Efficient Culture of Human Naive and Memory B Cells for Use as APCs Kuei-Ying Su, Akiko Watanabe, Chen-Hao Yeh, Garnett Kelsoe and Masayuki Kuraoka This information is current as of September 25, 2021. J Immunol 2016; 197:4163-4176; Prepublished online 10 October 2016; doi: 10.4049/jimmunol.1502193 http://www.jimmunol.org/content/197/10/4163 Downloaded from

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References This article cites 91 articles, 42 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/197/10/4163.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Efficient Culture of Human Naive and Memory B Cells for Use as APCs

Kuei-Ying Su,*,†,1 Akiko Watanabe,*,1 Chen-Hao Yeh,* Garnett Kelsoe,*,‡ and Masayuki Kuraoka*

The ability to culture and expand B cells in vitro has become a useful tool for studying human immunity. A limitation of current methods for human B cell culture is the capacity to support mature B cell proliferation. We developed a culture method to support the efficient activation and proliferation of naive and memory human B cells. This culture supports extensive B cell proliferation, with ∼103-fold increases following 8 d in culture and 106-fold increases when cultures are split and cultured for 8 more days. In culture, a significant fraction of naive B cells undergo isotype switching and differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved and, when recovered, retain their ability to proliferate and differentiate. Significantly, prolif- erating CD B cells express high levels of MHC class II, CD80, and CD86. CD B cells act as APCs and present alloantigens and Downloaded from microbial Ags to T cells. We are able to activate and expand Ag-specific memory B cells; these cultured cells are highly effective in presenting Ag to T cells. We characterized the TCR repertoire of rare Ag-specific CD4+ T cells that proliferated in response to tetanus toxoid (TT) presented by autologous CD B cells. TCR Vb usage by TT-activated CD4+ T cells differs from resting and unspecifically activated CD4+ T cells. Moreover, we found that TT-specific TCR Vb usage by CD4+ T cells was substantially different between donors. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual. The Journal of Immunology, 2016, 197: 4163–4176. http://www.jimmunol.org/

