key: cord-0810170-et46tnkq authors: Traggiai, Elisabetta title: Immortalization of Human B Cells: Analysis of B Cell Repertoire and Production of Human Monoclonal Antibodies date: 2012-05-23 journal: Antibody Methods and Protocols DOI: 10.1007/978-1-61779-931-0_10 sha: c14c4b667373f5b598e0d1f4073121fac7b51fcb doc_id: 810170 cord_uid: et46tnkq One of the major challenges in human B cell immunology field has been the objective to establish stable monoclonal cells lines that express the B cell receptor (BCR) on their cell surface and secrete antibodies. Such a system is extremely attractive not only for studying various aspect of BCR signaling but also for the generation of human monoclonal antibody and the analysis of the human B cell repertoire. This chapter describes an efficient method to immortalize and clone human B cells by Epstein–Barr Virus (EBV) transformation. In recent years, it has become evident that there is a great need to develop in vitro methods allowing to analyze human B cell responses in order to understand their regulation in normal as well as pathological situation such as autoimmune disorders, immunode fi ciency, and infection diseases ( 1 ) . One of the main limitations is that mature B cells, naïve as well as memory, cannot be maintained ef fi ciently in vitro. When B cells are cultured in vitro in the presence of CD40 ligand (CD40L), cytokines, BCR triggering and Toll-like receptor (TLR) agonists they do respond. However, as soon as the B cells respond they differentiate into terminal plasma cells ( 2 ) . This process is accompanied by cell cycle arrest precluding the generation of long-term B cell lines. Recently, transduction of peripheral memory B cells with the transcriptional factors involved in the control of B cell proliferation and differentiation, B cell lymphoma-extra large (BCL-xl) and BCL-6, respectively, has allowed to immortalize and generate stable cell lines expressing BCR and are able to synthesize and secrete immunoglobulins ( 3 ) . Several approaches have been developed to dissect and investigate the molecular regulation of the human B cell repertoire. One successful method has been to combine immunoglobulin (Ig) gene repertoire analysis and Ig reactivity at the single cell level ( 4 ) . This strategy has been successfully applied for autoimmunity and immunode fi ciency questions, and for the fi rst time in human for central and peripheral B cell tolerance ( 5 ) . Among the oldest techniques to generate stable human B cell lines is the immortalization with EBV ( 6 ) . In the past, one of the major limitations was the very low ef fi ciency of B cell infection and subsequent cloning ( 7 ) . Thus, alternative methods to produce human monoclonal antibodies have been developed, such as immunization of transgenic mice expressing human Ig loci, phage display library, and humanization of mouse antibodies via genetic engineering (8) (9) (10) . We recently described an improved method to infect and immortalize human B cells with EBV, in the presence of the TLR9 agonist, CpG 2006, during viral transformation and cloning ( 11 ) . We have used this method to isolate neutralizing as well as nonneutralizing antibodies against severe acute respiratory syndrome coronavirus (SARS-CoV) ( 11 ) . These antibodies have been generated from one individual, who recovered from SARS infection. The obtained antibodies display a high in vitro potency to neutralize viral replication. This approach is not only applicable to the area of infectious diseases but also other clinical conditions, such as autoimmune diseases and cancer, and allows exploiting the breadth and the avidity of the human B cell repertoire ( 12, 13 ) . The method consists of four sequential steps: (a) human B cell subset isolation, (b) EBV infection, (c) B cell cloning, and (d) screening (Fig. 1 ). 5. 6-Well tissue culture plates. The human peripheral B cell pool is composed of cells at different stage of development, characterized by different signal requirements to differentiate into immunoglobulin secreting cells and carrying different Ig isotypes on the cell surface: (a) immature transitional 12. Centrifuge at 330 × g for 10 min at 4°C and carefully remove the supernatants by aspiration. 13. Resuspend the pellet in 2 mL of PBS 2% FCS in 5 mL propylene conical tubes. 14. After this step, cells are sorted according to B cell subset populations. 15. Dilute mAbs in 400 mL of PBS 2% FCS to stain the cells (see Table 2 for the dilution factor). 22. Sort with a BD FACSAria according to the desired phenotype (see Note 3). 1. Prewarm complete culture medium at 37°C. 2. Thaw the cryo-preserved B95.8 cells by gently agitating the cryovial in a 37°C water bath. 3. In a sterile tissue culture hood spray the vial with 70% ethanol. 