Complement-activated interferon-γ–primed human endothelium transpresents interleukin-15 to CD8+ T cells

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

“…Human EC responses to complement were elicited using discarded and pooled preparations of high titer PRA sera obtained from the Yale HLA tissue typing laboratory to treat serially passaged human umbilical vein EC (HUVEC) cultures, as previously described. 6 HUVECs from a donor allogeneic to the peripheral blood mononuclear cells (PBMCs) were pretreated with human IFN-γ for 48 h prior to treatment with complement permissive gelatin veronal buffer (GVB), 25% PRA in GVB, IL-1 receptor antagonist (IL-1Ra) +25% PRA/GVB, or anti-IL-15 blocking antibody +25% PRA/GVB. Peripheral blood human CD8 + CCR7 − HLA-DR − and CD4 + CCR7 − HLA-DR − T EM lymphocytes were prelabeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) dye and cocultured with allogeneic HUVECs for 7 days (1:20:30 EC:CD4:CD8 ratio).…”

“…In this experiment, we combined the CD4 + and CD8 + T EM , isolated at a 3:2 ratio in the cocultures with ECs, extending our prior work in which these populations were tested separately. 6,9 Preventing recognition of surface IL-15/IL-15Rα with anti-IL-15 blocking antibody significantly reduced the augmentation of the CD8 + but not CD4 + T EM expansion. Furthermore, the alloantibody and complement-activated ECs had enhanced ability to induce augmented proliferation compared to normal sera and vehicle control-treated ECs, measured by CFSE dye dilution (Figure S1).…”

“…This was initially surprising as IL-1Ra blocked the translocation of IL-15/IL-15Rα complexes to the cell surface of isolated human ECs treated with PRA in culture. 6 Although activated T cells are not a major source of IL-1, they can produce TNF, which can replace IL-1 by acting on ECs, triggering NF-κB activation and subsequently IL-15/ IL-15Rα translocation to the EC surface. 6 To determine if TNF was present in the EC:T cell cocultures, we performed ELISA assays and readily detected TNF-α after 24 h of coculture, especially in the PRAactivated EC group compared to control and normal sera treated (Figure S2).…”

“…IL-1β then acts back on the ECs to increase their capacity to recruit and activate alloreactive CD4 + and CD8 + T EM , assessed as increased proliferation. 6,9 Among the IL-1β-induced effects is translocation of IL-15/IL-15Rα complexes from the nucleus, where they reside after IFN-γ-induction, to the cell surface where IL-15 can be transpresented to T EM , contributing to enhanced CD8 + but not CD4 + T EM proliferation. 6 These observations provide a putative mechanism through which DSA can augment T cell responses, contributing to mixed rejection.…”

“…6,9 Among the IL-1β-induced effects is translocation of IL-15/IL-15Rα complexes from the nucleus, where they reside after IFN-γ-induction, to the cell surface where IL-15 can be transpresented to T EM , contributing to enhanced CD8 + but not CD4 + T EM proliferation. 6 These observations provide a putative mechanism through which DSA can augment T cell responses, contributing to mixed rejection. We have supported our in vitro observations using a human immune system mouse model of allograft vasculopathy.…”