cells are key to adaptive immunity and are now rec- The Ag-presentation function of B cells has long been known ognized for their multifunctionality; B cells not only (9, 10), and B cells are recognized as professional APCs along B produce Abs, they also present Ags to T cells (1), se- with dendritic cells (DCs), macrophages, and thymic epithelial crete cytokines (2), and regulate other immunocytes (3). Ag cells (11). Ag-presenting B cells participate in the initiation and presentation by B cells is involved, to a significant extent, in continuation of autoimmune diseases, such as systemic lupus immunoprotection and the pathogenesis of autoimmune dis- erythematosus (12, 13), rheumatoid arthritis (14, 15), type 1 dia- eases (1, 4, 5). The effects of Ag presentation by B cells on betes (16), and multiple sclerosis (5), in humans and mice. Beyond by guest on September 25, 2021 T cells depend on the activation state of B cells. Studies show the scope of autoimmunity, B cells serving as APCs are charac- that CD154- or mitogen-activated B cells function as effective teristic of atherosclerosis (17), insulin resistance (18), allergy (19), APCs to induce T cell activation (6, 7), whereas resting B cells allorejection (20), infection, and even immune responses elicited are tolerogenic (8). by vaccination (21). On the whole, professional APCs initiate adaptive immune *Department of Immunology, Duke University, Durham, NC 27710; †Tzu Chi Med- cellular responses by processing and presenting Ags to T cells, as ical Center, Hualien 970, Taiwan; and ‡Human Vaccine Institute, Duke University, well as by providing costimulatory signals necessary for the ac- Durham, NC 27710 tivation of T cells. These functional properties of APCs were 1K.-Y.S. and A.W. contributed equally to this work. applied in the clinical assessment of T cell responses in vitro; for ORCIDs: 0000-0002-7927-2310 (K.-Y.S.); 0000-0003-4741-4705 (A.W.); 0000- example, to evaluate the efficacy of vaccination (22), to identify the 0001-5801-2918 (C.-H.Y.). causal allergens for patients (23), and to predict the compatibility Received for publication October 19, 2015. Accepted for publication August 30, of allografts (24). Generally, autologous APCs are loaded with 2016. target Ags and are cocultured with T cells; T cell proliferation or This work was supported in part by Duke Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery Grant AI100645-02 and Autoimmunity Center of Excel- function is then measured (25, 26). To develop effective vaccines lence Grant AI56363. that target T cells, epitope mapping of the vaccine Ags is inevi- The TCRb sequence data sets presented in this article have been submitted to the immu- table (22). This is because T cell responses are generally focused noSEQ Analyzer database (https://clients.adaptivebiotech.com/pub/9816555b-5673-4316- on only a few epitopes among the many present on microbial 85e2-244acb293f0b) under accession numbers 14_d0_CD4naive-2016881253, 14_d7_activatedCD4-2016881253, 14_d7_TTspecificCD4-2016881253, 6_d0_CD4naive- pathogens (27). With ample epitope candidates and multiple 2016881253, 6_d7_activatedCD4-2016881253, and 6_d7_TTspecificCD4-2016881253. rounds of screening, a thorough mapping of T cell epitopes re- Address correspondence and reprint requests to Prof. Garnett Kelsoe, Duke Univer- quires large numbers of APCs (22, 28, 29). sity Medical Center 3010, 117 Jones Building, Research Drive, Durham, NC 27710. Indeed, the availability of autologous APCs is often problematic in E-mail address: [email protected] studies of human T cell responses (22). Although tetramers of MHC The online version of this article contains supplemental material. molecules conjugated with peptides provides an alternative option for Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; CD, culture- derived; DC, dendritic cell; FSC, forward scatter; HA, recombinant influenza hem- measuring T cell responses to specific Ags (30), in practice, only limited agglutinin; MFI, mean fluorescent intensity; MHCII, MHC class II; rPA, recombinant numbers of Ags can be assessed using tetramers (31), restricting the Bacillus anthracis protective Ag; Td, tetanus–diphtheria toxoid; TT, tetanus toxoid; application of tetramers in large-scale evaluations of candidate epitopes. TT-PE, TT conjugated with PE. For this reason, autologous APCs are still the primary choice in T cell Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 epitope discovery. To overcome the low numbers of APCs in the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502193 4164 EFFICIENT CULTURE SYSTEM FOR HUMAN B CELLS circulating blood, usually the rate-limiting step for mapping PBS containing 2% FBS prior to analysis. Bound biotin-conjugated Abs human T cell epitopes, leukapheresis is often required to obtain were revealed by fluorochrome-labeled streptavidin. Doublets were ex- adequate numbers of APCs from a patient’s blood (28, 29). Alterna- cluded from our analysis and cell sorting by combination(s) of forward scatter (FSC)-A versus FSC-H, FSC-H versus FSC-W, and side scatter-H tively, APCs can be expanded in vitro. The low numbers of circulating versus side scatter-W gatings. Dead cells were excluded by positive DCs and macrophages in blood and their limited capacity for prolif- 7-aminoactinomycin D (7-AAD) staining (BD Biosciences). Labeled cells eration in vitro limit their applications (32–34). In contrast, B cells are were analyzed on a BD FACSCanto after fixation (BD Cytofix) or sorted on more abundant in circulating blood and are easier to expand in vitro a BD FACSAria using Diva software (BD Biosciences). compared with DCs and macrophages (35–37). To that end, B cells Isolation of mature naive B cells offer a useful and a potentially more convenient source of APCs; Human mature naive B cells were isolated from PBMCs by negative se- however, current methods for B cell culture still do not generate suf- lection with the EasySep Human Naive B Cell Enrichment Kit, according to ficient cell numbers (35–37). the manufacturer’s instructions (STEMCELL Technologies). The purity of In this study, we adapted the culture methods established by Luo mature naive B cells (CD19+CD272IgM+IgD+), as determined by flow et al. (38) to expand in vitro the numbers of naive and memory cytometry, was .94%. human B cells. This culture method efficiently induces the activa- CD culture system tion, proliferation, and differentiation of unselected or Ag-binding 3 3 B cells. Significantly, the culture-derived (CD) B cells express high Human B cells (1–6 10 ) were plated in six-well plates or 10-cm tissue culture dishes (BD Falcon) to achieve input cell densities of ∼100 B cells levels of accessory molecules necessary for effective APC function per cm2. These culture plates or dishes were preseeded overnight with (MHC class II [MHCII], CD80, and CD86) and effectively present CD154-expressing stromal cells (CD40Llow cell line, gift from David alloantigens and microbial Ags to human T cells. Expansion of Baltimore) (38). B cells were cultured in R5 medium (RPMI 1640 with 5% Downloaded from m Ag-specific human memory B cells in CD cultures results in the human serum [Sigma], 55 M 2-ME, 2 mM L-glutamine, 100 U/ml pen- icillin, 100 mg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, generation of Ag-specific APC activity that is significantly more and 1% MEM nonessential amino acids [all from Invitrogen]), supple- efficient for the cognate Ags than for unrelated Ags of comparable mented with recombinant human IL-2 (50 ng/ml), IL-4 (10 ng/ml), IL-21 mass. Using CD cultures, we are able to characterize, globally, the (10 ng/ml), and BAFF (10 ng/ml) (all from PeproTech, Rocky Hill, NJ) for TCR repertoire for Ag-specific T cells. Thus, this culture method 8 d, unless indicated otherwise. The final volume of B cell cultures was 2 ml per well for six-well plates and 6 ml per dish for 10-cm dishes. provides a platform for studying the BCR and TCR repertoires http://www.jimmunol.org/ Cells were fed with fresh R5 medium containing cytokines on days 4 and within a single individual. 6 by aspirating half of the old medium without touching the bottom of the wells and replacing the same volume with prewarmed fresh medium Materials and Methods containing cytokines. In some experiments when cultures were carried beyond 8 d, cells were split onto new feeder cells with fresh cytokines on Human blood samples day 8, and medium was changed on posttransfer days +4 and +6. At the end Blood samples were collected from healthy adult donors with informed of culture, CD B cells were harvested, counted, aliquoted, and cryo- consent in accordance with guidelines from the Duke Institutional Review preserved in liquid nitrogen until use. In experiments quantifying the ki- Board committee. Mononuclear cells were isolated by Ficoll-Paque PLUS netics of mature naive B cell proliferation, input cell numbers were (GE Healthcare Life Sciences) density gradient centrifugation with optimized to facilitate accurate cell counts: input cell numbers on day 0 were 3 4 3 3 3 3 SepMate-50 tubes (STEMCELL Technologies). Cells were cryopreserved 1 10 ,2.5 10 ,and1 10 per well in six-well plates for 4-, 6-, and by guest on September 25, 2021 in liquid nitrogen until use. For microbial Ag-specific T cell studies, blood 8-d cultures, respectively; beyond 8 d, cells were split onto new feeders, and the input cell numbers on day 8 were 4 3 104,13 104,2.53 103,and13 samples were collected 2–5 wk after tetanus–diphtheria toxoid (Td) boost 3 and/or influenza vaccination. 10 per well for 10-, 12-, 14-, and 16-d cultures, respectively. Cryopreservation of human cells Isolation and culture of Ag-specific human memory B cells Cells were cryopreserved based on a previous protocol, with modifications Human PBMCs recovered from tetanus–diphtheria vaccinees were incubated (39). Briefly, cells were suspended in RPMI 1640 medium (Invitrogen) or with a combination of flow cytometry mAbs and tetanus toxoid (TT) conju- neat FBS (FCS HyClone; Thermo) at a concentration of #2 3 107 cells gated with PE (TT-PE), which was generated using the R-Phycoerythrin 2 2 + per ml. An equal volume of cooled freezing medium containing 20% Labeling Kit-NH2 (Dojindo). Single live B cells (7AAD CD3 CD19 ) + high DMSO (Sigma) and 80% FBS was added drop-wise to the cell suspension were gated, from which IgG memory B cells were defined as CD27 CD24 2 2 + + 2 to a final concentration of 10% DMSO. Cells were aliquoted into cryovial IgM IgD IgG . IgG memory B cells that did (TT-PE )ordidnot(TT-PE ) 2 tubes and placed in a prechilled freezing container (Nalgene Mr. Frosty; bind TT were sorted into CD cultures at ∼100 cells per cm .Followingtheir + 2 Sigma). Cryovials were stored at 280˚C for 4–24 h and then were stored in activation and proliferation for 8 d, TT-PE and TT-PE CD B cells were liquid nitrogen until thawing for culture. harvested and frozen until use. A fraction of day-8 CD B cells was placed into new cultures (∼100 CD B cells per cm2)andallowedtoexpandforanother mAbs and flow cytometry 8 d; these day-16 CD B cells were harvested and cryopreserved until use. The following mouse mAbs specific for human surface Ags were used for Coculture of T and B cells flow cytometry and cell sorting in this study. Anti-human CD3 allophy- cocyanin (clone HIT3a), CD4 PE (clone A151A1), CD8 allophycocyanin- PBMCs were thawed and labeled with CFSE (Invitrogen). T cells were Cy7 (HIT8a), CD19 PE-Cy7 and allophycocyanin (HIB19), CD24 PE and isolated by negative selection with the EasySep Human T Cell Enrichment BV510 (ML5), CD27 BV421 (M-T271), CD38 BV510 (HIT2), CD45 Kit (STEMCELL Technologies) from CFSE-labeled PBMCs. The purities PE-Cy7 (HI30), CD80 PE (5D10), CD86 biotin (IT2.2), IgD FITC and of CD3+ T cells (as determined by flow cytometry) were .98% after allophycocyanin-Cy7 (IA6-2), IgM FITC and allophycocyanin-Cy7 enrichment. Frozen CD B cells were thawed and counted. T and CD B cells (MHM-88), and mouse IgG1 isotype-control PE, PE-Cy7, and biotin were suspended well before coculture. (MOPC-21) were purchased from BioLegend (San Diego, CA). CD38 Equal numbers (1 3 104 each) of T and CD B cells per well were biotin (HIT2) was purchased from eBioscience (San Diego, CA). IgM cocultured in 96-well U-bottom plates (Fisher Scientific) in 100 mlper PE-Cy5 (G20-127), IgG allophycocyanin (G18-145), and CD3 PE-Cy5 well of R5 medium without exogenous cytokines. For alloreactive T cell– (UCHT1), as well as mouse IgG1 isotype-control allophycocyanin proliferation studies, the plates were incubated at 37˚C in a 5% CO2 hu- (MOPC-21), BV510 (X40), and V450 (MOPC-21), were purchased from midified incubator for 5 d. For microbial Ag–specific T cell–proliferation BD Biosciences (San Jose, CA). MHCII FITC (TDR31.3) was purchased studies, the plates were incubated for 7 d. TT from Clostridium tetani from LifeSpan BioSciences. Streptavidin–Pacific Orange was purchased (List Biological Laboratories), recombinant influenza hemagglutinin (HA) from Invitrogen. Mouse IgG1k isotype control was purchased from (H3 A/Wisconsin/67/2005, kindly provided by S.C. Harrison), and Rockland. recombinant Bacillus anthracis protective Ag (rPA; BEI Resources) were Analysis of cell phenotypes and cell isolation were performed by flow used in Ag-specific T cell–proliferation studies. T cells were treated with cytometry. Briefly, cells were incubated with fluorochrome-conjugated equal numbers of anti-CD3/CD28 Dynabeads (Invitrogen) as positive con- mAbs specific for human surface Ags (listed above) and resuspended in trols in alloreactive and microbial Ag–specific T cell–proliferation studies. The Journal of Immunology 4165

