Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve … · 2019. 4. 16. · 229 230 231...

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ORIGINAL RESEARCHpublished: xx March 2019

doi: 10.3389/fimmu.2019.00611

Frontiers in Immunology | www.frontiersin.org 1 March 2019 | Volume 10 | Article 611

Edited by:

Aurelio Cafaro,

Istituto Superiore di Sanità (ISS), Italy

Reviewed by:

Michelle Angela Linterman,

Babraham Institute (BBSRC),

United Kingdom

Andreas Hutloff,

Deutsches Rheuma-

Q2

Forschungszentrum

(DRFZ), Germany

*Correspondence:

Stephen J. Kent

[email protected]

Adam K. Wheatley

[email protected]

Specialty section:

This article was submitted to

Viral Immunology,

a section of the journal

Frontiers in Immunology

Received: 21 January 2019

Accepted: 07 March 2019

Published: xx March 2019

Citation:

Tan H-X, Esterbauer R,

Vanderven HA, Juno JA, Kent SJ and

Wheatley AK (2019) Inducible

Bronchus-Associated Lymphoid

Tissues (iBALT) Serve as Sites of B

Cell Selection and Maturation

Following Influenza Infection in Mice.

Front. Immunol. 10:611.

doi: 10.3389/fimmu.2019.00611

Inducible Bronchus-AssociatedLymphoid Tissues (iBALT) Serve asSites of B Cell Selection andMaturation Following InfluenzaInfection in Mice

Q1Hyon-Xhi Tan1, Robyn Esterbauer 1, Hillary A. Vanderven2, Jennifer A. Juno1,Stephen J. Kent 1,3,4* and Adam K. Wheatley 1*

1 Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and

Immunity, Melbourne, VIC, Australia, 2 Biomedicine, College of Public Health, Medical and Veterinary Sciences, James Cook

University, Douglas, QLD, Australia, 3 Melbourne Sexual Health Centre and Department of Infectious Diseases, Alfred Hospital

and Central Clinical School, Monash University, Melbourne, VIC, Australia, 4 ARC Centre for Excellence in Convergent

Bio-Nano Science and Technology, University of Melbourne, Parkville, VIC, Australia

Seasonally recurrent influenza virus infections are a significant cause of global morbidity

and mortality. In murine models, primary influenza infection in the respiratory tract elicits

potent humoral responses concentrated in the draining mediastinal lymph node and the

spleen. In addition to immunity within secondary lymphoid organs (SLO), pulmonary

infection is also associated with formation of ectopic inducible bronchus-associated

tissues (iBALT) in the lung. These structures display a lymphoid organization, but their

function and protective benefits remain unclear. Here we examined the phenotype,

transcriptional profile and antigen specificity of B cell populations forming iBALT in

influenza infected mice. We show that the cellular composition of iBALT was comparable

to SLO, containing populations of follicular dendritic cells (FDC), T-follicular helper (Tfh)

cells, and germinal center (GC)-like B cells with classical dark- and light-zone polarization.

Transcriptional profiles of GC B cells in iBALT and SLO were conserved regardless of

anatomical localization. The architecture of iBALT was pleiomorphic and less structurally

defined than SLO. Nevertheless, we show that GC-like structures within iBALT serve as a

distinct niche that independently support the maturation and selection of B cells primarily

targeted against the influenza virus nucleoprotein. Our findings suggest that iBALT, which

are positioned at the frontline of the lung mucosa, drive long-lived, and unique GC

reactions that contribute to the diversity of the humoral response targeting influenza.

Keywords: germinal center (GC), influenza, iBALT, B cell, humoral immunity

INTRODUCTION

The generation of adaptive immune responses to infection requires the complex and elegant Q5

coordination of T and B lymphocytes, concentrated within secondary lymphoid organs (SLO), suchas the spleen and lymph nodes. SLO are highly organized and regulated structures, facilitatingantigen concentration, capture and presentation to naïve lymphocytes, thereby facilitating the

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Tan et al. B Cell Characterization in iBALT

activation, and differentiation of T and B cells into effectorpopulations. Further specialized zones of germinal centers (GC)drive maturation of the humoral response via the selection ofhigh affinity antibodies, providing an effective source of long-lived protection in the form of serum antibody and antigen-experienced memory B and T cell populations.

