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Abstract

Graft-versus-host disease (GVHD) remains a major complication following hematopoietic stem cell transplantation (HCT), affecting the skin as the earliest and most common target. Skin infiltrating alloreactive lymphocytes causing keratinocyte cell death are pathophysiological hallmarks of GVHD. In search for a therapeutic approach targeting skin localized alloreactive lymphocytes in acute GVHD (aGVHD), near-infrared photoimmunotherapy (NIR-PIT) was established as a strategy that has not been yet introduced in the field of dermatology. NIR-PIT combines the systemic application of a monoclonal antibody labelled with a photosensitizer. Upon exposure to near-infrared light, the conjugated agent causes rapid cell death along with its fluorescence emission. Initially, the selectivity of this approach was demonstrated in vitro when targeting CD8 T cells. For studying the therapeutic value of NIR-PIT in vivo, a humanized mouse model of cutaneous aGVHD was established. Here, CD8 specific NIR-PIT caused the entire and selective ablation of CD8 T cells from human skin grafts in which aGVHD was induced. At the same time, inhibition of histopathological signs and expression of the disease specific biomarker anti-leukoprotease elafin were observed. These studies assigned important effector functions to CD8 T cells in the context of cutaneous aGVHD. Hence, NIR-PIT can be emphasized to serve as a novel skin-selective therapeutic approach for clinical use. Up to this point keratinocyte cell death that occurs downstream to allorecognition was believed to result from either apoptosis or unspecific necrosis. As an extension of the first part of these studies, the exact mode of keratinocyte cell death in aGVHD was examined. To this end, we compared lesional and non-lesional skin obtained from aGVHD patients and observed a strong upregulation of molecules of the necroptotic signaling pathway: nucleic-acid (NA) sensor Z-DNA-binding protein 1 (ZBP1), phosphorylated receptor interacting protein kinase 3 (RIP3) and phosphorylated mixed-lineage kinase like transcription profile (MLKL). The chief role of interferon gamma (IFNγ) for inducing the activation and de novo expression of the necroptotic signaling pathway was initially demonstrated in keratinocyte cultures. These findings were both confirmed in ex vivo skin organ cultures and in vivo in a humanized mouse model of experimental human aGVHD. Finally, inhibition of STAT1 and RIP3 activities using tofacitinib and GSK’872, respectively, could prevent necroptosis, thus confirming the results. These studies uncovered how IFNγ-mediated necroptosis and the necroptotic signaling pathway play a central role in aGVHD related keratinocyte cell death. While in the first part of these studies the important role of alloreactive CD8 T cells in aGVHD and a novel therapeutic approach for a targeted therapy of skin diseases was introduced, subsequent studies identified necroptosis as a novel cell death pathway executed by IFNγ producing alloreactive T cells.