Interleukin-21 Drives Proliferation and Differentiation of Porcine Memory B Cells into Antibody Secreting Cells

Abstract

Immunological prevention of infectious disease, especially viral, is based on antigen-specific long-lived memory B cells. To test for cellular proliferation and differentiation factors in swine, an outbred model for humans, CD21+ B cells were activated in vitro with CD40L and stimulated with purported stimulatory cytokines to characterize functional responses. IL-21 induced a 3-fold expansion in total cell numbers with roughly 15% of all B cells differentiating to IgM or IgG antibody secreting cells (ASCs.) However, even with robust proliferation, cellular viability rapidly deteriorated. Therefore, a proliferation inducing ligand (APRIL) and B cell activating factor (BAFF) were evaluated as survival and maintenance factors. BAFF was effective at enhancing the viability of mature B cells as well as ASCs, while APRIL was only effective for ASCs. Both cytokines increased approximately two-fold the amount of IgM and IgG which was secreted by IL-21 differentiated ASCs. Mature B cells from porcine reproductive and respiratory virus (PRRSV) immune and naïve age-matched pigs were activated and treated with IL-21 and then tested for memory cell differentiation using a PRRSV non-structural protein 7 ELISPOT and ELISA. PRRSV immune pigs were positive on both ELISPOT and ELISA while naïve animals were negative on both assays. These results highlight the IL-21-driven expansion and differentiation of memory B cells in vitro without stimulation of the surface immunoglobulin receptor complex, as well as the establishment of a defined memory B cell culture system for characterization of vaccine responses in outbred animals.

The PRRSV-Specific Memory B Cell Response Is Long-Lived in Blood and Is Boosted During Live Virus Re-exposure

“…The normalization of collected data across different tissue types and lymph nodes was achieved by dividing the number of live (fixable viability dye − ), CD21 + , nsp7 tetramer + , and decoy tetramer − cells by the total number of live CD21 + B cells that were incubated with the nsp7 tetramer prior to pulldown enrichment and is presented as nsp7-specific B cells/million B cells. The frequency of nsp7specific memory B cells has been shown previously to be very low and this was confirmed in the tissues examined here (22). Single negative control animals were used for each time point and tissue which were used to compare with vaccinated animals' memory cell response.…”

“…Solid tissue was processed as previously described with the following modifications (22). Lymph nodes and tonsil were scored with a sterile razor blade prior to cellular dissociation using the plunger of a syringe.…”

“…Once bound, there are three other antigens which may be bound by additional surface immunoglobulins of the B cell, resulting in a highly avid reagent. However, because nsp7-specific B cells within tissue are very rare, ∼1 in 100,000 B cells, a magnetic bead enrichment step is necessary to concentrate and enumerate tetramer bound cells (22).…”

Interleukin (IL)-21 Promotes the Differentiation of IgA-Producing Plasma Cells in Porcine Peyer's Patches via the JAK-STAT Signaling Pathway

“…IL-21 binds to the receptor IL-21R dominantly expressed on B cells and enhances B cell proliferation, maturation, plasma cell and memory B cell differentiation, and immunoglobulin class switching in mouse models and human studies (27)(28)(29). A previous study showed that IL-21 drove the proliferation and differentiation of porcine spleen B cells into antibody-secreting cells (30). In this study, we found that exogenous IL-21 combined with CD40L significantly upregulated the IgA production level in porcine B cells through the promotion of proliferation, PC differentiation and IgA CSR of B cells, which was consistent with the findings in humans (31,32).…”

Molecular Dissection of the Antibody Response: Opportunities and Needs for Application in Cattle

“…This knowledge has been applied to successfully culture activated porcine B cells, taking advantage of the cross-reactivity of the human reagents with porcine B cells (60). Addition of IL-21 plus CD40L to purified pig B cells resulted in activation and proliferation over a 4-day period, and inclusion of BAFF and APRIL maintained the viability of the cells for 7 days.…”