CD B cell–immunophenotypic analysis and T cell–proliferation promote continued proliferation, we split and transferred cultured analysis cells (100 cells per cm2) into fresh cultures that contained fresh cyto- Analysis of CD B cell phenotypes and T cell proliferation was performed by kines and feeder cells. These newly expanded populations formed flow cytometry. Briefly, cells were incubated with fluorochrome-conjugated B cell clusters as early as 2 d after transfer and continued to pro- mAbs specific for human surface Ags (listed above) in PBS containing 2% liferate to confluence by posttransferday8(Fig.1A).Thisculture FBS. Bound biotin-conjugated mAbs were revealed using Streptavidin– system is capable of supporting vigorous B cell proliferation for Pacific Orange. Analysis was performed with FlowJo software (TreeStar, $16 d. Indeed, CD B cells are capable of continued proliferation Ashland, OR). CD B cells were phenotyped using flow cytometry. Absolute cell counts for at least another week in fresh cultures (data not shown). were performed with CaliBRITE beads, according to the manufacturer’s To quantify the proliferative capacity of human B cells in the CD instructions (BD Biosciences), or with a hemocytometer and trypan blue culture system, mature naive B cells (CD19+CD272CD24highIgM+ exclusion of dead cells. CD B cells were incubated with mAbs for surface IgD+) from frozen human peripheral blood (Fig. 1B) were cultured, staining of CD19, CD45, CD3, MHCII, CD80, CD86, CD27, CD24, CD138, IgG, and IgM. Dead cells were excluded from analysis by 7-AAD as described above, with the adjustment of input cell numbers (see staining. Materials and Methods) to obtain accurate kinetics of B cell pro- T cell proliferation was assessed as described previously (24). Briefly, liferation. With an input of 103 B cells per well in culture, B cells cells were washed and resuspended in R5 medium without exogenous expanded to $106 cells per well after 8 d. Subsequently, cultured cytokines. CFSE dissolved in DMSO was added at a final concentration of m B cell populations were split and transferred to fresh cultures (see 5 M. Cells were mixed well with CFSE and incubated in the dark at room 3 temperature for 5 min. Cells were washed three times with warm medium above) at 10 B cells per well on day 8; these cultured B cells 6 to remove excess CFSE and were resuspended in culture medium. After continued to proliferate and reached $10 cells at 8 d after trans- culture, cell proliferation was assessed by flow cytometry. Briefly, cells fer (day 16). A similar expansion capacity was seen in cells cultured Downloaded from were harvested and incubated with mAbs for CD3, CD4, CD8, CD19, and for 4, 6, 10, 12, and 14 d. CD cultures supported logarithmic ex- 7-AAD. Single, live (7AAD2)CD192CD3+ leukocytes were gated as the pansion of purified mature naive B cells with $103-fold increases T cell population. The frequency of T cell proliferation was determined by 6 CFSE dilution. by day 8 of culture and 10 -fold increases by day 16 (Fig. 1C). Deep sequencing for TCRb repertoire analysis Naive B cells become activated and differentiated in CD + cultures

