Division of Immunology and Allergology
Geneva University Hospitals
Human rapamycin and IL-10-induced tolerogenic dendritic cells partially inhibit xenogeneic NK and CD8+ T cell responses in vitro
Natacha Madelon1, Gisella Puga Yung1, Jörg D Seebach1.
1Department of Medical Specialties, Geneva University Hospitals, Medical Faculty, Geneva, Switzerland
Laboratory of Transplantation and Immunology.
Introduction: Xenograft rejection mediated by natural killer (NK) cells and cytotoxic T lymphocytes (CTL) represents an obstacle for successful xenotransplantation. Dendritic cells (DC) are antigen presenting cells and play a major role in xenograft rejection by leading to NK cell and CTL activation. On the other hand, the potential of human tolerogenic DC to protect pig endothelial cells (pEC) from cell-mediated xenogeneic responses has not yet been investigated. Here, we evaluated the potential of human DC in vitro differentiated with rapamycin (Rapa-DC) or IL-10 (IL-10-DC) to specifically inhibit human anti-pig NK and CTL cytotoxicity and cytokine production.
Methods: Human monocytes were purified from the adherent fraction of peripheral blood mononuclear cells and cultured for 6 days in the presence of GM-CSF and IL-4 to differentiate into DC, or in combination with rapamycin or IL-10, to obtain tolerogenic Rapa-DC and IL-10-DC, respectively. IL-2-expanded human NK cells were cultured overnight with autologous DC, Rapa-DC or IL-10-DC at a ratio 5:1 in medium supplemented with LPS. To generate xenoantigen-specific CTL in vitro, purified human CD8+ T cells were stimulated weekly with autologous DC, Rapa-DC or IL-10-DC in combination with lethally 60Gy γ-irradiated pEC. The CTL:DC:pEC ratio in these co-cultures was 10:1:1 in medium supplemented with LPS, IL-7 and IL-2. DC phenotype, xenogeneic NK cell and CTL responses towards pEC were evaluated by measuring a panel of cell surface markers, intracellular IFNγ production and CD107a cell surface expression by flow cytometry. Lysis of pEC by NK cells and xenoantigen-specific CTL was evaluated using direct cytotoxicity assays. Cytokine production by DC was analyzed by ELISA
Results: Upon LPS stimulation, both human Rapa-DC and IL-10-DC expressed lower levels of the co-stimulatory molecules CD83 and CD86, while IL-10-DC expressed higher levels of the inhibitory molecule PD-L1. Culture supernatants from Rapa-DC and IL-10-DC exhibited reduced levels of IL-10 and IL-12p70, respectively. Both Rapa-DC and IL-10-DC significantly inhibited IFNγ production and degranulation by NK cells in response to pEC. This reduced IFNγ production by NK cells was partially caused by the defective IL-12p70 production by IL-10-DC as shown by the normalization of IFNγ production in NK-IL-10-DC co-cultures upon supplementation with IL-12. Only IL-10-DC reduced IFNγ production and degranulation of CTL towards pEC. Finally, both Rapa-DC and IL-10-DC partially inhibited lysis of pEC by xenoantigen-specific CTL in a MHC-restricted manner, as shown by a respective reduction of xenogeneic lysis of 17% and 40%.
Conclusion: In vitro differentiated tolerogenic human Rapa-DC and IL-10-DC inhibited NK and CTL-mediated cellular xenogeneic responses suggesting that they could be a useful tool to promote xenograft tolerance.
Lyssia Gruaz. Joel Pimenta. Laura Padayachy. This work was supported by SNF grant # 320030_138376.
07:00 - 08:00
|Cell & Tissue Xenotransplantation: Tolerance||Human tolerogenic dendritic cells partially inhibit xenogeneic NK and CD8+ T cell responses in vitro||Room 109|
11:00 - 12:30
|Alternative Sources of Beta Cells: Xenoislet, Stem Cells, Tissue Engineering||Protective potential of murine tolerogenic dendritic cells in rat-to-mouse islets xenotransplantation model||Room 110|