Outflanking immunodominance to target subdominantbroadly neutralizing epitopesDavide Angelettia,b,1, Ivan Kosika, Jefferson J. S. Santosa, William T. Yewdellc, Carolyn M. Boudreaud,e,Vamsee V. A. Mallajosyulaf, Madeleine C. Mankowskia, Michael Chambersg, Madhu Prabhakarang,Heather D. Hickmanh, Adrian B. McDermottg, Galit Alterd, Jayanta Chaudhuric, and Jonathan W. Yewdella,1

aLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; bDepartment ofMicrobiology and Immunology, Institute of Biomedicine, University of Gothenburg, 40530 Gothenburg, Sweden; cImmunology Program, Memorial SloanKettering Cancer Center, New York, NY 10065; dRagon Institute of MGH, MIT, and Harvard University, Cambridge, MA 02139; ePhD Program in Virology,Division of Medical Sciences, Harvard University, Boston, MA 02115; fInstitute for Immunity, Transplantation and Infection, Stanford University, Stanford, CA94305; gVaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and hLaboratoryof Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved May 29, 2019 (received for review September 20, 2018)

A major obstacle to vaccination against antigenically variable virusesis skewing of antibody responses to variable immunodominantepitopes. For influenza virus hemagglutinin (HA), the immuno-dominance of the variable head impairs responses to the highlyconserved stem. Here, we show that head immunodominancedepends on the physical attachment of head to stem. Stem immuno-genicity is enhanced by immunizing with stem-only constructs or byincreasing local HA concentration in the draining lymph node.Surprisingly, coimmunization of full-length HA and stem altersstem-antibody class switching. Our findings delineate strategiesfor overcoming immunodominance, with important implicationsfor human vaccination.

influenza | immunodominance | B cell | hemagglutinin | antibodies

Seasonal influenza remains a significant public health burden,with vaccines requiring frequent reformulation yet providing

limited protection (1, 2). Broadly neutralizing antibodies (Abs)binding viral hemagglutinin (HA) have sparked the hope ofdeveloping a universal influenza vaccine (3). Most Abs target thehighly variable globular head of HA (3–5). The conserved HAstem is much more cross-reactive between strains and a target forprotective Abs, but is poorly immunogenic following infection orvaccination (6). HA and other immunogens activate naïve B cellspresent in lymph nodes (LNs) or spleen. Epitopes with sufficientavidity for B cell receptors (BCRs) trigger signaling events thatlead to B cell seeding of germinal centers (GCs). Here, B cellsproliferate and experience somatic BCR hypermutation andclass-switch recombination. B cell clones with increased BCRavidity for immunogen are selected for proliferation and candifferentiate into antibody-secreting plasma cells and memoryB cells (7).

Although Abs can potentially bind to all surfaces of immunogenicproteins, Ab responses focus on a limited number of immuno-dominant antigenic sites. This phenomenon, termed immunodomi-nance, is just now being defined and mechanistically dissected at thelevel of serum Abs and B cell responses (8). Two recent studiessuggest that B cell precursor frequency and BCR avidity contributeto the subdominance of conserved HIV GP160 epitopes (9, 10).Other studies suggest, however, that after initial B cell seeding, GCsare more permissive than previously thought, allowing B cells withBCRs of even 100-fold differences in avidity to emerge from thesame LN (11, 12). The contribution of these factors to HA stemsubdominance in primary responses and how stem-specific Abs canbe efficiently induced with vaccination in the context of full-lengthHA remain to be determined. Here, we mechanistically dissect de-terminants of B cell immunodominance to HA, providing evidencethat stem subdominance is due to competition between head andstem naïve B cells that can be overcome by simply increasing im-munogen delivery to the draining lymph node.

