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ABSTRACT Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection1. However, the outcome of interactions between an influenza

haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the

haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind

equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of

virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h.

In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the

draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the

initiation of an effective adaptive response. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS ACTIVATION AND MATURATION OF ANTIGEN-SPECIFIC B CELLS IN NONECTOPIC LUNG INFILTRATES ARE

INDEPENDENT OF GERMINAL CENTER REACTIONS IN THE DRAINING LYMPH NODE Article Open access 11 April 2025 HUMAN NAÏVE B CELLS SHOW EVIDENCE OF ANERGY AND CLONAL REDEMPTION FOLLOWING VACCINATION

Article Open access 14 May 2025 INFLUENZA VIRUS INFECTION EXPANDS THE BREADTH OF ANTIBODY RESPONSES THROUGH IL-4 SIGNALLING IN B CELLS Article Open access 18 June 2021 ACCESSION CODES

PRIMARY ACCESSIONS GENBANK/EMBL/DDBJ * KF419287 * KF419288) REFERENCED ACCESSIONS GENBANK/EMBL/DDBJ * KF419287 * KF419288 DATA DEPOSITS Sequences of the mouse _V_, _D_ and _J_ genes were

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protein micelles from amphiphilic membrane proteins. _Proc. Natl Acad. Sci. USA_ 75, 5306–5310 (1978) Article  ADS  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS S.K.D. and C.G.

were funded by the Cancer Research Institute. J.J.C. was funded by the Human Frontiers Science Program. F.W.A., R.J. and H.L.P. are funded by grants from the National Institutes of Health.

F.W.A. is a Howard Hughes Medical Institute investigator. S.K.D. and H.L.P. are funded by the American Association for Cancer Research-Pancreatic Cancer Action Network. We are grateful to P.

Wisniewski for cell sorting, to J. Jackson for mouse husbandry, to G. Bell for statistical analysis, to N. Watson for electron microscopy and to M. Witte for sortase nucleophiles. AUTHOR

INFORMATION Author notes * Stephanie K. Dougan and Joseph Ashour: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Whitehead Institute for Biomedical Research, 9

Cambridge Center, Cambridge, 02142, Massachusetts, USA Stephanie K. Dougan, Joseph Ashour, Roos A. Karssemeijer, Maximilian W. Popp, Ana M. Avalos, Marta Barisa, Arwen F. Altenburg, Jessica

R. Ingram, Juan Jose Cragnolini, Rudolf Jaenisch & Hidde L. Ploegh * Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA Maximilian W. Popp

 & Hidde L. Ploegh * Boston Children’s Hospital, Karp Family Research Building, One Blackfan Circle Boston, 02115, Massachusetts, USA Chunguang Guo & Frederick W. Alt Authors *

Stephanie K. Dougan View author publications You can also search for this author inPubMed Google Scholar * Joseph Ashour View author publications You can also search for this author inPubMed

 Google Scholar * Roos A. Karssemeijer View author publications You can also search for this author inPubMed Google Scholar * Maximilian W. Popp View author publications You can also search

for this author inPubMed Google Scholar * Ana M. Avalos View author publications You can also search for this author inPubMed Google Scholar * Marta Barisa View author publications You can

also search for this author inPubMed Google Scholar * Arwen F. Altenburg View author publications You can also search for this author inPubMed Google Scholar * Jessica R. Ingram View author

publications You can also search for this author inPubMed Google Scholar * Juan Jose Cragnolini View author publications You can also search for this author inPubMed Google Scholar *

Chunguang Guo View author publications You can also search for this author inPubMed Google Scholar * Frederick W. Alt View author publications You can also search for this author inPubMed 

Google Scholar * Rudolf Jaenisch View author publications You can also search for this author inPubMed Google Scholar * Hidde L. Ploegh View author publications You can also search for this

author inPubMed Google Scholar CONTRIBUTIONS S.K.D. and J.A. contributed equally. Somatic cell nuclear transfer was performed by S.K.D.; S.K.D., J.A., R.A.K., M.W.P., M.B. and A.F.A.

performed experiments. A.M.A. generated hybridomas. J.R.I. and J.J.C. generated flu-specific VHHs. C.G. sequenced the BCR loci. F.W.A. and R.J. provided advice and reagents. S.K.D., J.A. and