TCR repertoires of CD4 T cells were analyzed using the immunoSEQ http://www.jimmunol.org/ + Analyzer (Adaptive Biotechnologies, Seattle, WA). CFSE-labeled CD4 In CD cultures, human mature naive B cells soon acquire an ac- T cells isolated from recent Td vaccinees (donors A and D) were cultured with anti-CD3/CD28 Dynabeads or with autologous CD B cells in the tivated phenotype that promotes effective Ag presentation, and they presence of TT (described above). After 7 d of culture, we sorted CFSEdim eventually differentiate into Ab-secreting plasmablasts and plas- CD4+ T cells and isolated genomic DNA from the sorted CD4+ T cells by macytes. To characterize the activation and differentiation of CD phenol/chloroform extraction (40). Isolated genomic DNA was sent to B cells during culture, we observed the expression of MHCII, Adaptive Biotechnologies, which performed amplification of rearranged CD80, CD86, membrane IgG, CD27, and CD138 on CD B cells TCRB genes using multiplex PCR, high-throughput sequencing for the identification of V, D, and J gene segments using the Illumina HiSeq plat- over 16 d of culture. Within 4 d, CD B cells exhibited an acti- form, and characterization of the TCRb repertoire using the immunoSEQ vated phenotype that manifested in increased expression of MHCII, human TCRb assay (41, 42). CD80, and CD86 (Fig. 2A, 2B, upper panels). Elevated levels of by guest on September 25, 2021 Data deposition MHCII, CD80, and CD86 were generally sustained through the 16-d culture period although CD80 expression declined somewhat b All TCR sequence data sets are available at https://clients.adaptivebiotech. by days 14–16 (Fig. 2A, 2B, upper panels). com/pub/9816555b-5673-4316-85e2-244acb293f0b. Later, CD cultures supported IgM → IgG class-switch recombi- Image acquisition nation and differentiation to plasmablasts/plasmacytes. In these → Images of cultured mature naive B cells were taken with a Canon EOS 20D cultures, IgM IgG class-switch recombination first became ob- camera through the eyepiece lens of an Olympus CKX41 microscope at vious on day 8, with 15–20% of cultured naive mature B cells original magnification 3200. expressing membrane IgG; this increased gradually and peaked at Data analysis 30–40% of CD B cells by day 14 (Fig. 2A, 2B, upper panels, Supplemental Fig. 1). Expression of CD27, a differentiation marker Graphs were compiled and statistical analysis was performed using one-way linked to the human B cell memory compartments (43, 44), accu- or two-way ANOVA, followed by a multiple-comparison test, with GraphPad Prism software, version 6 (GraphPad, San Diego, CA). Results mulated slowly on CD B cells until day 12 and then sharply in- are presented as mean 6 SD or mean 6 SEM. Differences between creased on days 14–16 (Fig. 2A, 2B, lower panels, Supplemental TT-binding enriched and unenriched CD B cells in inducing T cell pro- Fig. 1). CD138 expression, a marker of specialization for Ab se- liferation were considered significant at p , 0.05. cretion (45), came later, increasing abruptly at days 14–16. Thus, the CD culture system efficiently activates human mature naive Results B cells and induces B cell proliferation and differentiation. Extensive proliferation of human B cells in vitro Given that many human samples are routinely cryopreserved, we To generate large numbers of activated human B cells in vitro, we also cryopreserved CD B cells and then recultured them to test developed a B cell culture system in which B cell populations are whether they retained their proliferative and differentiative ability expanded on feeder cells that express low levels of CD154 in after the freeze-thaw process. Frozen aliquots of day-8 CD B cells medium containing the recombinant human cytokines IL-2, IL-4, were thawed and cultured at 103 cells per well for 6 d. Like IL-21, and BAFF (38). harvested B cells from cryopreserved peripheral blood (Fig. 1C), To evaluate the proliferation of these CD B cells, mature naive the frozen day-8 CD B cells proliferated 2 3 102-fold upon B cells from frozen peripheral blood samples were introduced reculture and maintained high expression of activation markers (day 0) and maintained in cultures for as long as 16 d. B cell (data not shown). Thus, CD B cells are readily cryopreserved and numbers increased substantially in this culture system (CD cul- retain their activation status and ability to proliferate. ture system); starting with B cell densities of ∼100 cells per cm2, These findings indicate that the CD culture system supports the we routinely observed clusters of B cells by day 4 of culture that extensive proliferation of human mature naive B cells and up- became confluent by day 8 (Fig. 1A). To avoid overcrowding and regulates MHCII, CD80, and CD86 on cultured B cells. Taken 4166 EFFICIENT CULTURE SYSTEM FOR HUMAN B CELLS Downloaded from http://www.jimmunol.org/ FIGURE 1. Vigorous proliferation of human mature naive B cells in vitro. Mature naive human B cells were isolated from frozen PBMCs and cultured, as described in Materials and Methods, for as long as 16 d in CD cultures. (A) B cell proliferation was assessed by microscopy; representative images of cultured B cells show substantial proliferation over time (original magnification 3200). Initial plating densities were 6000 cells per dish (∼100 cells per cm2) on days 0 and 8. Cultured cell populations were split and transferred to new cultures that contained new feeder cells and fresh cytokines on day 8 and allowedtoexpandforanother8d.(B) Representative flow cytometry profiles of mature naive B cells placed into CD cultures. Single live B cells (7AAD2CD32CD19+) that were CD272CD24+ and expressed surface IgM and IgD were defined as mature naive B cells. Typically, .94% of starting B cell populations expressed this mature naive phenotype. (C) The kinetics of B cell proliferation are shown as fold increases in viable B cell (7AAD2CD45+CD19+) numbers compared with the number of input cells (day 0). Input cell numbers were optimized to facilitate accurate cell counts, and B cell numbers were determined by flow cytometry (Materials and Methods). Each symbol represents a donor (n = 3); duplicate cultures were established for each donor. by guest on September 25, 2021 together, our results suggest that CD B cells may be capable of tion and to anti-CD3/CD28 treatment. In contrast, CD B cells acting as potent APCs for autologous and heterologous T cells. from all donors have a similar ability to induce allogeneic T cell proliferation (CFSEdim 47 6 8%, 46 6 10%, 43 6 14%, CD B cells effectively activate allogeneic T cell proliferation 50 6 15%, and 40 6 9% by CD B cells from donors A, B, C, D, To test whether CD B cells function as APCs to induce Ag-specific and E, respectively). Collectively, in vitro–expanded CD B cells T cell proliferation, we first examined their ability to elicit pro- efficiently presented alloantigens to induce allogeneic T cell pro- liferation of allogeneic T cells in MLRs. T cells from five unrelated liferation but did not activate T cell proliferation nonspecifically. donors (demographic data in Table I) were cocultured with their own (autologous) CD B cells or with CD B cells from the other CD B cells effectively activate Ag-specific autologous T cells donors. CFSE-labeled T cells and unlabeled day-8 CD B cells (104 To determine the ability of CD B cells to process and present mi- cells each) were cocultured for 5 d, and T cell proliferation was crobial Ags, we cocultured CD B cells with autologous T cells from measured by CFSE dilution at the end of coculture. T cells cul- recent vaccinees in the presence of priming and control (unexposed) tured alone did not proliferate (∼0% CFSEdim), whereas intro- vaccine Ags and determined T cell proliferation by CFSE dilution duction of anti-CD3/CD28 beads resulted in proliferation of most after coculture for 7 d. Day-8 CD B cells (originating from mature T cells through multiple divisions (64–89% CFSEdim) (Fig. 3A, naive B cells) were cocultured with autologous T cells from donors Table II). Autologous CD B cells did not induce T cell prolifer- recently immunized with Td vaccine (donors A and B, 2–5 wk ation (∼1% CFSEdim); in contrast, every CD B cell cohort induced postimmunization), trivalent influenza vaccine (donor C), or both strong proliferation in allogeneic T cells (26–67% CFSEdim) (donor D). Corresponding protein Ags (TT or HA) or an irrelevant (Fig. 3B, Table II). Of note, allogeneic T cell proliferation was Ag (rPA) (10 mg/ml each) were added to individual cocultures observed in the CD4+ and CD8+ compartments (Fig. 3B, Table II). (Fig. 4). In the absence of added Ag, little or no T cell proliferation Interestingly, the relative intensities of the allogeneic T cell was observed (,5% CFSEdim), whereas cultures containing anti- responses corresponded with those found during treatment with CD3/CD28 beads supported vigorous (.80% CFSEdim) T cell anti-CD3/CD28 beads (Fig. 3B, Table II); for example, T cells proliferation. Significantly, Ag-dependent autologous T cell prolif- from donor E had the highest proliferation rate (CFSEdim 60 6 5% eration correlated well with each donor’s recent vaccination history. [mean 6 SD]) in response to allogeneic stimulation by CD B cells TT triggered CD3+ T cell proliferation (#45% CFSEdim) in donors among all donors (32 6 6%, 41 6 5%, 46 6 9%, and 47 6 5% A, B, and D, and HA induced T cell proliferation (13–30% CFSEdim for donors A, B, C, and D, respectively), as well as had CFSEdim) in all donors; however, the highest frequencies of the most vigorous T cell division in response to anti-CD3/CD28 CFSEdim T cells were observed in donors C and D (22 and 30% treatment (89 6 3%). Conversely, T cells from donor A had the CFSEdim, respectively), who were recently immunized with in- lowest proliferation frequency in response to allogeneic stimula- fluenza vaccine. In contrast, rPA did not induce T cell proliferation The Journal of Immunology 4167

FIGURE 2. Activation and differ- entiation of naive B cells in CD cultures. Activation of cultured B cells was evident based on increased expression of MHCII, CD80, and CD86 during the early culture period and was followed by differentiation into CD27+ and CD138+ populations of class-switched, IgG+ B cells. Representative line graphs (A) and frequencies of B cells expressing el- Downloaded from evated levels of MHCII, CD80, CD86, IgG, CD27, and CD138 dur- ing culture shown as a percentage of CD19+ cells (B). Elevated expression of these surface molecules was de- fined based on representative flow http://www.jimmunol.org/ histograms that quantified expression in input B cells (day 0) and cultured B cells on days 8 and 16; dashed black lines indicate thresholds for elevated expression. Each symbol represents a single donor (n = 3). by guest on September 25, 2021

(#5% CFSEdim), with the single exception of donor D (15% (IgM2IgD2); 57% of the class-switched memory B cells express CFSEdim). On inquiry, we discovered that this individual is exposed surface IgG (Fig. 5A). Among circulating IgG memory B cells, ∼2% to rPA as a result of his occupation. In all cases, T cell proliferation were TT-PE+ (Fig. 5A). The frequency of the memory phenotype in was most evident in CD4+ T cells: 64–90% of the CFSEdim Tcells another donor (donor B) is ∼35% of circulating B cells, and ∼53% were CD4+ (Fig. 4). CD B cells efficiently take up, process, and of these memory B cells are IgM2IgD2; IgG memory cells repre- present protein Ags to autologous CD4+ T cells and, thereby, induce sent 35% of the class-switched memory pool. The TT-PE+ frequency Ag-specific T cell activation and proliferation. among IgG memory B cells is ∼12%, which is higher than in donor A. Combining data from both donors, circulating B cells are com- Ag-specific human memory B cells are activated and posed of 38.4 6 2.3% (mean 6 SD) memory B cells; 47.4 6 8.5% proliferate in CD cultures of these memory B cells are class-switched, and 46.5 6 15.4% of Ag-specific B cells are supremely efficient APCs for their cognate them are IgG+.ThefrequencyofTT-PE+ cells in the IgG memory Ags (9). Consequently, we determined whether Ag-specific memory B cell pool varies between donors (12% versus 2%). Overall, the B cells might proliferate and differentiate in our culture system and average frequency of TT-PE+ IgG memory B cells is 0.5% among whether cultured memory B cells present specific Ags more effi- circulating CD19+ B cells in these two recent Td vaccinees. ciently than do unselected CD B cells. IgG memory B cells (CD19+ Isolated memory B cells were seeded at ∼100 cells per cm2 in CD CD27+CD24hiIgM2IgD2IgG+) from the peripheral blood of Td cultures and allowed to expand for 8 d; subsequently, the prolifer- vaccinees were sorted based on their ability to avidly bind TT-PE. ating cultured cells were reseeded in fresh cultures (∼100 cells per cm2) TT-binding (TT-PE+) and non–TT-binding (TT-PE2) IgG memory for another 8 d (total 16 d). In CD cultures, TT-PE+ and TT-PE2 IgG B cell populations were expanded separately in CD cultures. memory B cells proliferated comparably, with ∼103-fold increases by A representative example of our sorting strategy to identify day 8 and 2 3 105-fold increases by day 16 over input cell numbers TT-specific memory B cells shows that ∼40% of peripheral blood (Fig. 5B). We measured TT-binding enrichment by comparing the B cells from donor A exhibit the CD27+CD24high memory phe- frequencies of positive TT-PE labeling on these CD B cells using notype, of which ∼41% underwent class-switch recombination flow cytometry. In donor A, after subtracting the PE-TT signal 4168 EFFICIENT CULTURE SYSTEM FOR HUMAN B CELLS