While SLO develop during embryogenesis at discreteanatomical locations (1), the induction of immunologicalstructures resembling SLO has been reported at peripheral sitesin response to inflammatory stimuli or infection with pathogenicmicroorganisms (2–5). Variably described as ectoptic lymphoidtissue or tertiary lymphoid organs, such lymphocyte aggregationsalso tend to form proximal to the bronchi within the lung and aretermed inducible bronchus-associated lymphoid tissues (iBALT).Induction of iBALT has been extensively reported followingpulmonary infections by bacterial (6, 7) or viral agents (8–10).iBALT can also form in response to exposure to particulates (11,12) or pro-inflammatory mediators (13, 14), and chronic allergicor inflammatory diseases such as COPD and asthma (15). Despiteconservation across numerous species and a wide number ofcontexts, the contribution of iBALT to humoral immunity againstinfluenza is not fully understood.

While it is unlikely that a single pathway for iBALTgenesis exists given the wide range of originating stimuli, thedevelopmental models proposed for ectoptic lymphoid tissueshave commonalities (16). Based on these models, cytokine andchemokine production from innate cells such as ILCs andγδT cells recruit pro-inflammatory granulocyte populations,which in turn drive the CXCL13-dependent recruitment ofmature B cells into the lung (14, 17–19). Similar to themaintenance of conventional lymphoid tissues, T and B cellssupport the differentiation of stromal cells into mature folliculardendritic cells (FDC) and fibroblastic reticular cells (FRC),with homeostatic maintenance allowing these lymphoid-likestructures to persist for months in the absence of ongoinginflammation or infection (20–22). While iBALT is decidedlypleiomorphic (16), several groups have reported the presenceof B-cell clusters, networks of FDC, T cell areas, and zonesresembling germinal centers, with iBALT supporting theproliferation of both B and T cells (8–10, 13, 23). Notably,iBALT can initiate protective humoral and T cell responsesfollowing influenza infection in mice lacking SLO (8, 9),although the relative importance of lung-generated responses inimmunocompetent animals is unclear.

Using the influenza infection model in mice, we sought toaddress two questions. First, to what extent do the GC-likestructures in the lung resemble GC in SLO? In particular, doiBALT GC-like structures facilitate B cell selection and affinitymaturation? Second, to what extent is the lung a primingsite for humoral immunity to pulmonary influenza infectionin immunocompetent animals? We show GC responses ininfluenza infection-induced iBALT were comparable to GCin SLO in terms of cellular composition, phenotype, andtranscriptional profile. However, the architecture of iBALTGC was non-classical, lacking discrete light and dark zonepolarization and T cell compartmentalization to the light zone.Nevertheless, using labeled recombinant influenza antigens

to probe B cell specificities, we find that influenza-specificGC responses within iBALT predominantly targeted the viralnucleoprotein, constituted a unique clonal repertoire relative toSLO, and demonstrated a comparable capacity to drive somaticmutagenesis and B cell selection requisite for affinity maturation.Our work has implications for the development of improvedhumoral immunity to influenza by establishing long-lived andunique GC reactions at the frontline of the lung mucosa.

MATERIALS AND METHODS

Animal InfectionAll animal procedures were approved by the University Q7

of Melbourne Animal Ethics Committee. C57BL/6 mice at6–10 weeks of age were used. Mice were anesthetizedby isoflurane inhalation prior to infection. For intranasalinfections, mice were instilled with 50 µL volume of 50TCID50 of A/Puerto Rico/8/34 (PR8).

Confocal MicroscopyFresh tissues were snap-frozen in O.C.T. compound (SakuraFinetek USA) and stored at −80◦C. Tissues were sectioned at7µm thickness (Leica). Prior to staining, sectioned tissues werefixed in cold acetone solution (Sigma) for 10min then rehydratedwith PBS for 10min and blocked with 5% (w/v) bovine serumalbumin (Sigma) and 2% (v/v) normal goat serum (NGS).Cell staining was performed using the following antibodies:IgD (11-26c.2a; Biolegend), B220 (RA3-6B2; BD), GL7 (GL7;Biolegend), CD35 (8C12; BD), CD3 (17A2; Biolegend), CD169(3D6.112; BD), CD86 (GL1, Biolegend), CXCR4 (2B11; BD),CD4 (GK1.5; Biolegend), Ki67 (11F6, Biolegend), BCL6 (K112-91; BD). To amplify the CXCR4-PE signal, tissues were stainedsequentially with rabbit anti-PE antibody (polyclonal; NovusBiologicals) and a secondary goat anti-rabbit IgG Alexa Fluor555 antibody (polyclonal; Life Technologies). Slides were sealedwith ProLong Diamond Antifade Mountant (Life Technologies).Tiled Z-stack images covering were captured on a Zeiss LSM710microscope. Post-processing of confocal images was performedwith ImageJ v2.0.0.