4. Very gently transfer the content of the vial in a 15 mL tube and add drop by drop up to 10 mL of warm complete medium. 5. Centrifuge at 330 × g per 10 min at room temperature (RT). 6. Carefully aspirate the supernatants. Do not disturb the pellet. 7. Resuspend the cells in warm complete culture medium and transfer them in a 6-well plates at 1 × 10 6 cell/mL. Monitor cells for growth (see Note 4). The screening strategy to select the "good" antibodies is a crucial part of the human antibody development. Humoral responses to vaccination or infection can be quantitatively but also qualitatively different. This re fl ects various level of protection in vivo, related to the differences in antibody function, which can be measured in vitro. All antibodies, which bind to a given antigen can be measured by enzyme-linked immunoabsorbent assay (ELISA). antibodies utilized to select B cells, such as anti-surface Ig could provide a signal to the B cell that can in fl uence the EBV infection. Indeed in all the antibodies combinations mentioned in Table 1 the surface Ig is used to negatively select the B cell population. Moreover, it is crucial to use anti-CD22 or CD20 microbeads to enrich B cells, instead of anti-CD19 microbeads. The latter induces the internalization of CD19 surface and consequently also of CD21, which is responsible for EBV entry into B cells ( 14 ) . 4 . Make sure that B95.8 do not grow in big clusters, some cells adhere to the plastic, and are intolerant to acid conditions. It is crucial to split them every 2 days with a 1/2 or 1/3 dilution factor. The goal is to gradually expand these cells to obtain the needed amount, which depends on the size of the infection you plan to perform. When the desired volume is reached, all cells maintenance procedures (including medium change) are stopped. Complete culture medium (see Subheading 2.1 ) Sorted B cells (see Subheading 2.1 ) TLR9 agonist (CpG 2006, 5 ¢ TCg TCg TTT TgT CgT TTT gTC gTT 3 ¢ (phosphotio bonds)) (Invivogen EBV virus (see Subheading 2.2 ) Ficoll-Hypaque density gradient Complete culture medium (see Subheading 2.1 ) TLR9 agonist (CpG 2006, 5 ¢ TCg TCg TTT TgT CgT TTT gTC gTT Irradiated allogeneic PBMCs isolated from peripheral blood from normal donors EBV virus (see Subheading 2.2 ) 384-Well plates (Corning-Costar, Corning Incorporated Life Sciences Multichannel for 384 plates 5, 15, and 50 mL propylene conical tubes (Falcon) Freezing media: 90% FCS, 10% DMSO Cryovials. 1. ELISA plates, medium binding Blocking solution: PBS 10% FCS Developing buffer: 1.59 g Na 2 CO 3 , 2.93 g NaHCO 3 , and 0.2 g NaN 3 in 1 L, pH 9 ELISA substrate 104, conc. 1 mg/mL in developing buffer (cat. no. N2765, Sigma-Aldrich) B-lymphocytes effector functions in health and disease Characterization of a transitional preplasmablast population in the process of human B cell to plasma cell differentiation Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming Ef fi cient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning B-cell tolerance checkpoints in health and autoimmunity EB virus-induced B lymphocyte cell lines producing speci fi c antibody Requirements for the establishment of high-titered human monoclonal antibodies against tetanus toxoid using the Epstein-Barr virus technique Phage antibodies: fi lamentous phage displaying antibody variable domains Human antibodies from combinatorial libraries Replacing the complementarity-determining regions in a human antibody with those from a mouse An ef fi cient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus Generation of anti-NAG-2 mAb from patients' memory B cells: implications for a novel therapeutic strategy in systemic sclerosis Understanding and making use of human memory B cells Epstein-Barr virus receptor of human B lymphocytes is the C3d receptor CR2 Alternatively, if the speci fi c antigen can be expressed in the plasma membrane of a given cell line upon transfection of the relevant gene the binding of the antibody to the cell surface can be evaluated in fl ow cytometry. The ability to neutralize the speci fi c pathogen in vitro is another possible measurement of antibody function. 2. These columns separate 1 × 10 8 magnetically labeled cells from up to 10 9 . Smaller or larger column are also available.Alternatively an automated column separator (e.g., autoMACS, Miltenyi) can be used.3. In order to obtain a pure B cell population, it is necessary to use a positive selection method with immunomagnetic beads, followed by cell sorting. The positive selection method has the advantage that B cells are isolated with high degree of purity. However, unless Fab antibodies are used there is also the theoretical disadvantage that cells bearing high af fi nity Fc receptors (monocytes), may bind to the antibodies. In addition, the