Table I. Demographic data for five unrelated donors greatly: we observed a 77 and 94% reduction in the MFI of IgG in day-8 and day-16 cultured IgG memory B cells, respectively, com- Time Between pared with the MFI in input cells (data not shown). The expression Vaccination and Donor Age (y) Gender Recent Vaccination Blood Collection levels of surface MHCII, CD80, CD86, and IgG in CD B cells from sorted TT-PE+ and TT-PE2 IgG memory B cell populations cultured a A 47 Female Td toxoid 5wk for the same duration were comparable (data not shown). B 48 Female Td toxoida 5wk C 38 Female TIVb 16 d b The presentation efficiency of CD B cells is increased by D 39 Male Td and TIV 16 d preselecting for Ag-specific BCRs E 26 Male None Not applicable aThese donors received Td vaccination at least once prior to the vaccination To evaluate the ability of CD B cells enriched for TT binding to listed. induce T cell proliferation in response to cognate Ags, cultured IgG b These donors received the trivalent influenza vaccine. memory B cells were recovered and cocultured with equal numbers (104) of CFSE-labeled autologous T cells from donors A or B, (∼1%) on cultured TT-PE2 memory B cells, we found that ∼8and who had recently received a Td booster. Both day-8 and day-16 6% of CD B cells from the TT-PE+ population were positively la- CD B cells from TT-PE+ and TT-PE2 IgG memory B cells were beled with TT-PE after 8 and 16 d of culture, respectively (data not tested for their ability to induce T cell proliferation in the absence shown). In donor B, ∼30 and 15% of CD B cells from 8- and 16- or presence of TT (seven tested concentrations, 5-fold serial di- d cultures, respectively, were positively labeled with TT-PE (data not lutions from 10 mg/ml), HA (2 or 10 mg/ml), or the irrelevant Ag shown), indicating a successful enrichment of TT-binding memory rPA (10 mg/ml) in the cocultures. After 7 d of coculture, total Downloaded from B cells resulting from cell sorting. T cell proliferation was determined by CFSE dilution (Fig. 6). To evaluate whether the cultured cells derived from IgG memory Without the addition of microbial Ags, both CD B cell pop- B cells also acquire the APC phenotype in CD cultures, the expression ulations (enriched or not for TT binding) induced little or no levels of surface MHCII, CD80, and CD86 on day-8 and day-16 autologous T cell proliferation (CFSEdim ∼3 and ∼5% for TT-PE+ cultured IgG memory B cells were compared with ex vivo unse- and TT-PE2 CD B cells, respectively, from donor A and CFSEdim + 2 ∼ ∼ lected IgG memory B cells. TT-PE and TT-PE IgG memory B cells 4and 3.5% from donor B). A coculture well using day-8 http://www.jimmunol.org/ became activated, increasing the expression of MHCII, CD80, and TT-PE2 CD B cells (CFSEdim ∼30%) was an exception, which CD86following8dinculture;theseexpressionlevelsdecreasedby may have been due to the effect of a culture medium component day 16 but remained higher than the baseline levels (Fig. 5C). Ap- on the T cells. With the addition of rPA, little or no T cell pro- proximately 92 and 64% of these cells had elevated expression of liferation was induced by either CD B cell population (CFSEdim MHCII by days 8 and 16, respectively; similar trends were seen with ∼6 and ∼2% for donor A and donor B, respectively). With the regard to the expression of CD80 (74 and 31% by days 8 and 16) and addition of irrelevant Ag or no Ag, cells derived from IgG CD86 (92 and 61% by days 8 and 16) (Fig. 5C). The expression levels memory B cells in CD cultures induced little or no T cell pro- of MHCII, CD80, and CD86, measured as mean fluorescent index liferation, indicating that the CD B cells from the memory pool,

(MFI) by flow cytometry, decreased ∼45, 70, and 65%, respectively, similar to those from the naive mature pool, do not activate T cells by guest on September 25, 2021 in day-16 cultured cells compared with the levels in day-8 cells nonspecifically (Figs. 3, 4, 6). (Fig. 5C). Surface IgG expression in CD B cells was also assessed. With the addition of TT Ag, T cell proliferation was more robust in After culture for 8 d, ∼75% of cultured IgG memory B cells retained the cocultures with CD B cells enriched for TT binding than in those surface IgG expression, and the frequency decreased to ∼35% on day with CD B cells not enriched for TT binding, indicating that Ag-driven 16; however, the total expression levels of surface IgG decreased T cell proliferation corresponded with the Ag specificity of CD B cells

FIGURE 3. CD B cells effectively acti- vate allogeneic T cell proliferation. Frozen aliquots of cultured B cells (day 8) were thawed and cocultured with equal numbers (104) of CFSE-labeled allogeneic or autol- ogous T cells. (A) Representative flow plots of T cell proliferation in cultures alone (T only) or in the presence of anti-CD3/ CD28 beads (aCD3/CD28) or in cocultures with autologous or allogeneic CD B cells. (B) CFSE-labeled T cells from five unre- lated donors (A, B, C, D, and E) were cocultured with their own (autologous) or each other’s CD B cells. Matched CFSE- labeled T cells were similarly cultured in the presence of anti-CD3/CD28 beads or alone as positive or negative controls, re- spectively. Five days later, T cell prolifera- tion was estimated by CFSE dilution. Results for T cells cocultured with CD B cells from donor A are illustrated as proliferation of CD3+ (left panel), CD4+ (middle panel), and CD8+ (right panel) T cell populations. Summarized results are shown in Table II. Data are mean 6 SD. n = 5; two independent experiments. The Journal of Immunology 4169

Table II. Percentage of T cell proliferation determined by CFSE dilution in response to autologous or allogeneic CD B cells

CD B Cells

ABCDET Onlya aCD3/CD28a CD3+CFSEdim (% of CD3+)A 36 5b 39 6 5266 6356 2286 206 0646 4 B476 706 0396 3416 10 36 6 606 0796 8 C366 8436 506 0586 7476 406 0766 7 D526 4416 7496 526 1486 706 0846 2 E556 5606 6586 8676 336 106 0896 3 CD4+CFSEdim (% of CD3+)A 36 5286 5226 5276 1216 206 0536 6 B296 706 0226 5226 7176 406 0676 4 C236 4256 206 0316 2206 206 0626 6 D246 5196 3246 416 1286 306 0616 3 E366 5406 5366 7506 416 106 0686 3 CD8+CFSEdim (% of CD3+)A 06 196 636 376 276 106 096 2 B176 606 0166 4196 6186 406 0106 3 C126 4176 306 0256 6256 406 0126 1 D266 5216 4246 306 0186 606 0216 5 E156 4186 2186 1156 316 106 0166 5 CFSE-labeled T cells from five unrelated donors (A, B, C, D, and E) were cocultured with their own (autologous) or each other’s CD B cells. aMatched CFSE-labeled T cells were cultured similarly in the presence of anti-CD3/CD28 beads (aCD3/CD28) or alone (T only) as positive or negative controls, respectively. Downloaded from bPercentage of CFSEdim cells (6 SD) among CD3+ cells.