HA ProteinsRecombinant HA protein for use as flow cytometry probeswas derived for A/Puerto Rico/08/1934 as previously described(24). Briefly, expression constructs were synthesized (GeneArt)and cloned into mammalian expression vectors. Proteinswere expressed by transient transfection of Expi293 (LifeTechnologies) suspension cultures and purified by polyhistidine-tag affinity chromatography and gel filtration. Proteins werebiotinylated using BirA (Avidity) and stored at −80◦C.Prior to use, biotinylated HA proteins were labeled by thesequential addition of streptavidin (SA) conjugated to BV421,phycoerythrin (PE), or allophycocyanin (APC), and stored at4◦C. Recombinant influenza A H1N1 NP protein (11675-V08B;Sino Biological) was labeled with PE or APC fluorochromes usingcommercial conjugation kits, as per manufacturer’s protocol(AB102918, AB 201807; Abcam).


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Flow CytometryFor experiments requiring discrimination of blood and tissuepopulations, mice were intravenously labeled with 3 µg ofCD45.2 antibody (104; eBioscience) in a 200 µL volume priorto tissue collection. Tissues were mechanically homogenizedinto single cell suspensions in RF10 media (RPMI 1640, 10%FCS, 1 × penicillin-streptomycin-glutamine; Life Technologies).Except for MLN, red blood cell lysis was performed withPharm LyseTM (BD). Isolated cells were Fc-blocked with aCD16/32 antibody (93; Biolegend). For all B cell experiments,cells were surface stained with Live/dead Aqua viability dye(Thermofisher) and B220 (RA3-6B2; BD), IgD (11-26c.2a;BD), CD45 (30-F11, BD), GL7 (GL7; Biolegend), CD38 (90;Biolegend), Streptavidin (BD), CD3 (145-2C11; Biolegend),and F4/80 (BM8; Biolegend). For LZ/DZ experiments cellswere also stained with CD86 (GL1, Biolegend) and CXCR4(2B11; BD). Antigen-specific B cells were detected basedupon dual staining with HA or NP probes conjugated toAPC and PE. For Tfh cell characterization, cells were stainedin Transcription Factor Buffer Set (BD) with Live/deadRed viability dye (Life Technologies), BCL6 (IG191E/A8;Biolegend), CD3 (145-2C11; Biolegend), PD-1 (29F.1A12;Biolegend), CXCR5 (L138D7; Biolegend), CD4 (RM4-5; BD),B220 (RA3-6B2; BD), and F4/80 (T45-2342; BD). Stainedcells were washed twice and fixed with 1% formaldehyde(Polysciences) for acquisition on a BD LSR Fortessa, orresuspended in Optimem (Life Technologies) for sorting on aBD FACSAria III.

RNAseq and Data AnalysisSingle cell suspensions were processed and stained as describedabove. Memory B cells (B220+ IgD- CD38hi GL7-), GCB cells (B220+ IgD- CD38lo GL7+), and T cells (CD3+)were sorted into RLT buffer (Qiagen) containing 0.14Mβ-mercaptoethanol (Sigma). Post-sort, cells were suspendedin a volume of RLT buffer representing 3.5x the sortingsolution volume. Genomic DNA was removed using a gDNAeliminator (Qiagen), according the manufacturer’s protocol.RNA was extracted by addition of 100% ethanol in a 5:7volume ratio to flow-through solution and processed withthe RNeasy Plus Micro Kit (Qiagen), according to themanufacturer’s instructions. The Australian Genome ResearchFacility (Melbourne, Australia) performed the RNAseq withan Illumina HiSeq 2500 and obtained 100bp single reads.The library was prepared with a TruSeq Stranded mRNALibrary Prep Kit (Illumina). RNAseq analysis was performedusing the web-based Galaxy platform maintained by MelbourneBioinformatics (25). Reads were mapped to the Mus musculusreference genome (mm10) using HISAT2 (26) and reads werequantified using HTSeq (27). Count matrices were generatedand inputted into Degust (http://degust.erc.monash.edu) fordata analysis and visualization with the Voom/Limma methodselected for data processing. Heat maps were generated usingMorpheus (The Broad Institute; https://software.broadinstitute.org/morpheus/). Raw sequence reads can be accessed with GEOcode: (pending approval).

Sequencing and Analysis of Murine B CellReceptor GenesSequencing of murine heavy chain immunoglobulin sequenceswas performed as previously described (28). Briefly, single Bcells stained with the panel above and NP-PE or HA-BV421probes were sorted into 96-well plates and cDNA preparedusing SuperScript III Reverse Transcriptase (Life Technologies)and random hexamer primers (Life Technologies). Heavy chainimmunoglobulin sequences were amplified by nested PCR usingHotStar Taq polymerase (Qiagen) and multiplex primers bindingV-gene leader sequences or the immunoglobulin constantregions. PCR products were Sanger sequenced (Macrogen) andVDJ recombination analyzed using High V-QUEST on IMGT(29). Clonality was determined based upon shared gene usage andCDR-H3 length and sequence similarity. Circular layout graphicswere generated using the Circlize package in R (30).

