Expression of TRAIL (tumor necrosis factor–related apoptosis–inducing ligand) by immune cells can lead to the induction of apoptosis in tumor cells. However, it becomes increasingly clear that the interaction of TRAIL and its death receptors (DRs) can also directly impact immune cells and influence immune responses. Here, we review what is known about the role of TRAIL/DRs in immune cells and immune responses in general and in the tumor microenvironment in particular.

Macrophages are highly plastic cells that can polarize into functionally distinct subsets in vivo and in vitro in response to environmental signals. The development of protocols to model macrophage polarization in vitro greatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers in in vitro experiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs) in vitro, we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1β, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGFβ) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markers in vitro. Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.

Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca2+ 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 Ca2+ concentrations. 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 Ca2+ 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 Ca2+ concentrations. Furthermore, elevated Ca2+ 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 Ca2+ concentration. In summary, our data demonstrate that the Ca2+ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca2+ levels can boost cytokine production by both mouse and human lymphoid cells.