Complement-activated human endothelial cells stimulate increased polyfunctionality in alloreactive T cells

Antibody-mediated deposition of complement membrane attack complexes (MACs) on IFN-γ-primed human endothelial cells (ECs) triggers autocrine/paracrine IL-1β-mediated EC activation and IL-15 transpresentation to alloreactive effector memory T cells (T_EM ), changes that enable ECs to increase T cell proliferation and cytokine release. Here, we report the use of single-cell microchip 32-plex proteomics to more deeply assess the functionality of the activated T cells and dependence upon EC-derived signals. Compared to control ECs, MAC-activated human ECs increase both the frequency and degree of polyfunctionality among both CD4⁺ and CD8⁺ -proliferated T_EM , assessed as secreted proteins. IFN-γ and TNF-α remain the predominant cytokines made by alloreactive T_EM , but a few CD4⁺ T_EM also made IL-4 while more CD8⁺ T_EM made perforin and granzyme B. Increased polyfunctionality was attenuated by treatment of the MAC-activated ECs with anti-IL-15 blocking antibody more effectively than IL-1 receptor blockade. The increased polyfunctionality of T cells resulting from interactions with MAC-activated ECs may further link binding of donor-specific antibody to T cell-mediated allograft pathologies.