StatisticsData are generally presented as the median ± interquartilerange or the mean ± SD. Flow data was analyzed in FlowJo v9(FlowJo) and all statistical analyses were performed using Prismv7 (GraphPad).

RESULTS

Dynamics of iBALT Induction FollowingIntranasal Influenza InfectionTo study lung B cell responses to influenza, we first performedintranasal infection of mice with A/Puerto Rico/08/1934 (PR8)virus. Consistent with previous reports (8, 10, 31, 32),intranasal infection resulted in a pronounced infiltration ofB cells into the lung. Lung-infiltrating B cells, distinguishedfrom blood-circulating populations by intravenous CD45.2labeling, displayed peak numbers 14 days (d14) post-infectionand persisted up to d112 (Figure 1A; gating Figure S1). Asubpopulation of B cells expressing a GC-like phenotype (B220+IgD- GL7+ CD38lo) were evident in lungs at d14 post-infection and detectable up to d112, albeit waning at thelatter timepoint (Figure 1B). In comparison, mediastinal LN(MLN) displayed the largest proportion of GC B cells withcontinued maintenance at high frequencies up to d112 post-infection, in line with our previous observations (33). SplenicGC B cells frequencies peaked at d14, rapidly waned andwas proportionally lowest amongst the tissues analyzed fromd28 onward.

When visualized using confocal microscopy, lung tissuesfrom infected mice similarly showed B cell infiltration andclustering (Figure 1C). Although B cell infiltration was observedat d7, cells were not structurally organized and were mostlydispersed around the bronchi regions. GC-like structures,demarcated by GL7 staining, were apparent at d14, peakingin size between d28 and d56, and subsequently waning insize by d112. When analyzed by flow cytometry, lung Bcells gradually acquired a canonical GC phenotype in latertimepoints, with full differentiation into GL7+ CD38lo and peakabsolute numbers of GC B cells observed by d35 (Figure S2).


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FIGURE 1 | iBALT formation and characterization following intranasal influenza infection in mice. (A) Lung B cell infiltration (B220+ intravenous (IV) CD45.2-) and (B)Q4

Q3 frequency of GC B cells (B220+ IgD- CD38lo GL7+) across various tissues were measured in mice infected intranasally with A/Puerto Rico/08/1934. Data representtwo independent experiment (n = 6). Error bars represent mean ± SD. (C) Induction and maturation of iBALT across various time-points visualized by composite

images comprising B220 (orange), IgD (gray), GL7 (green), and CD35 (cyan); scale bar−100µM. (D) GC cellular composition of lungs and spleen visualized by

immunofluorescence staining at d35 post-infection; scale bar−50µM. (E) Frequency and (F) visualization of light- and dark-zone GC B cells in lungs and SLO at d35

post-infection. Data represent a single experiment (n = 6). Error bars represent mean ± SD. Scale bar−50 µM.



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As mature persistent GC responses are well-established inboth iBALT and the draining MLN by d35, we selectedthis as a suitable timepoint for subsequent GC analysis. Wecontrasted the structural characteristics of putative lung GC withcanonical splenic GC from infected mice at d35 post-infection(Figure 1D). Despite a pleiomorphic appearance (Figure S3),both FDC (CD35+) and highly clustered B cells (B220+)expressing GL7 were detected in iBALT analogous to thespleen. In contrast, T cells (CD3+) were dispersed throughoutthe lung tissues and CD169 staining, which demarcatessubcapsular sinus macrophages in the spleen, was negligible iniBALT structures.

GL7 expression in SLO is tightly linked to GC localization(34). The distributed GL7 staining throughout iBALT ledus to question if the observed GC-like B cell structureswere functionally analogous to actual germinal centers. Wetherefore examined the differential expression CXCR4 andCD86,previously described for the delineation of dark- and light-zoneresident cells in both human and murine GC (35). GC B cellsfrom the lung displayed the characteristic differential expressionprofiles of CXCR4 and CD86, analogous to staining in MLNand spleen (Figure 1E). However, CXCR4 and CD86 stainingvisualized by immunofluorescence microscopy on lung residentB cell clusters was diffuse, lacking the discrete localization oflight- and dark-zone staining seen in splenic samples (Figure 1F).Light and dark zone polarization requires differential chemokineexpression by stromal cells, including FDC (36). Comparedto the expected focused and GC-polarized FDC staining inMLN and spleen, FDC staining within the lung was diffusewith little indication of light-zone localization (Figure S4). Thus,while GC-like structures within influenza-induced iBALT displaymany phenotypic characteristics consistent with conventionalGC within SLO, they appear morphologically unconventional.

