The Ca2+ concentration in vitro impacts the cytokine production of mouse and human lymphoid cells and the polarization of human macrophages

Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca 2+ concentration of at least 1.5 mM during in vitro stimulation is needed for optimal cytokine production by conventional αβ T cells. Here we extend these findings by showing that also unconventional T cells (invariant Natural Killer T cells, mucosal-associated invariant T cells, γδ T cells), as well as B cells, show an increased cytokine response following in vitro stimulation in the presence of elevated Ca 2+ concentrations (approx. 1.8 mM). This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence their survival. A similarly increased cytokine response due to elevated Ca 2+ levels was observed with primary human monocytes. In contrast, primary human monocyte-derived macrophages, either unpolarized (M0) or polarized into M1 or M2 macrophages, displayed increased cell death in the presence of elevated Ca 2+ concentrations. Furthermore, elevated Ca 2+ concentrations promoted phenotypic M1 differentiation by increasing M1 markers on M1 and M2 macrophages and decreasing M2 markers on M2 macrophages. However, the cytokine production of macrophages, again in contrast to the lymphoid cells, was unaltered by the Ca 2+ concentration. In summary, our data demonstrate that the Ca 2+ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca 2+ levels can boost cytokine production by both mouse and human lymphoid cells.