ResultsStem-Only Immunogen Elicits a Robust GC B Cell Response. To betterunderstand the immunodominance of the HA head domain, weimmunized mice intramuscularly (i.m.; the typical route for humanvaccination) with full-length H1 HA- or stem-only recombinantpurified proteins (13, 14) in different combinations (Fig. 1A).Twenty-one days after a single boost, we quantitated antigen-specificGC B cells in draining LNs, specific for HA head or HA stem, viacombinatorial flow cytometry staining using H1 HA and H5 HA (twoproteins with different heads but semiconserved stems) (SI Appendix,Fig. S1A). GCs formed in the ipsilateral draining LNs in similarnumbers independent of the immunogen (SI Appendix, Fig. S1 B andC). As expected (5, 15, 16), HA i.m. immunization mostly inducedhead-specific GC B cells. Importantly, stem immunization generateda stem-specific B cell response of similar magnitude to the head-specific response, demonstrating that our stem construct is not in-trinsically of low immunogenicity (Fig. 1B). After immunization withHA and stem in separate legs, immunogen-specific B cells developedonly in the ipsilateral LN (Fig. 1C and SI Appendix, Fig. S1 D and E).Notably, this lack of competition in the draining LN also occurredwhen we mixed intact HA and stem (Fig. 1C and SI Appendix, Fig. S1D and E), strongly suggesting that head immunodominance resultsfrom naïve B cell competition for full-length HA. Immunization with

Significance

The most promising target for a universal influenza A vaccine isthe conserved hemagglutinin (HA) stem domain. However, ininfected or immunized individuals, the response to HA stem islimited due to HA head immunodominance. To understandfactors hampering the development of stem B cells, we haveimmunized mice with full-length HA and HA stem alone or indifferent combinations and shown that physical attachment ofHA head to stem severely hinders stem responses. By increasinglocal antigen concentration of full-length HA, we were able torescue stem-specific B cells and serum antibodies. Our resultsprovide a template for improving vaccination by overcomingimmunodominance.

Author contributions: D.A., I.K., C.M.B., G.A., and J.W.Y. designed research; D.A., I.K.,J.J.S.S., W.T.Y., C.M.B., V.V.A.M., M.C.M., M.C., and M.P. performed research; V.V.A.M.,M.C., M.P., H.D.H., A.B.M., G.A., and J.C. contributed new reagents/analytic tools; D.A.,I.K., J.J.S.S., W.T.Y., C.M.B., M.C.M., and J.W.Y. analyzed data; and D.A. and J.W.Y. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: davide.angeletti@gu.se or jyewdell@niaid.nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1816300116/-/DCSupplemental.

Published online June 18, 2019.

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unrelated protein or denatured HA elicited strong GC formation (SIAppendix, Fig. S1F) but no B cells specific to folded HA or stem (SIAppendix, Fig. S1G). To assess the contribution of N-linked glycans inmasking stem subdominance (since the stem-only immunogen is of bac-terial origin, it is not glycosylated), we removed glycans on full-length HA(SI Appendix, Fig. S2A) by endoglycosidase treatment. Despite increasingaccessibility of Abs to stem (SI Appendix, Fig. S2B), we still could notdetect stem-specific GC B cell responses (SI Appendix, Fig. S2C).

Mixing Full-Length HA with Stem-Only Immunogen Affects the Qualityof the Serum Ab Responses. We next measured head- vs. stem-specificserum Abs in four immunization groups (Fig. 2A and SI Appendix, Fig. S3A and B). Stem was highly immunogenic as a standalone immunogen, butfull-length HA failed to elicit a stem response in 11 of 12 mice. Stem Abswere not induced upon immunization with deglycosylated HA (SI Ap-pendix, Fig. S2D). Mice generated an Ab response toward the His tag and/or the foldon tag, albeit generally at much lower levels relative to the

Fig. 1. Immunodominance of the B cell responsesdepends on physical attachment of the HA head tostem. (A) Schematic of the immunization strategy.Group 1 was immunized with full-length HA in theleft hind leg, group 2 with stem in the left hind leg,group 3 with full-length HA and stem in equimolaramounts in the left hind leg, and group 4 with full-length HA in the left hind leg and stem in the righthind leg. (B) Representative flow cytometry plotshowing swIg GC B cells, gated as live CD3− B220+

GL7+ CD38− IgD− IgM−, and ability to bind HA head(H1 single positive), and HA stem (H1+H5+). (C) Enu-meration of head vs. stem swIg GC B cells for the fourdifferent groups 21 d after challenge. Three inde-pendent experiments with 4 mice each (pooled for thefirst experiment; individual for the other 2 experiments)(n = 9). Bars represent mean ± SEM; statistical analysiswas performed using two-way ANOVA with Holm–

Sidak’s multiple comparison test. *P < 0.05, ***P <0.001, ****P < 0.0001.