(Fig. 6, donors A and B). Using day-8 CD B cells from donor A, the lasted until the concentrations of TT were ,0.016 mg/ml. Simi- population of CD B cells enriched for TT binding induced more larly, day-16 CD B cells enriched for TT binding induced more T cell proliferation (∼20% more CFSEdim T cells) compared with CD T cell proliferation (∼10–37% more CFSEdim T cells) than did CD B cells not enriched for TT binding in response to 10 mg/ml TT B cells not enriched for TT binding (p , 0.0001), and the http://www.jimmunol.org/ (CFSEdim ∼45 and ∼25%, respectively). By serially reducing TT advantage remained until TT was ,0.4 mg/ml. Day-8 and day-16 concentrations, we observed ∼10–38% more CFSEdim T cells in the TT-binding–enriched CD B cells from donor B also exhibited an cocultures with day-8 CD B cells enriched for TT binding than enhanced ability to induce T cell proliferation, with ∼10–36% in those not enriched for TT binding in the presence of TT $ more CFSEdim T cells observed at TT $ 0.08 mg/ml (p , 0.01 and 0.016 mg/ml (p , 0.0001) (Fig. 6, donor A); the superior ability of p , 0.001 for days 8 and 16, respectively, for CD B cells enriched TT-binding–enriched day-8 CD B cells to induce T cell proliferation or not for TT binding). by guest on September 25, 2021

FIGURE 4. Ag presentation by autologous CD B cells results in T cell proliferation. Frozen aliquots of cultured B cells (day 8) were thawed and cocultured with equal numbers (104) of CFSE-labeled autologous T cells from recent vaccinees (2–5 wk postvaccination). Donors A and B received a tetanus–diphtheria booster immunization, donor C received the trivalent influenza vaccine, and donor D was injected with both vaccines simultaneously. TT, HA (H3/Wis- consin), or the irrelevant Ag rPA (each, 10 mg/ml) was added to indi- vidual cocultures; cultures without added Ag (unstim) or anti-CD3/ CD28 beads served as negative and positive controls, respectively. After 7 d of culture, T cell proliferation was estimated by CFSE dilution among all (CD3+) T cells and CD4+ and CD8+ T cell subsets. Results are compiled from two to four indepen- dent experiments and are presented as mean 6 SEM. 4170 EFFICIENT CULTURE SYSTEM FOR HUMAN B CELLS

FIGURE 5. In vitro proliferation and activa- tion of Ag-specific human memory B cells. (A) Representative sorting strategy to identify TT-specific memory B cells (from donor A, a tetanus–diphtheria vaccine recipient). Single live B cells (7AAD2CD32CD19+) were gated on the CD27+CD24highIgM2IgD2IgG+ cell population (IgG memory B cells). IgG memory B cells that did (TT-PE+) or did not (TT-PE2) bind TT were sorted into CD cultures. (B) Expansion of TT- binding and TT-nonbinding memory B cells. On days 8 and 16, cell numbers were determined by flow cytometry (Materials and Methods)

B cell expansion is shown as viable B cell Downloaded from (7AAD2CD45+CD19+) numbers over input cells. Data are shown as mean fold increase 6 SD; n =2.(C) Representative line graphs of MHCII, CD80, and CD86 on cultured IgG memory B cells, as well as ex vivo IgG+ memory B cells. http://www.jimmunol.org/

Furthermore, the population of CD B cells enriched for TT CFSEdim T cells detected using day-8 and day-16 CD B cells, binding induced measurable T cell proliferation with the addition respectively) (Fig. 6), suggesting that these B cells can acquire of the noncognate Ag, HA, at 10 mg/ml (13–23% CFSEdim T cells Ags through a BCR-independent pathway; however, the by guest on September 25, 2021 observed using day-8 CD B cells and 4–9% CFSEdim T cells using HA-presenting ability of TT-PE+ enriched CD B cells was less day-16 CD B cells, both donors) and at 2 mg/ml (5–17 and 2–5% efficient than their presenting ability for the TT cognate Ags

FIGURE 6. TT-specific cultured memory B cells are more efficient than their non- specific counterparts at inducing the prolif- eration of TT-specific T cells. Frozen aliquots of day-8 and day-16 cultured cells derived from TT-PE+ and TT-PE2 IgG memory B cells were thawed and cocul- tured with CFSE-labeled autologous T cells for 7 d, and T cell proliferation was esti- mated by CFSE dilution among all (CD3+) T cells. Experiments were performed using cells from two healthy donors (donor A and donor B) with a recent tetanus–diphtheria booster. TT (10 mg/ml and 5-fold serial dilutions into six additional doses), HA (3H/Wisconsin) (2 or 10 mg/ml), or rPA (10 mg/ml) was added to individual cocul- tures; cultures without added Ag (unstim) served as negative controls. Results are compiled from two or three independent experiments and are shown as box-and- whisker plots with 5th and 95th percentiles. **p , 0.01, ***p , 0.001, ****p , 0.0001, two-way ANOVA and multiple- comparison test. ns, no significant difference. The Journal of Immunology 4171

(Fig. 6). In contrast, CD B cells not enriched for TT binding rearrangements from TT-activated cells represented 25% (24% exhibited an Ag-presenting capability that was similar to that of for donor D and 26.9% for donor A) (Fig. 7C). Rearrangements of TT-binding–enriched cells in the presence of 10 mg/ml HA (14–22 Vb2-1 are particularly represented (among the top 10 TCRb se- and 7–9% CFSEdim T cells observed using day-8 and day-16 quences in both donors; in donor A, Vb2-1 rearrangements are unenriched CD B cells, respectively) or 2 mg/ml HA (10–12 and ranked third and eighth in abundance, whereas in donor D they are 2–3% CFSEdim T cells observed using day-8 and day-16 unen- ranked first) (Supplemental Table I). The increased frequencies of riched CD B cells, respectively) (Fig. 6). Taken together, we found a handful of TCRb rearrangements are consistent with clonal comparable capacities for CD B cell populations, regardless of proliferation and dominance in response to TT; in donor A, two TT-binding enrichment, to present non-TT Ags (HA and rPA) to Vb2-1 rearrangements account for 56% of all Vb2-1 rearrange- autologous T cells (p $ 0.59 and p $ 0.49 for donor A and B, ments in the TT-activated cohort, whereas in donor D, a single respectively). From the above T cell–proliferation results for all rearrangement accounts for 47% of all Vb2-1 rearrangements. tested Ags (Fig. 6), we conclude that the expression of TT-specific This Ag-specific structural selection is also evident in CDR3 BCRs on TT-PE+ enriched CD B cells contributes to their superior sequences of the dominant top 10 TCRb rearrangements. The two TT presentation compared with TT-PE2 enriched CD B cells. most frequent Vb2-1 rearrangements from TT-activated T cells for Taken together, these results indicate that cells expanded from donor A (third and eighth) share a virtually identical CDR3 amino IgG memory B cells in vitro can function as APCs that take up Ags acid sequence motif (ASRPGQPPYEQY and ASSGGQPPYEQY, through BCR-dependent and BCR-independent pathways. Fur- respectively). This near identity implies convergent selection, thermore, the BCR-dependent Ag-uptake pathway significantly presumably for a common peptide/MHCII epitope. We conclude enhances the Ag-presenting function of these CD B cells in in- that TCR sequences from TT-activated CD4+ T cells reflect the Downloaded from ducing autologous T cell proliferation. structural restriction inherent in Ag-specific receptors preferen- tially expanded in response to TT presented by autologous CD b + TCR V gene segment usage of TT-specific human CD4 Bcells. T cells To demonstrate the usefulness of our CD culture system in ana- Discussion