Transcriptional Profile of GC-Like B CellsFrom iBALTTo facilitate an in-depth comparison of GC B cells fromdifferent anatomical sites, memory, and germinal center B cellswere sorted from the lung, spleen, MLN and blood of miceat d35 post-infection (gating Figure S1), and differential geneexpression profiles were generated by RNASeq. Based uponmulti-dimensional scaling, iBALT GC-like B cells clusteredclosely with GC B cells from SLO and were distinct from bothmemory B cell populations and T cell controls (Figure 2A).This was similarly evident within a heatmap generated usingthe top 200 differentially expressed genes, where memory andGC-like populations displayed discrete expression profiles acrosslung-, MLN-, and spleen-localized cells (Figure 2B). Notably,iBALT GC-like B cells, but not memory B cells, expressed themaster transcription factor bcl6, and canonical GC B cell markersfas, aidca, gcsam, and s1pr2 in a manner comparable to GC Bcells from SLO (Figure 2C). Conversely, we observed limitedexpression of genes associated with the memory phenotype inGC B cell populations (ccr7, s1pr1, ly6d, cd38, sell). Consistentwith previous BrdU incorporation data (8), iBALTGC-like B cellsappeared to be proliferating (expressing ki67).

iBALT-Resident Tfh Cells Express Bcl6and Ki67Follicular T helper cells (Tfh) are critical for the generation andmaintenance of germinal centers in SLO (36), but the presence ofTfh in lung iBALT induced by influenza infection is understudied.We found comparable frequencies of Tfh cells (CD3+ CD4+CXCR5++ PD1++) were evident within the lungs and MLNof mice at d35 and d56 post-infection, encompassing close to10% of the total CD4+ T cell population (Figure 3A; gatingFigure S5A). Tfh from both MLN and lungs expressed elevatedlevels of the lineage-defining transcription factor BCL6 (37,38) compared to conventional CD4+ T cells (CXCR5- PD1-)(Figure 3B), althoughwith higher comparative expression (basedupon fluorescence intensity) observed within MLN-derived Tfhcells. Intracellular Ki67 expression suggested that lung Tfh cellscontained a similar proportion of recently proliferating cellscompared to MLN at d35, but marginally higher proliferationthan MLN Tfh cells at d56 (Figure 3C). B220+ cells expressinghigh levels of Ki67 and BCL6 were also present in both lungs andMLN of infected mice, although with frequencies higher in in thelatter tissue (Figure S5B).

Ki67 (Figure 3D) and BCL6 (Figure 3E) expression in spleenand lungs of mice at d35 post-infection was visualized byconfocal microscopy. As expected, the spleen showed distinctspatial segregation of T and B cell zones. In contrast, we foundCD4T cell and B cell clusters to be substantially smaller in thelungs and their spatial segregation was not clearly delineated.Mirroring expression measured by flow cytometry, Ki67, andBCL6 expression was detected in B220 and CD4 cells of bothspleen and lungs, although expression of these markers was moreevident in B220 cells. Overall, while we observed differences inanatomical localization, Tfh within iBALT-resident GC structuresappear phenotypically analogous to SLO-resident Tfh cells.

Specificity of GC-Resident B Cells forMajor Influenza AntigensThe antigen specificity of lung- or SLO-localized GC B cellswas analyzed using recombinant influenza HA and NP probes(Figure 4A). NP-specific GC B cells were evident in all infectedmice at both d35 and d56 post-infection at all sites, whereasHA-specific GC B cells were observed almost exclusively in theMLN and spleen, with little to no HA-specific GC B cells in thelungs (Figure 4B). In terms of absolute numbers of HA- and NP-specific GC B cells at both timepoints, the majority of HA-specificGC B cells were found in the MLN, a minor population in thespleen, and little to no HA-specific GC B cells observed in thelungs (Figure 4C). In contrast, NP-specific GC B cell were foundat high frequencies and counts in all tissues, withMLN and spleenshowing similar total cell counts while less cells were detectedin lungs.

GC Within iBALT Support Maturation ofInfluenza-Specific B CellsWe next examined if iBALT GC structures display a functionalcapacity to drive maturation of the influenza-specific B cellresponse. NP- and HA-specific B cell populations were sorted



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FIGURE 2 | Transcriptional profile of GC-like B cells in iBALT. GC (B220+ IgD- GL7+ CD38lo) and memory B cells (B220+ IgD- GL7- CD38hi) were sorted from SLO

and iBALT in mice at d35 post-infection with PR8. mRNA expression profiles were compared using RNAseq data from 2 to 3 independent sequencing experiments.