Fig. 2. Serum immune responses of immunizedmice are impaired uponmixed immunization. (A and B) Antibody end point titers (A) and IgG subclass response (B) toHA head (following stem Ab absorption) and to stem for the different immunization groups (n = 12 for groups 1, 2, and 4; n = 10 for group 3). Bars represent mean ±SEM; statistical analysis was performed using two-way ANOVA with Holm–Sidak’s multiple comparison test. (C) Sera were tested for the ability to induce ADCD onstem-conjugated beads. Data are presented as the area under the curve (AUC) of geometrical mean fluorescence intensity (GMFI) of 1:5 and 1:10 dilutions,and each data point is the mean of two technical replicates. (D) Ability of the sera to induce ADCP on stem-conjugated beads by primary monocytes. Each datapoint is the mean of 2 technical replicates. Three independent experiments with four mice each (n = 12 for groups 1, 2, and 4; n = 10 for group 3). Bars representmean ± SEM; statistical analysis was performed using one-way ANOVAwith Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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specific response to HA (SI Appendix, Fig. S3C). Stem-induced Absbound to H5 HA (SI Appendix, Fig. S3D) and inhibited binding of twowell-characterized anti-stem mAbs (SI Appendix, Fig. S3E) (17, 18). Abtiters only partially correlated with GCB cell numbers (SI Appendix, Fig.S3F), suggesting bottlenecks between GC B cells, plasmacell (PC)differentiation, and serum Ab production. Since anti-stem Ab-based

protection in mice acts via Fc-mediated functions (19), the Ab heavy-chain class is critical in anti-stem Ab-mediated protection. Re-markably, we found that head-specific Abs induced by HA immuni-zation were biased to IgG1 while stem-induced Abs were IgG2-biased,a pattern that was maintained when immunizing with each immuno-gen in opposite legs (Fig. 2B). Importantly, however, stem Abs in-duced by HA/stem mixed immunization did not switch toward IgG2,providing an important potential caveat for mixing immunogens in asingle site. Stem-induced sera were unable to neutralize PR8 in vitro(SI Appendix, Fig. S3G) (13, 19), despite binding cell-surface HA (SIAppendix, Fig. S3H). Rather, in vitro serum neutralization titers highlycorrelated with head-specific Ab ELISA titers (SI Appendix, Fig. S3I).We tested sera for Ab-dependent complement deposition (ADCD) ac-tivity and Ab-dependent cellular phagocytosis (ADCP) by primarymonocytes (20). We found that stem-only immunization inducedhigh levels of stem-dependent ADCD and ADCP, and that mixedimmunization significantly reduced stem-mediated effector functions(Fig. 2 C and D). This revealed a positive correlation with IgG2 anti-stem titers but not total IgG titers (SI Appendix, Fig. S4 A and B). Whensera were tested on H5 HA-conjugated beads, ADCD activity mirroredthe stem results (SI Appendix, Fig. S4C); however, H1 HA ADCD andADCP results highlighted the important contribution of anti-head Abs(SI Appendix, Fig. S4 D and E). Taken together, these findings indicatethat mixing immunogens at the same immunization site or immunizingat distant sites can influence the magnitude and quality of Ab responses.

Stem Abs in Serum Are Durable and Not Influenced by Immunizationwith Full-Length HA. Most of the human population has serum Abstoward the variable head. We therefore wanted to test whether micewith preexisting immunity to full-length HA would be able to mounta sizable immune response after stem-only immunization. We alsowanted to determine the durability of stem-specific Ab responsesafter full-length HA challenge. Therefore, we primed mice with full-length HA or stem constructs and boosted after 21 d with stem orfull-length HA, respectively. In both cases, we obtained robust GCresponses in the draining iliac LN, with the majority of B cells beingspecific for the boosting protein (SI Appendix, Fig. S5A). Impor-tantly, after stem priming and HA boosting, levels of stem-specific Bcells in GCs were higher than after stem immunization. Remarkably,stem Abs in serum were comparably high between the two groups (SIAppendix, Fig. S5B), albeit at a lower level compared with stem-onlyimmunization (see Fig. 2A for comparison). Taken together, these

Fig. 3. Naïve B cell precursor frequency is similar between head and stem.(A) Representative flow cytometry plot showing background stain of naïve Bcells (CD3− CD43− B220+) on streptavidin (SA) conjugated to PE and APC andused in combination and staining of the same cells with H1 HA. (B) Gatingstrategy for total mature naïve B cell population (CD3− CD43− B220+), andfollicular (FO) (CD3− CD43− B220+ CD23+ CD21low IgMlow IgDhigh) and mar-ginal zone (MZ) (CD3− CD43− B220+ CD23− CD21l+ IgMhigh IgDlow) B cells.Overlay shows IgM and IgD expression in FO vs. MZ B cells. (C) Precursorfrequency as % of parent population for mature naïve, FO, and MZ B cells.(n = 8). Three independent experiments with 2 or 4 mice each. Bars representSEM; statistical analysis was performed using two-sided unpaired t test.

