ and λ light chains were cloned into respective expression vectors. Paired heavy and light chain expression vectors were co-transfected into 293F cells, and mAbs were purified from culture supernatant by protein A affinity chromatography, then screened for QIV specificity by ELISA. b) Minimum positive concentrations of clonally unique mAbs generated from singly sorted PBs as determined by QIV ELISA; positive binding defined as greater than 3× background. c) Distance-to-nearest-neighbor plots for choosing a distance threshold for inferring clones via hierarchical clustering. After partitioning sequences based on common V and J genes and junction length, the nucleotide Hamming distance of a junction to its nearest nonidentical neighbor from the same participant within its partition was calculated and normalized by junction length (blue histogram). For reference, the distance to the nearest      FNA (right) scRNAseq samples pooled from all timepoints from participant 05. Each dot represents a cell, colored by phenotype as defined by the gene expression profile. Total numbers of cells are below clusters. e) Representative flow cytometry gating of CD20 hi CD38 int population in FNA. Cells pre-gated IgD lo CD19 + CD4 − live singlet lymphocytes. f) Kinetics of total (closed circles) and HA + (open circles) GC B cells in FNA, as defined by flow cytometry gates in e and Extended Data Fig. 2k. Symbols at each timepoint represent one sample (n=7). Daggers denote samples excluded from analysis due to low cell recovery or blood contamination. Dendrograms of clonal families of H1 HA-binding day 60 GC mAbs 1A06 (f) and 1C10 (g). Horizontal branch length represents the expected number of substitutions per codon in V -region genes, corresponding to the key in the lower left of each panel. Colored symbols represent sequences from cells isolated at day 5 unless otherwise specified, corresponding to the indicated phenotype and isotype. a) Binding of group 1-binding mAbs generated from singly sorted PBs and GC B cells that overlapped clonally (purple) or did not overlap (red and blue) for PB and GC B cells, respectively, from participant 05 using an influenza virus protein microarray (IVPM). Scale bar is median fluorescence intensity. Vaccine strains in bold type; underlined strains circulated in humans in participants' lifetimes. b) Percentages of mAbs that bound two or more HA strains from participants 04, 05, and 11 from GC clones that did not participate in the early PB response (blue) and from PB and shared clones (red). P-values from Fisher's exact test. The number of mAbs is indicated in the middle of the charts. c) Polyclonal epitopes of Fabs from plasma at indicated timepoints from participants 04, 05, and 11 with HA from A/Michigan/45/2015. Epitopes were determined by 3D reconstructions and/or 2D class averages (images to bottom right of 3D reconstructions). HA proteins shown in grey; Fabs shown in multiple colors. d) Monoclonal and polyclonal epitopes of immune complexes with HA from A/Michigan/45/2015 and Fabs generated from the indicated GC mAbs (blue) and plasma pAbs (red). Fabs with dashed outlines have predicted epitopes due to limited particle representation. e) Protection of GC mAbs 1B05 and 2C09 in a mouse influenza virus challenge model. Mice received 5 mg/kg of the indicated mAb intraperitoneally 1 day before intranasal challenge with A/California/04/2009 E3 (H1N1), and were weighed daily; 7 mice were used for 1B05 and 1G01, 6 for 2C09 and isotype control, and 5 for uninfected. Error bars indicate mean ±SEM.