lyzing the human TCR repertoire, we characterized TCR Vb usage The value of the CD culture system in evaluating T cell specificity http://www.jimmunol.org/ of TT-specific CD4+ T cells from two donors. We isolated geno- and the TCR repertoire is demonstrated by the efficient and large mic DNA from CFSEdim CD4+ T cells that proliferated in re- yield of CD B cells that are capable of acting as APCs to induce sponse to TT presented by autologous CD B cells and then T cell proliferation against alloantigens or pathogenic Ags. Ex- amplified TCRb VDJ rearrangements by PCR. For comparison, panded Ag-specific human CD4+ T cells in the CD culture system we amplified TCRb VDJ rearrangements from genomic DNA of were subsequently analyzed for the use of TCR Vb gene seg- unstimulated CD4+ T cells and CFSEdim CD4+ T cells activated by ments. Furthermore, we showed that human Ag-specific memory anti-CD3/CD28 from the same donors. From the two unrelated B cells can be expanded efficiently in vitro and function as highly donors (A and D), we obtained a total of 31,019, 41,265, and effective APCs, which should allow them to serve as a valuable 8,791 productive TCRb VDJ rearrangements by deep sequencing tool for studying the interaction between cognate T and B cells. by guest on September 25, 2021 from freshly isolated CD4+ T cells, anti-CD3/CD28–activated The usefulness of CD B cells includes evaluating a B cell rep- CD4+ T cells, and TT-activated CD4+ T cells, respectively. ertoire that changes during exposure to Ags in chronic inflammatory Vb gene segment usage of freshly isolated CD4+ T cells was conditions (46–49). Mapping the alterations in B cell repertoires diverse in both donors (Fig. 7A, 7B; gray bars). As expected for during the course of disease may provide insights into the patho- unspecific expansion of CD4+ T cells, Vb gene segment usage was genesis of these diseases and, subsequently, potential therapeutic virtually identical between freshly isolated CD4+ T cells and anti- strategies for them. For example, the existence of B cells that se- CD3/CD28–stimulated CD4+ T cells in both donors (Fig. 7A, 7B; crete broadly neutralizing Abs against HIV demonstrates coevolu- gray and blue bars). In contrast, Vb usage of TT-activated CD4+ tionary changes in the B cell repertoire and viral variants (46) and T cells was clearly distinct from freshly isolated CD4+ T cells and suggests that the timing of humoral immune responses does not from unspecifically activated CD4+ T cells (Fig. 7A, 7B; red bars). correspond well with the progression of mutations in HIV Ags. In donor A, Vb2-1, Vb4-3, Vb5-4, Vb6-1, and Vb19-1 gene Furthermore, B cell repertoire studies in patients with autoimmune segments were particularly frequent in TT-activated CD4+ T cells diseases revealed that autoreactive B cell clones are generated as a compared with resting or unspecifically activated CD4+ T cells result of defective tolerance checkpoints, as well as persistent Ag (Fig. 7A). In donor D, Vb2-1, Vb5-1, Vb18-1, and Vb29-1 gene stimulation (47–49). In our laboratory, we are currently studying segments were particularly enriched in TT-activated CD4+ T cells human B cell repertoires using the CD culture method. (Fig. 7B). Along with humoral responses, the repertoire of T cells theo- In general, although Vb usage by TT-activated CD4+ Tcells retically changes upon persistent exposure to Ags (14, 50). This is differed between donors A and D, the common use of the Vb2-1 because T cells are interacting with B cells that can function as gene segment may represent a general structural solution for the APCs during chronic inflammatory diseases (51–53). Cognate TCR of TT-specific CD4+ T cells (Fig. 7A, 7B, red bars). The T and B cells interact and provide reciprocal help that is required frequency of Vb2-1 in resting and unspecifically activated CD4+ for activation and differentiation of both cell types, which results T cells was 5.2 and 4.6%, respectively, for donor A and 8.8 and in alterations in T and B cell populations (9, 52). In contrast to 6.6%, respectively, for donor D (Fig. 7A, 7B). In TT-activated co- studies of B cell repertoire dynamics, relatively few studies of horts, the frequency of Vb2-1 rearrangements was doubled in both coevolutionary changes in human T cell repertoires and chronic donors (9.9% in donor A and 14.1% in donor D) (Fig. 7A, 7B). pathogenic Ags have been reported (54, 55). Furthermore, studies This structural selection is evident in the decreased diversity of directly determining TCR specificity have been rare (56), at least independent TCRb rearrangements recovered from TT-activated in part because sufficient numbers of autologous APCs are not T cells (Fig. 7C). Although the 10 most common TCRb rear- frequently available. In this study, we developed and explored a rangements in resting and unspecifically activated CD4+ T cells method that provides abundant autologous APCs using CD B cells constituted ,6% (1.1–5.5%) of all TCRb sequences, the top 10 and could be used to study T cell repertoire progression. 4172 EFFICIENT CULTURE SYSTEM FOR HUMAN B CELLS Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. Vb gene segment usage of TT-specific CD4+ T cells. (A and B) Distributions of Vb gene segment usage for freshly isolated CD4+ T cells (day 0, gray), anti-CD3/CD28–activated CD4+ T cells (blue), and TT-activated CD4+ T cells (TT-specific, red) isolated from PBMCs of donor A (A) and donor D (B). CFSE-labeled T cells were cocultured with equal numbers of autologous CD B cells in the presence of TT or were cultured with anti-CD3/ CD28 beads in the absence of CD B cells for 7 d (see also legend for Fig. 4). After culture, CFSEdim CD4+ T cells were purified for isolation of genomic DNA. Characterization of TCRb was performed by Adaptive Technologies using the immunoSEQ human TCRb assay. (C) The top 10 unique VDJ rearrangements recovered most frequently from each T cell group were selected, and the percentage of individual rearrangements among all productive VDJ rearrangements is shown. ***p , 0.001, ****p , 0.0001, one-way ANOVA. ns, no significant difference.