Fig. 4. Early B cell affinity is different for head vs. stem. (A) Representative flow cytometry plot showing total (CD3− B220+ GL7+ CD38−), swIg (CD3− B220+

GL7+ CD38− IgD− IgM−), and IgM (CD3− B220+ GL7+ CD38− IgD− IgM+) GC B cells and the gating selection for the AC50 at 10 d post HA or stem i.m. im-munization. (B) Pooled iliac LNs from three mice at 10 d after immunization with full-length H1 HA (orange) or stem (blue) were stained with graded amountsof H1 HA (orange) or H5 HA (blue) and plotted against the frequency of GC B cells stained (n = 3). (C) Nanomolar concentration of HA giving half-maximalbinding (AC50) was derived from individual experimental curves (n = 3). (D) MFI of HA+ B cells at the highest concentration (66 nM) at 10 d postimmunization.Three independent experiments with 3 pooled LNs each. Bar graphs represent mean and bars represent SEM; statistical analysis was performed using two-sided unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.

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data show that serum stem Abs are durable and that after stempriming, stem-specific B cells are present in the GC after boostingwith full-length HA.

Head- and Stem-Specific Naïve B Cells Exhibit Comparable Frequencies.Why does the physical attachment of the head to the stem suppress Abresponses to the stem?We hypothesized that head immunodominanceresults from sequestration of full-length HA by naïve head-specific Bcells, preventing stem-specific B cell activation due to differences innaïve B cell numbers or BCR avidity (8, 9). We first measured thefrequencies of head- and stem-specific naïve B cells. Using a methoddeveloped for quantitating naïve T cells (21), we detected naïve HA-specific B cells at low frequencies, despite their low avidity. HA-specificB cells were detected by double staining with the same probe labeledseparately with different fluorophores (Fig. 3A). Double stainingallowed for the exclusion of naïve B cells specific for the fluorophores.This revealed that HA and stem precursor frequencies are similar:∼250/300 cells per million naïve mature B cells (Fig. 3 B and C), or∼15,000 cells per mouse, given a naïve B cell population of ∼5 × 107

cells (22).

Early Head-Specific GC B Cells Have Higher-Avidity BCRs. We nextmeasured GC B cell avidity at day 10 after full-length HA or stemimmunization using the AC50 method (23). This was the first daypostimmunization with sufficient expansion of GC B cells to identifyantigen-specific class-switched (swIg) and IgM cells in draining LNs(Fig. 4A). Consistent with our prior findings (23), head-specific Bcells exhibited AC50 values in the low-nanomolar range. Critically,the avidity of stem-specific B cells was ∼10-fold lower (Fig. 4 B and

C). Consistent with their higher avidity, the median fluorescenceintensity of HA+ cells was significantly higher for head-specific Bcells (Fig. 4D). By 3 wk postboosting, the avidity of both head- andstem-specific GC B cells had increased 5- to 20-fold, as expectedfrom affinity maturation (SI Appendix, Fig. S6).

Modifying the Immunization Route Overcomes Stem Subdominance.Previous studies suggested that GCs are more permissive in allowingentry of B cells with a wide range of affinities (11, 12). Importantly,these findings derived from footpad (f.p.) immunization, while weused i.m. immunization. To determine how the route of immunizationinfluences antigen concentration in the draining LN, we immunizedmice i.m. or f.p. with 10 μg R-phycoerythrin (PE) in adjuvant and 24 hlater measured the amount of fluorescent PE present in LN extracts(Fig. 5A). Strikingly, after f.p. immunization, we could detect PE in78% of the popliteal LNs and 61% of the iliac LNs tested, while afteri.m. immunization, PE was detectable only in 22% of the LNs analyzed(for both inguinal and iliac) (P = 0.0022; Fisher’s exact test). Theaverage amount of PE detected (with our limit of detection being0.1 ng/mL) in the popliteal LN after f.p. immunization was at least14- and 30-fold higher, respectively, than in draining inguinal andiliac LNs after i.m. immunization. We next immunized mice withvirion-derived H1 HA either f.p. or i.m. (with a dose of 10 μg), ori.m. with a dose of 25 μg followed by an additional 50 μg on day 1 andon day 2, to increase immunogen concentration in the LN (repeatedi.m.). Three weeks postimmunization, GC frequencies in the iliac LNwere similar but nodal cellularity significantly expanded with increasingantigen dose, with the average number of GC B cells per LN comparedwith i.m. immunization nearly doubling or tripling, respectively, with f.p.