(A) Multi-dimensional scaling plot showing clustering of memory and GC populations relative to T cell controls. (B) Heatmap of the top 200 differentially expressed

genes, with shading indicating expression relative to the average (red–overexpressed, blue–underexpressed). (C) Differential expression of selected genes previously

described to demarcate memory and GC subsets. Error bars represent mean ± SD.



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FIGURE 3 | Characterization of Tfh cells in iBALT. (A) Frequency of Tfh cells (CD3+ CD4+ CXCR5++ PD1++) in MLN and lungs of mice at d35 and d56

post-infection with PR8. (B) Intracellular BCL6 staining measured by mean fluorescence intensities in Tfh and non-Tfh CD4T cells (CD3+ CD4+ CXCR5- PD1-)

isolated from MLN and lungs. (C) Frequency of proliferation marker Ki67 expressed in Tfh and non-Tfh CD4T cells isolated from MLN and lungs. Data represent a

single experiment (n = 4–5). Error bars represent mean ± SD. Visualization by immunofluorescence microscopy of (D) Ki67 and (E) BCL6 expression in T and B cells

of spleen and lung iBALT; scale bar−50µM.



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FIGURE 4 | Antigen specificity of GC B cells in lung iBALT and SLO. (A) Representative HA and NP probe staining of GC B cells (B220+ IgD- CD38lo GL7+) isolated

from lung iBALT, MLN, and spleen of mice at d35 post-infection with PR8. (B) Frequency of HA-, NP-specific, and undefined GC B cells isolated from individual mice

at d35 and d56 post-infection. (C) Absolute counts of HA-specific and NP-specific GC B cells in lung iBALT, MLN, and spleen of mice at d35 and d56 post-infection.

Data represent two independent experiment (n = 4–5). Error bars represent mean ± SD.

from GC in the lung and MLN at d14, d35, and d56 and heavychain B cell receptor sequences were recovered, aligned andgrouped into clonal families. Antigen-specific B cell populations

in the lung (NP) and MLN (NP and HA) consisted of numerousclonally-expanded lineages (Figure 5A), indicating proliferationconsistent with the observed Ki67 expression. We observed



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FIGURE 5 | BCR sequencing of GC B cells in the lung and mediastinal lymph node. Heavy chain immunoglobulin sequences were recovered from NP- and

HA-specific GC B cells sorted from the lung and mediastinal lymph nodes of PR8-infected mice on d14, d25, and d56. Data represents a single experiment with 5

mice per timepoint. (A) Circular plots detailing the clonal distribution of NP- and HA-specific populations from each site. Each segment represents a unique B cell

clonotype, the width proportional to the number of clonal family members recovered. Shared clonotypes between sites are indicated by the linking arcs. Number of

unique clonal families and BCR sequences are indicated below. (B) The degree of V-gene somatic mutation in recovered heavy chain immunoglobulin sequences from

each site. NP- and HA-specific B cells are indicated in blue and red, respectively. Error bars represent median ± interquartile range.

discrete compartmentalization of the humoral response, withminimal sharing of clonotypes and different clonal hierarchiesseen between the lung and MLN, indicating B cell recruitmentto each of these sites was highly segregated. This pattern wasconserved across all animals and timepoints analyzed, and isconsistent with previous reports that the lung hosts a uniqueB cell repertoire (39). The observed rates of V-gene somaticmutation in the MLN were comparable for both NP- and HA-specific GC B cells, increasing over time from a median 3.1% to5.2% to 5.3% from d14 to d35 to d56 respectively (Figure 5B).

Similarly within the lung, while few HA-specific B cells wererecovered, somatic mutation in B cells that were mostly NP-specific increased from 2.4% to 4.5% to 4.6% across the same

timepoints. Notably, the mutation load of influenza-specific GCB cells within iBALT remained consistently lower (∼0.7%) incomparison to theMLN at each timepoint sampled. However, therate at which somatic mutations accumulated was comparable tothe MLN suggesting an equivalent capacity to drive maturationof humoral responses exists at both sites. Differences observedbetween the tissues are likely due to the delayed biogenesis ofiBALT-localized GC.

DISCUSSION

There is considerable interest in the generation of local immunitywithin the lungs to protect from influenza and other respiratory



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pathogens. iBALT represent a focus of lymphoid tissues that hasthe potential to improve immunity within the lower respiratorytract. Despite the iBALT being pleiomorphic and less structurallydefined than SLO GC structures, we found GC B cells in iBALTand SLO were transcriptionally similar and show that iBALTserve as a distinct anatomical niche for the maturation andselection of B cells primarily targeted against the influenzavirus nucleoprotein.