In our CD culture system, cytokines and CD40:CD154 inter- was observed every 2 d; the population doubling time was ∼15 h in action between B cells and feeder cells support activation, vigorous day 4–16 CD B cells (calculated using the logarithmic least-squares expansion (Figs. 1, 5) (57–59), and APC function of human naive fitting technique [http://www.doubling-time.com/compute.php]). and memory B cells (Figs. 4–7) (6, 60, 61). Among the cytokines We believe that our CD culture may be the most efficient system present in our culture medium (IL-2, IL-4, IL-21, and BAFF), for inducing primary human B cell division in vitro (6, 60, 61). IL-21 is crucial to induce robust B cell proliferation (Supplemental In the CD culture system, mature naive human B cells are ac- Fig. 2), an observation that is consistent with other studies (62– tivated and express elevated levels of MHCII, CD80, and CD86 as 64). BAFF supports B cell survival (65), the differentiation of early as day 4 (Fig. 2A), undergo IgM → IgG class-switch re- human memory B cells into Ab-secreting cells (66), and class- combination (days 8–16), and later differentiate into plasmablasts/ switch recombination in human B cells (67, 68). Because IL-4 and plasmacytes at around day 14–16 after culture (Fig. 2B). By IL-21 differentially regulate class-switching to certain Ig isotypes day 16, the frequency of CD138+ plasmablast/plasmacyte pop- (64, 69), one might consider the combinations of cytokines for CD ulations reached 10–30% in the CD culture system (Fig. 2B). cultures depending on their own purposes. We note that our estimate of plasmablast/plasmacyte frequen- Although studies also used CD40-mediated activation to induce cies in the CD culture system might vary, depending on the use of proliferation of human B cells (6, 60, 61, 63), our cultures supported surface markers (e.g., CD27highCD38high) to define plasmablasts/ more robust and sustained proliferation of B cells than did previous plasmacytes (44, 62). culture methods. In the CD culture system, B cells expanded by The observation that the CD culture system supports activation ∼106-fold after 16 d of culture; the rate of expansion was stable and extensive proliferation of memory B cells (Fig. 5B) is con- over a 16-d culture period after an initial lag phase (Fig. 1C). sistent with previous reports that human memory B cells can be Beginning at day 4 of culture, a nearly log increase in cell numbers activated and differentiate through a pathway that bypasses BCR The Journal of Immunology 4173 signaling (62, 63). CD B cells originating from memory B cells against pathogens with a complex set of epitopes (30, 82). Second, exhibited slightly less cell expansion and more frequent CD138 tetramers are not primarily designed to measure the breadth of expression compared with those originating from naive B cells, cellular immune responses induced by vaccination (83). Third, each although neither difference was statistically significant (Figs. 1C, individual needs an MHC test to find MHC-matched tetramers 5B). Furthermore, the elevated expression of MHCII, CD80, and (if available) (84). In contrast, CD B cells would allow for the CD86 on cultured memory B cells declined faster than in cultured identification (and subsequent isolation) of T cells against patho- naive B cells (Figs. 2A, 5C). These findings may be due to fun- gens using a complex set of Ags. Because the tested Ags would not damental differences in the activation capacity and differentiation be limited to peptide forms (Figs. 4, 6, 7), CD B cells would allow potential of naive and memory B cells (63, 70). more Ags to be tested and would provide a suitable method for CD B cells from day-8 cultures displayed more efficient Ag- determining the intensity and breadth of vaccine responses. More- presenting functions than did CD B cells from day-16 cultures over, an MHC test would not be necessary because T and B cells (p # 0.0067 and p # 0.0011 for TT-PE+ and TT-PE2 enriched CD could be isolated from the same individuals (Figs. 4, 6, 7). In ad- B cells, respectively) (Fig. 6). This loss of function correlated with dition to specific T cell isolation, CD B cells could be valuable in the higher levels of MHCII, CD80, and CD86 expressed by day-8 defining the characteristics of Ag-specific memory T cells, such as CD B cells (Fig. 5C). Although plasmacytes may retain surface their expression of surface molecules and effector molecules. expression of MHCII, CD80, and CD86 and function as APCs Using autologous CD B cells and CD4+ T cells, we character- (71), the expression of mRNA encoding MHCII, CD80, and CD86 ized the TCR repertoire for TT-reactive CD4+ T cells in two un- molecules is very low in plasmacytes (71, 72). Thus, plasmacytic related donors. Analysis of 8800 rearranged TCRB gene sequences differentiation of CD B cells is also likely to contribute to losses in from TT-reactive CD4+ T cells showed that, in each donor, dis- Downloaded from APC function. tributions of TCR Vb usage of TT-activated CD4+ T cells was Potent alloresponses by T cells are induced by CD B cells from distinct from resting CD4+ T cells or from CD4+ T cells unspe- allogeneic donors (Fig. 3, Table II). We observed alloreactivity cifically activated by anti-CD3/CD28 (Fig. 7). Although TCR Vb among CD4+ and CD8+ T cell subsets, indicating that the Ag- usage of TT-activated CD4+ T cells differed substantially between display functions of MHC class I and MHCII are normal in CD donors, presumably as the result of dissimilar HLA types and/or

B cells (27). This suggests that CD B cells acquire Ags derived differences in vaccination and exposure histories (85), we noted http://www.jimmunol.org/ from dying cells and/or through autophagy in an MHCII- selective expansion of small sets of Vb rearrangements, such that dependent pathway (27, 73); conversely, the Ags for MHC class the top 10 rearrangements in either donor constituted $25% of all I–dependent presentation can be acquired from the intracellular TCRb amplicands. This decreased diversity is consistent with TT- space for canonical presentation and/or from the extracellular specific clonal expansion. space for cross-presentation (27, 74). Unlike allogeneic cocul- Although genetically diverse, human TT-specific TCRab may tures, little or no T cell proliferation was observed in the autolo- share some general structural(s) characteristics, because we observed gous T–B cocultures (Figs. 3, 4, 6, Table II). These responses may overrepresentation of Vb2-1 rearrangements in both donors (Fig. 7, have contained some minor component of xenoactivation, because Supplemental Table I). Frequencies of Vb2-1 usage were doubled in we did not isolate CD B cells from the CD154-expressing mouse the TT-activated cohorts of donor A and D (Fig. 7), and this increase by guest on September 25, 2021 stromal cells after culture. Nonetheless, as expected (75), xenoreactivity was the consequence of clonal expansion. Interestingly, in donor A, of T cells against the mouse stromal cell line appeared to be negligible two Vb2-1 TCR rearrangements, ranked third and eighth among the in these conditions (Figs. 3, 4, 6, Table II). top 10, shared a CDR3 amino acid sequence that was independently CD B cells efficiently induced the proliferation of autologous generated by distinct VDJ rearrangements (Supplemental Table I). T cells against microbial Ags when these cells were prepared from We hypothesize that this convergence represents a common structural donors who had recently been vaccinated with (components of) the solution for a single peptide/MHCII epitope. same microbial Ags (Fig. 4). Presumably, this reflects the in- One unique feature of Ag-presenting B cells is that B cells uptake creased numbers of specific T cells elicited by homologous vac- Ag in BCR-independent and -dependent manners (1, 9, 86). Although cination (56, 76). CD B cells expanded from nonselected naive nonantigen-specific B cells uptake Ags in a BCR-independent mature B cells are capable of acquiring Ags through BCR- manner, Ag-specific B cells uptake Ags more efficiently through their independent fluid-phase endocytosis (77) and presenting these high-affinity BCRs (9). Our results (Fig. 6) indicate that TT-binding Ags to T lymphocytes. As expected (78, 79), the presentation of memory CD B cells could present TT more efficiently than TT exogenous Ags (TT and HA) by CD B cells to T cells occurs nonbinding memory CD B cells, which is consistent with previous primarily through the MHCII-dependent pathway that induces reports using EBV-transformed B cells (9). Our data show that HA CD4+ T cell responses (Fig. 4). Interestingly, we observed T cell presentation was comparable in cultured memory B cells with or responses against rPA in the individual (donor D) who had a without enrichment for TT binding (Fig. 6). In addition, the com- history of occupation-related exposure to rPA (Fig. 4); a low level parable expression levels of MHCII, CD80, CD86, and IgG on both of Abs against rPA also was detected in this individual’s plasma CD B cell groups (data not shown) suggest that BCR-independent (data not shown). Together with the coculture results, this indi- Ag-presenting function was similar in these cell groups. Therefore, cates the presence of humoral and cellular immune responses we conclude that the superior ability of TT-binding–enriched CD against rPA in donor D (80, 81). B cells to induce T cell proliferation in the presence of TT was re- It is appropriate to use CD B cells with sets of Ags to determine lated to their harboring of TT-specific BCRs, which uptake TT more the Ag specificity of T cells and to evaluate postvaccine cellular efficiently. responses. The identified Ag-responding T cells can be isolated for Consistent with our own and earlier observations (9), TT-binding subsequent determination of the TCR repertoire (Fig. 7) (56). memory CD B cells induced T cell proliferation more efficiently Alternatively, many studies used tetramers of MHC molecules than did naive CD B cells in the presence of TT (Supplemental conjugated with peptides and fluorochromes to separate the target Fig. 3). 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