H.L.P. designed experiments, analysed data and wrote the paper. CORRESPONDING AUTHOR Correspondence to Hidde L. Ploegh. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing financial interests. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 FLU MICELLES STAIN HA-SPECIFIC B CELLS. A, Schematic for preparation of glycoprotein micelles from

HA–SRTAlexa 647 virus. B, Immunoprecipitation of HA–Alexa 647 with anti- Alexa 647 monoclonal antibody. Triton X100-disrupted virions were incubated with 400 μg anti-Alexa 647 overnight and

HA–Alexa 647 was then recovered using protein G-Sepharose. Bound proteins were eluted with 0.1 M glycine pH 2.8. W, wash; E, elution. C, Typhoon image of the fractions obtained from a linear

sucrose gradient after 20 h centrifugation (107,900_g_). D, Fraction 8 from the sucrose gradient was concentrated and sucrose-depleted by centrifugation over a 30 kDa filter (Amicon

UltraCel). The preparation was stained with phosphotungstate and examined by transmission electron microscopy (×150,000 magnification). E, Splenocytes from mice infected with A/WSN/33 or

control mice were stained with anti-CD19 and HA–Alexa 647 micelles and analysed by cytofluorometry. Plots are representative of 6 mice per group. EXTENDED DATA FIGURE 2 FLUBI ANTIBODY IS OF

THE IGG2B SUBCLASS. ELISA plates were coated with A/WSN/33-infected MDCK cell lysate and exposed to 1:100 diluted serum from a single C57BL/6 (wt), FluBI, FluBI;_Rag2_−/−, or wild-type mouse

infected with A/WSN/33. Plates were washed and probed with isotype-specific secondary antibodies. Uninfected wild-type mice have flu-reactive antibodies of the IgM subclass. Flu-specific

IgE was not detected in any sample. Error bars are s.d. of samples analysed in triplicate. EXTENDED DATA FIGURE 3 SEQUENCE OF THE VDJ AND VJ SEGMENTS OF THE FLUBI ANTIBODY. Genomic DNA was

prepared from tails of FluBI mice. The heavy and light chain rearrangements were first identified by amplifying and sequencing of the segments with degenerate primers: for heavy chain:

forward 5′-ARGCCTGGGRCTTCAGTGAAG-3′ and reverse 5′-AGGCTCTGAGATCCCTAGACAG-3′; for light chain: forward 5′-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3′ and reverse

5′-ATGCGACGTCAACTGATAATGAGCCCTCTCC-3′. Then the full sequences of the rearranged heavy and light chain segments were obtained using specific primers: forward 5′-TTACTGAGCACACAGGACCTC-3′ and

reverse 5′-AGGCTCTGAGATCCCTAGACAG-3′; for light chain: forward 5′-CAGCCCATATTCTCCCATGT-3′ and reverse 5′-ATGCGACGTCAACTGATAATGAGCCCTCTCC-3′. Amplified products were agarose gel-purified and

sequenced. Sequences were aligned to the NCBI mouse _V_, _D_ and _J_ genes using IgBlast. Sequences were deposited in GenBank (accession numbers KF419287 and KF419288). EXTENDED DATA FIGURE

4 FLUBI MICE LACK B-1A B CELLS, BUT SHOW NEAR-NORMAL DEVELOPMENT OF FOLLICULAR B CELLS. Cells were isolated from spleen, lymph node (LN, pooled mesenteric and cervical), peritoneal cavity

and bone marrow of FluBI, FluBI _Rag2_−/− or C57BL/6 mice. Erythrocytes were lysed and cells were stained with the indicated antibodies and 7-AAD viability dye. LN plots were gated on total

live cells. All other populations were gated on CD19+ live cells. Numbers indicated the percentage of cells in the indicated gates. B-1a B cells (CD5+) are absent and B-1b B cells

(CD5−CD11b+) are reduced in the peritoneal cavity of FluBI and FluBI _Rag2_−/− mice. Plots are representative of 5 mice per group. EXTENDED DATA FIGURE 5 FLUBI B CELLS ARE INFECTED BY

