Upon priming by antigens, B cells undergo activation, proliferation, and differentiation into antibody-secreting cells. During thymus-dependent (TD) antibody responses, the proliferation and differentiation of antigen-primed B cells essentially rely on the helper function from CD4+ T cells. Follicular helper T (Tfh) cells constitute a specialized Th subset that localizes in close proximity to B cells and supports B cell proliferation and differentiation through co-stimulatory receptors and cytokines. Impaired Tfh-mediated B cell proliferation and differentiation were observed in patients with immunodeficiency, while overactivation of this process may lead to dysregulated immune responses seen in autoimmune disorders. Here, we describe an ex vivo coculture assay using circulating Tfh cells and B cells isolated from human blood. This method can be used to examine the function of patients' B cells for proliferation, differentiation, and antibody secretion, mediated by the physiological help from Tfh cells.
Calcineurin inhibitors (CNI), used frequently in solid organ transplant patients, are known to inhibit T cell proliferation, but their effect on humoral immunity is far less studied. Total and naive B cells from healthy adult donors were cultured in immunoglobulin (Ig)A- or IgG/IgE-promoting conditions with increasing doses of cyclosporin, tacrolimus, rapamycin or methylprednisolone. The effect on cell number, cell division, plasmablast differentiation and class-switching was tested. To examine the effect on T follicular helper (Tfh) cell differentiation, naive CD4(+) T cells were cultured with interleukin (IL)-12 and titrated immunosuppressive drug (IS) concentrations. Total B cell function was not affected by CNI. However, naive B cell proliferation was inhibited by cyclosporin and both CNI decreased plasmablast differentiation. Both CNI suppressed IgA, whereas only cyclosporin inhibited IgE class-switching. Rapamycin had a strong inhibitory effect on B cell function. Strikingly, methylprednisolone, increased plasmablast differentiation and IgE class-switching from naive B cells. Differentiation of Tfh cells decreased with increasing IS doses. CNI affected humoral immunity directly by suppressing naive B cells. CNI, as well as rapamycin and methylprednisolone, inhibited the in-vitro differentiation of Tfh from naive CD4(+) T cells. In view of its potent suppressive effect on B cell function and Tfh cell differentiation, rapamycin might be an interesting candidate in the management of B cell mediated complications post solid organ transplantation.
