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

Abstract

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 have developed a culture method to support the efficient activation and proliferation of both naïve and memory human B cells. This culture supports extensive B-cell proliferation, with approximately 103-fold increases following 8 days in culture, and 106-fold increases when cultures are split and cultured for 8 more days. In culture, a significant fraction of naïve 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, proliferating CD B cells express high levels of MHCII, CD80, and CD86. CD B cells act as APCs and present both alloantigens and microbial antigens to T cells. We are able to activate and expand antigen-specific memory B cells; these cultured cells are highly effective in presenting antigen to T cells. We have characterized the TCR repertoire of rare antigen-specific CD4+ T cells that proliferated in response to tetanus toxoid (TT) presented by autologous CD B cells. TCR Vβ usage by TT-activated CD4+ T cells differs from both resting and unspecifically activated CD4+ T cells. Moreover, we found that TT-specific TCR Vβ 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.

Self-tolerance curtails the B cell repertoire to microbial epitopes

“…To characterize human B cell repertoires before and after the second tolerance checkpoint (1, 2), we isolated single transitional B cells (CD19 + CD27 -CD38 hi CD10 + IgD + ) and mature, naive B cells (CD19 + CD27 -CD38 lo CD10 -IgD + ) from PBMCs of healthy donors (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.122551DS1) (23), and cultured individual B cells in Nojima cultures, which support proliferation and plasmacytic differentiation of human B cells (16,17). After 25 days of culture, we harvested culture supernatants containing clonal IgG produced by the differentiated progeny of single transitional or mature B cells and screened them on a panel of foreign and self-antigens (17,24).…”

“…To survey for tolerance-induced holes in the human BCR repertoire before and after the second tolerance checkpoint, we cultured single human B cells on stromal cell layers that support B cell proliferation and differentiation to IgG-secreting plasmablasts and plasmacytes (16,17). In this way, we obtained 2331 clonal IgGs from individual transitional and mature B cells representing the BCR repertoires before and after the second tolerance checkpoint (1,2).…”

B Cell-Based Cancer Immunotherapy

“…The ability of human CD40B cells to expand antigen-experienced CD4+ T cells, but also to prime naïve CD4+ T cells was demonstrated in several studies [15, 17, 22, 24, 26, 35, 37, 44, 45]. Lapointe et al [37] showed that when pulsed with tumor lysates, CD40B cells expanded and activated tumor antigen-specific memory CD4+ T cells from the blood of cancer patients.…”

Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek’s Disease Virus

“…Moreover, activation of lymphocytes by soluble CD40L and anti-TCR-␣␤ antibody alters cell metabolism (30), which can potentially mask the effects of MDV infection on metabolism. Unactivated human or chicken lymphocytes undergo apoptosis in vitro, and only cell activation by various ligands can reduce cell death in lymphocyte culture models (31,32). Therefore, the effects of MDV infection on glycolysis and glutaminolysis were initially studied in CEFs, but these data should be confirmed in immune cells in future studies.…”