A/WSN/33. CD40-activated OBI or FluBI B cells were incubated with A/WSN/33 virus at an MOI of 1.0 for 30 min on ice. Cells were then washed and incubated at 37 °C in RPMI (0.2% BSA). At 2 

h.p.i., cells were fixed, permeabilized and stained with anti-IgG and TAMRA-conjugated anti-NP (VHH54, derived from alpaca; see Extended Data Fig. 9). A, Cells were visualized by confocal

microscopy. B, Cells from A were scored as VHH54-positive or -negative. Error bars represent s.d. of positive cells counted per field (3 fields counted; ∼200 total cells were counted per

group). EXTENDED DATA FIGURE 6 ANTIBODY SECRETED BY FLUBI B CELLS DOES NOT CROSS-REACT WITH OTHER STRAINS OF INFLUENZA VIRUS. ELISA plates were coated with A/WSN/33 (H1N1), A/Udorn/307/1972

(H3N2) or A/Puerto Rico/8/1934 (H1N1) overnight at 4°. Plates were then washed, blocked with 10% fetal bovine serum and exposed to FluBI hybridoma supernatant or WSN-infected serum at the

indicated dilutions. Bound antibody was detected using horseradish peroxidase-coupled anti-IgG2b secondary reagent. EXTENDED DATA FIGURE 7 FLUBI B CELLS ARE NOT INFECTED WITH A/PUERTO

RICO/8/1934 VIRUS _IN VIVO_. C57BL/6 mice were administered 5 × 106 MHCII–GFP+ FluBI B cells 2 h before intranasal infection with 2 × 105 p.f.u. per mouse of either A/WSN/33 (WSN) or

A/Puerto Rico/8/1934 (PR8). Mice were euthanized 3 days post-infection, and lung resident cells were stained with anti-CD19 and TAMRA-conjugated VHH68 (anti-HA) or TAMRA-conjugated VHH52/54

(anti-NP). A, Representative plots gated on CD19+ cells. B, Quantification of flu-antigen positive cells as shown in A. _n_ = 3. Error bars are s.d. p = 0.06 using two-sided _t_-test.

EXTENDED DATA FIGURE 8 PROLIFERATING FLUBI CELLS IN THE MEDIASTINAL LYMPH NODE ARE PLASMABLASTS. A, Mediastinal lymph node cells from day 6 post live infection mice described in Fig. 4 were

analysed by confocal microscopy. GFP+ cells displayed a morphology consistent with plasmablasts. B, MSLN cells from day 6 post live infection mice described in Figure 4 were analysed by

cytofluorometry. Proliferating (violet low) cells were B220low and CD138+. EXTENDED DATA FIGURE 9 ALPACA-DERIVED VHHS RECOGNIZE HA AND NP FROM A/WSN/33. A, An alpaca was immunized with

ethanol-fixed influenza virus. Phage display libraries were constructed from selectively amplified VHH-specific complementary DNA using peripheral blood lymphocytes as starting material, and

panned twice against sortase labelled influenza HA–SRTbiotin virus bound to streptavidin coupled beads. VHH sequences obtained from specific binders were expressed with a sortase

recognition motif to allow direct conjugation of biotin or fluorophores. B, VHH54 and VHH68 conjugated directly to agarose beads were used to precipitate lysates of A/WSN/33 infected,

[35S]cysteine/methionine-labelled MDCK cells. EXTENDED DATA FIGURE 10 FLU-SPECIFIC VHHS CAN STAIN INFECTED FLUBI B CELLS. B cells from OBI or FluBI mice were cultured for 24 h in RPMI

containing anti-CD40 (1 μg ml−1) before exposure to A/WSN/33. OBI B cells, FluBI B cells and MDCK cells were incubated with A/WSN/33 at an MOI of 1.0 for 30 min on ice, washed once with PBS,

and transferred to 37 °C in RPMI (0.2% BSA). At 5 h post infection, cells were washed, permeabilized, fixed and stained using TAMRA-conjugated flu-specific VHHs (1 μg in 50 μl). Infected

MDCK cells were analysed in parallel as a positive control. Cells were analysed by cytofluorometry using a BD Fortessa. POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR

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_et al._ Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus. _Nature_ 503, 406–409 (2013). https://doi.org/10.1038/nature12637 Download citation * Received:

24 May 2013 * Accepted: 04 September 2013 * Published: 20 October 2013 * Issue Date: 21 November 2013 * DOI: https://doi.org/10.1038/nature12637 SHARE THIS ARTICLE Anyone you share the

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