Fig. 5. Stem subdominance can be subverted by increasing local antigen concentration. (A) Amount of PE detected in draining LNs following f.p. or i.m.immunization (n = 17–18). Boxplot showing median and min to max. Statistical analysis was performed using one-way ANOVA with Tukey’s multiplecomparison test. (B and D) Representative flow cytometry plots showing swIg GC B cells (gated as live CD3− B220+ GL7+ CD38− IgD− IgM−) (B) and IgM GC Bcells (gated as live CD3− B220+ GL7+ CD38− IgD− IgM+) (D), and ability to bind HA head (H1 HA single positive) and HA stem (H1+H5+) after i.m., footpad, orrepeated i.m. immunization. (C and E) Enumeration of head vs. stem swIg GC B cells (C) and IgM GC B cells (E) for the three different groups 21 d afterimmunization (n = 12 for i.m. and repeated i.m.; n = 24 for f.p.). (F) Antibody end point titers for total Ig, IgG, and IgM to H1-PR8 or stem for the differentimmunization groups. For f.p., red symbols indicate mice that are stem-seropositive, while open pink indicates stem-seronegative (“low”). Three independentexperiments with 4 mice each for i.m. and repeated i.m., and 5 independent experiments with 5 or 4 mice each for f.p. immunization. Bars represent mean ± SEM;statistical analysis was performed using two-way ANOVA with Holm–Sidak’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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or repeated i.m. immunization (SI Appendix, Fig. S7 A–C). Interestingly,IgM+ GC B cells increased to nearly equal to switched Ig after repeatedi.m. immunization (SI Appendix, Fig. S7B). This suggests both sustainedrecruitment of B cells to the GC due to increased immunogen deliveryand extended cycling of IgM+ GC B cells that are not able to receiveadequate signals from T follicular helper cells to exit the GC reaction.All immunization groups generated large numbers of head-specificIgG+ and IgM+ GC B cells on day 21. In contrast, i.m. immunizationgenerated a low number of stem-specific IgG+ and IgM+GCB cells andundetectable stem-specific serum responses. F.p. immunization in-creased stem-specific switched B cells 100-fold compared with i.m. (Fig.5 B and C and SI Appendix, Fig. S7 D and E), comparable to stem-onlyimmunization (Fig. 1C). Repeated i.m. immunization also increasedswitched and IgM+ stem-specific B cells (Fig. 5 D and E).

We were unable to detect IgG or IgM anti-stem Abs in i.m. immu-nized mice (Fig. 5F). Footpad immunization generated stem-specificIgG and IgM serum responses in the ∼50% of mice with the highestlevels of GC B cells (on average 20× more abundant than seronegativemice; Fig. 5C). This is consistent with a functional threshold of GCresponses to generate sufficient numbers of plasma cells for a detectableserum Ab response. Based on a cell-surface phenotype by flow cytom-etry, head- and stem-specific GC B cells had a similar dark zone/lightzone distribution (Fig. 6 A and B). Consistent with the B cell analyses,repeated i.m. immunization elicited significant levels of H5 cross-reactiveIgG in 3/12 mice, although 8/12 mice generated IgM responses. Althoughstem titers only partially correlated with H5 titers (SI Appendix, Fig. S8A),all H5 reactivity was removed by competition with anti-stem mAbs. Im-munization route also shaped the GC B cell immunodominance hierarchy