The cellular composition of iBALT can vary, dependingupon the stimuli used for induction (16). Consistent withprevious reports, we observed cellular infiltration concentrated atbronchial junctions that included FDC (8), Tfh and conventionalT cells (8, 9, 32), and germinal center B cell populations(10) following pulmonary influenza infection. In contrast, LPS-induced iBALT exhibits lung-infiltrating T cells with Tfh-likefunction but lacking classical CXCR5/BCL6 expression (13).Further, inoculation with LPS or whole Pseudomonas aeruginosabacteria results in ectopic lung GC structures that lack FDC,in contrast to the CXCL13-expressing FDC observed followinginfection with modified vaccinia virus Ankara (7, 13, 40).Nuances in iBALT biogenesis appear attributable to the extentand nature of inflammation induced by infectious diseasescompared to alternative pulmonary stimuli.

Germinal centers are highly specialized foci facilitating Bcell proliferation, competition, and selective differentiationin order to drive high affinity serological antibody responses.We find that clusters of GC-like B cells observed withiniBALT appear analogous to GC B cells in SLO with respect tocellular composition and phenotype. This includes evidence ofproliferation, division into light- and dark-zone phenotypes,expression of canonical GC transcription factors, and thepresence of Tfh cells. Here we extend previous studies(8, 40) by transcriptional profiling lung GC-like B cells byRNAseq. We found a conserved transcriptional signaturewas shared by GC B cells independent of anatomicallocation, with both lung- and SLO-resident populationsexpressing canonical transcription factors (e.g., bcl6), mediatorsof affinity maturation (e.g., aidca), and key chemotacticfactors (e.g., s1pr2).

One key area of difference between GC within SLO vs. iBALTwas the variant morphology, with a lack of highly distinct Tand B cell zones observed in iBALT consistent with previousreports (8, 9, 41), and a lack of spatial segregation of B cellsphenotypically characteristic of light- and dark-zone B cells. Theimpact of atypical GC architecture upon the development of Bcell immunity in the lung is unclear. Previous studies have shownlight- and dark-zone B cells exhibit largely overlapping geneexpression patterns (42). Experiments using CXCR4-deficientmice (43) have established that B cells can cycle between these twostates, and that discrete light- and dark-zone architecture is nota requisite for affinity maturation nor plasma cell differentiation(44). Based upon recovered BCR sequences from GC-residentB cells, we find GC in the lung maintain a capacity to drivematuration of the influenza-specific B cell response, with thecontinued accumulation of somatic mutations observed over56 days post-infection. While overall mutation frequencieswere consistently higher in the draining MLN, the rate of

accumulation was comparable between the two sites suggestingno detectable defect in the functionality of iBALT-localized GC.

Given the importance of anti-HA antibody for protectionagainst influenza (45), the contribution of iBALT to serologicalHA-specific antibody seems minimal. Certainly, non-neutralizing antibody responses, such as those targetingNP, can contribute to immunity via Fc-mediated functions orantibody-dependent cellular cytotoxicity, and further provideprotection against heterosubtypic influenza virus challengein mice (46–50). iBALT might conceivably act as a source ofmucosal-resident antibody-secreting cells (ASCs) that contributeto ongoing barrier protection in the lung. We observed strongcompartmentalization of B cell receptor repertoires withinanatomical sites, implying that alternative B cell clonotypes arerecruited to and maintained in lung GC compared to SLO. Thisadditional clonotypic diversity may be advantageous duringrecurrent exposure to antigenically diverse pathogens suchas influenza. The extended persistence of iBALT, reported forup to 3 months (9, 10, 31, 40), may also serve as a broaderniche to position B cells proximal to a site of recent mucosalbarrier damage. Interestingly, we find a significant proportion oflung-resident GL7+ B cells, comprising up to half of total cells,were of undefined specificity. This corroborates previous studiesdemonstrating that the iBALT can act as a general priming site,thus supporting the proliferation of GC B cells binding antigensunrelated to the primary inducing agent (23, 51). While these GCB cells may also simply represent B cells specific for alternativeantigenic targets of the influenza virus not captured by ourprobes (52), or non-specific bystander B cells adopting a GCphenotype, they may also be elicited by antigenic translocationof microorganisms as a result of lung damage during infection.Additional definition of the specificities of iBALT-resident GC Bcells is warranted.

In summary we find GC-like structures in iBALT arephenotypically, transcriptionally and functionally comparableto those within SLO, yet recruit a distinct subset of Bcell clonotypes into the humoral immune response. WhileiBALT can drive B cell diversification and may facilitateresidence of B and T lymphocytes proximal to the mucosa,the protective value of iBALT within immunocompetenthosts remains unclear, and in the context of our influenzainfection model, seems largely restricted to targeting theviral nucleoprotein.

DATA AVAILABILITY

The datasets generated for this study can befound in Gene Expression Omnibus, NCBI (Accession Q10

Q11number pending).

