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.