among the five major head antigenic sites (SI Appendix, Fig. S8 B andC). At the most extreme, Ca1-specific responses nearly doubled in f.p.vs. i.m. immunization, consistent with our previous findings of routedependence of immunodominance (16). Viral challenge studies withheterologous virus, of animals immunized one single time, revealed atendency toward higher protection after stem i.m. but no differencebetween the other groups (SI Appendix, Fig. S8D), indicating the needfor further boosting to achieve protection. Finally, we determined theinfluence of f.p. vs. i.m. HA immunization on the ratio of T follicularregulatory (Tfr) cells to T follicular helper (Tfh) cells, which has beenreported to regulate Ab responses (24). Although f.p. immunizationgenerated higher frequencies of both Tfh and Tfr cells compared withi.m. (Fig. 6 C–F), the Tfr/Tfh ratio was doubled after i.m. immuni-zation, suggesting an additional contribution to the suppression ofstem-specific responses (Fig. 6D).

DiscussionThere is great hope for broad antiinfluenza vaccination based oninducing stem Abs, which cross-neutralize in vitro and cross-protectanimals. However, in humans, despite repeated seasonal infectionand immunization, anti-stem plasma cells are transient in blood andserum Abs only rarely achieve a likely functional threshold. Notably,anti-stem memory B cells seem to be common in humans but canrarely outcompete anti-head memory B cells after vaccination (6).Interestingly, with the introduction of the H1N1 2009 pandemic,where the HA head was remarkably different from previous in-fluenza seasons, there was a more sustained reactivation of stem-specific memory B cells (25). Here, we investigated the ability ofstem-only immunogens to elicit functional B cell and Ab responsesand mechanistically dissected the subdominance of the stem in thecontext of full-length HA immunization.

The stem-only construct we used elicited a robust anti-stem GC Bcell and Ab response, suggesting that stem subdominance in HA isdependent on its physical attachment to the globular domain. Usingdeglycosylated HA, we show that the lack of glycosylation of thebacterially synthesized stem construct is not responsible for itsgreatly enhanced immunogenicity vs. stem in full-length HA (SIAppendix, Fig. S2). This is consistent with reports that stem immu-nogenicity is enhanced by blocking head epitopes with glycans (26)but not by removing stem glycans from intact HA (27). Althoughmixing stem with HA does not interfere with head-specific re-sponses, it reduces stem-specific polyclonal titers 10-fold and inter-feres with class switching, decreasing anti-stem ADCD and ADCP.This suggests possible competition for help from T follicular cells(28) and follicular dendritic cells (29) essential for PC differentia-tion. Diminished immunogenicity from mixing of viral vaccines (30,31) or following immunization in different sites (32) clearly occurs inhumans, but had not previously been mechanistically dissected.

We show that head- and stem-specific B cells are present atsimilar precursor frequencies and the major detectable differencelies in B cell avidity which, due to limitations in B cell numbers, wecan first measure at 10 d postimmunization. Recent reports showedthat responding GC B cells can secrete mAbs with 10- to 100-folddifferences in avidity for immunogen (11, 12), which is at odds withour findings. This discrepancy may be due to differences in meth-odology in the studies. First, while we directly measure BCR avidity,the other studies use soluble mAbs. Therefore, BCR multivalencymight contribute to the higher avidity we measure. Second, by im-munizing with stem only, we allow stem B cells to affinity maturewithout competition for T cell help, which may allow them to attain ahigher affinity. Further, a study shows that only higher-affinity pre-cursors can enter GCs for monomeric antigen (as the ones usedhere) and, even for multimeric antigen, a 30-fold naïve aviditydifference was sufficient to exclude low-avidity B cells from GCs(9). Finally, we cannot completely exclude the influence of compe-tition events at early GC stages, related to competition for limitedT cell help.

While elegant previous studies used transgenic mice to evaluatethe contribution of B cell precursor frequency and avidity to B cellmaturation (9, 10), they did not measure serum Abs, the key func-tional output of the humoral immune response. Here, after footpad