AUTHOR CONTRIBUTIONS

H-XT, SK, andAWdesigned the study. H-XT, RE, HV, JJ, andAW Q9

performed the experiments. H-XT, RE, JJ, SK, and AW wrote themanuscript. All authors read and revised the manuscript.



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ACKNOWLEDGMENTS

This study was supported by an NHMRC program grantQ6 ID1052979 (SK) and NHMRC project grant ID1129099 (AW).

JJ and SK are supported by NHMRC fellowships.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fimmu.2019.00611/full#supplementary-material Q12

Q8

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https://www.frontiersin.org/articles/10.3389/fimmu.2019.00611/full#supplementary-materialhttps://doi.org/10.1084/jem.184.5.1999https://doi.org/10.4049/jimmunol.178.9.5643https://doi.org/10.1128/IAI.71.6.3572-3577.2003https://doi.org/10.1093/rheumatology/ket401https://doi.org/10.1172/JCI28993https://doi.org/10.1086/508894https://doi.org/10.1084/jem.20131737https://doi.org/10.1038/nm1091https://doi.org/10.1016/j.immuni.2006.08.022https://doi.org/10.1073/pnas.1115369109https://doi.org/10.1080/01902140390240140https://doi.org/10.1164/rccm.200504-594OChttps://doi.org/10.1038/ncomms10875https://doi.org/10.1038/ni.2053https://doi.org/10.1164/rccm.200308-1167OChttps://doi.org/10.3389/fimmu.2016.00258https://doi.org/10.4049/jimmunol.1400909https://doi.org/10.1016/j.immuni.2009.08.001https://doi.org/10.1084/jem.20072713https://doi.org/10.1182/blood-2012-03-416859https://doi.org/10.1084/jem.189.2.403https://doi.org/10.1084/jem.187.7.997https://doi.org/10.1084/jem.20091472https://doi.org/10.1128/JVI.03422-13https://doi.org/10.1371/journal.pone.0140829https://doi.org/10.1038/nmeth.3317https://doi.org/10.1093/bioinformatics/btu638https://doi.org/10.1038/nprot.2016.102https://doi.org/10.1007/978-1-61779-842-9_32https://doi.org/10.1093/bioinformatics/btu393https://doi.org/10.1073/pnas.0800003105https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orghttps://www.frontiersin.org/journals/immunology#articles

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2019 Tan, Esterbauer, Vanderven, Juno, Kent and Wheatley. This is anopen-access article distributed under the terms of the Creative Commons AttributionLicense (CC BY). The use, distribution or reproduction in other forums is permitted,provided the original author(s) and the copyright owner(s) are credited and that theoriginal publication in this journal is cited, in accordance with accepted academicpractice. No use, distribution or reproduction is permitted which does not complywith these terms.


https://doi.org/10.1371/journal.pone.0040733https://doi.org/10.1172/JCI123366https://doi.org/10.1128/MCB.02047-06https://doi.org/10.1182/blood-2012-03-415380https://doi.org/10.1146/annurev-immunol-020711-075032https://doi.org/10.1016/j.immuni.2009.07.002https://doi.org/10.1126/science.1175870https://doi.org/10.1084/jem.20142284https://doi.org/10.3389/fimmu.2018.01707https://doi.org/10.1084/jem.20090410https://doi.org/10.1016/j.cell.2010.10.032https://doi.org/10.1016/j.immuni.2007.07.009https://doi.org/10.1016/j.immuni.2013.08.038https://doi.org/10.1186/1471-2288-10-18https://doi.org/10.1016/j.coviro.2016.12.002https://doi.org/10.1128/JVI.00150-11https://doi.org/10.4049/jimmunol.1003057https://doi.org/10.1099/jgv.0.000518https://doi.org/10.4049/jimmunol.181.6.4168https://doi.org/10.1016/j.immuni.2016.02.010https://doi.org/10.1038/ni.3680http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orghttps://www.frontiersin.org/journals/immunology#articles

Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in MiceIntroductionMaterials and MethodsAnimal InfectionConfocal MicroscopyHA ProteinsFlow CytometryRNAseq and Data AnalysisSequencing and Analysis of Murine B Cell Receptor GenesStatistics

ResultsDynamics of iBALT Induction Following Intranasal Influenza InfectionTranscriptional Profile of GC-Like B Cells From iBALTiBALT-Resident Tfh Cells Express Bcl6 and Ki67Specificity of GC-Resident B Cells for Major Influenza AntigensGC Within iBALT Support Maturation of Influenza-Specific B Cells

DiscussionData AvailabilityAuthor ContributionsAcknowledgmentsSupplementary MaterialReferences

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