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 Supplementary http://www.jimmunol.org/content/suppl/2016/10/07/jimmunol.150219 Material 3.DCSupplemental 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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.