Fig. 6. Footpad immunization does not shift stem GC B cells to DZ but altersTfr/Tfh ratio. (A) Representative flow cytometry plot showing dark zone (DZ)(CD3− B220+ GL7+ CD38− IgD− IgM− IgG+ CXCR4+ CD86−) vs. light zone (LZ)(CD3− B220+ GL7+ CD38− IgD− IgM− IgG+ CXCR4− CD86+) distribution of head-or stem-specific GC B cells at 11 d after f.p. immunization. (B) Quantification ofthe frequency of head or stem GC B cells in the DZ (n = 9). Two independentexperiments with 5 and 4 mice each. Bars represent SEM; statistical analysiswas performed using two-way unpaired t test. (C) Gating strategy for T fol-licular (Tf) cell population (B220− CD4+ CXCR5+ PD1+) and further classificationinto T follicular helper (Tfh) and T follicular regulatory (Tfr) cells based on FoxP3expression. (D) Quantification of Tfr contribution to total Tf (CD4+ CXCR5+ PD1+)(Tfr/Tfh ratio) in draining LNs (iliac for i.m. and popliteal for f.p.) 8 d post-immunization. (E and F) Tfh (E) and Tfr (F) expressed as frequency of CD4+ T cells(n = 10 for i.m.; n = 7 for f.p.). Two independent experiments with 5 mice eachfor i.m. andwith 4 and 3mice for f.p. immunization. Bars represent mean ± SEM;statistical analysis was performed using two-sided unpaired t test. *P < 0.05,**P < 0.01, ****P < 0.0001.

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immunization, we show that while all of the mice demonstrated asizable stem-specific GC B cell population, not all exhibited de-tectable levels of stem Abs in sera. In mice, we could bypass affinity-based head immunodominance by immunizing in a small anatomicspace (the footpad) draining to a single lymph node. In humans,practical realities would dictate either single immunization with asubdominant immunogen, immunization in different sites, or engi-neering subdominant immunogens to increase LN delivery, either bymodifying delivery (e.g., needle patches or slow-release vaccines) orattaching immunogens to particles that enhance delivery to B cellareas of draining LNs (33).

In summary, our findings demonstrate that the route of immuni-zation governs the immunodominance of the head vs. the stem re-gion. Stem subdominance is driven by differential B cell affinityunder conditions of limiting immunogen. To circumvent immuno-domination, immunizing with a subdominant protein is a promisingapproach for universal vaccination.

Materials and MethodsDetailed methods can be found in SI Appendix, Text.

Animals. C57BL/6 mice were purchased from Taconic Farms. For all experi-ments, female 8- to 12-wk-old mice were used and randomly assigned toexperimental groups. All mice were held under specific pathogen-free con-ditions. All animal procedures were approved and performed in accordancewith National Institute of Allergy and Infectious Diseases (NIAID) Animal Careand Use Committee guidelines.

Proteins and Immunization. Recombinant A/Puerto Rico/8/34 (PR8) HAwith theY98F mutation (18) and H1HA10-foldon stem-only construct derived from

PR8 (14) were used for the initial immunization studies. Groups 1 and 4received 10 μg of HA (0.8 μM) in the left hind leg, group 2 received 4 μg ofstem (0.8 μM) in the left hind leg, while group 4 received the same amountin the right hind leg. Group 3 received an equimolar mixture of the twoproteins in the left hind leg. Other animals were immunized i.m. or via thehock f.p. with 10 μg of virion-derived HA (0.8 μM) mixed with adjuvant. Forrepeated i.m. immunization, animals were immunized i.m. with 25 μg of HAwith adjuvant on day 0, followed by 50 μg of HA without adjuvant on day 1and 50 μg of HA without adjuvant on day 2.

Statistical Analysis. Prism (GraphPad Software) was used for statistical anal-ysis. For comparison between two groups, two-sided unpaired Student’s t testwas performed. For comparison of one variable between multiple groups,one-way ANOVA with Tukey’s multiple comparison test was performed. Forcomparison between multiple variables across multiple groups, two-wayANOVA with Holm–Sidak’s multiple comparison test was performed. Whendata did not pass the normality test, they were log-transformed. For all fig-ures, data points indicate individual mice. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001.

ACKNOWLEDGMENTS. We thank the NIAID Comparative Medicine Branchfor maintaining the mice used in this study, and Raghavan Varadarajan(Molecular Biophysics Unit, Indian Institute of Science) for assistance withstem protein design and production. This work is supported by the Divisionof Intramural Research, National Institute of Allergy and Infectious Diseases.D.A. was partially supported by a grant from the Swedish Research Council(Vetenskapsrådet 2017-01439) and the Institute of Biomedicine at the Univer-sity of Gothenburg. W.T.Y. was supported by a Special Fellow award from theLeukemia & Lymphoma Society and an NIH T32 training grant (CA009149). J.C.was supported by grants from the National Institutes of Health (1R01AI072194,1R01AI124186, and P30CA008748).

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