Targeting the Hypoxic Microenvironment in Cutaneous Chronic Graft-Versus-Host Disease through PI3Kδ Inhibition
Authors: Xu Yuxi , Hu Ziyi , Yang Guancui , He Yuxuan , Xu Qing , Zhou Ya , Wang Qi , Xie Xiaodong , Wang Rui , Yang Shijie , Wang Xiaoqi , Song Qingxiao , Zhang Xi 
Published: December 2024

Cutaneous chronic graft-versus-host disease (cGVHD) is a major challenge following allogeneic stem cell transplantation (allo-HSCT), negatively impacting patient quality of life and long-term survival. cGVHD pathogenesis involves complex interactions between donor immune cells and recipient tissues. Central to this inflammatory milieu are T cells, B cells, their associated autoantibodies, macrophages, and various cytokines. However, the precise cellular interactions within the immune microenvironment of cGVHD are not fully understood.

To explore this, we first established a murine model of Scl-cGVHD. Lethally irradiated BALB/c mice were transplanted with a combination of splenocytes (15x106) and bone marrow (BM) cells (10x106) from minor histocompatibility antigen (miHA)-mismatched B10.D2 donors. Compared to syngeneic controls, the allo-HSCT recipients showed body weight loss, hair loss, and an 80% mortality rate by day 60. H&E and Masson staining showed immune cell infiltration and fibrosis in the cGVHD skin. We then used spatial transcriptomics, multiplex immunofluorescence staining, western blot analysis, and flow cytometry to characterize the skin immune landscape and microenvironment in both cutaneous cGVHD patients and Scl-cGVHD mice. Our studies revealed that cGVHD skin lesions feature a hypoxic immune environment driven by IL-13, activating the PI3K-AKT signaling pathway, and hypoxia-repurposed transcriptional programs typically associated with tertiary lymphoid structures (TLSs). The TLSs followed distinct spatial patterns and involved genes linked to the PI3K-AKT pathway, cytokine and chemokine signaling, and extracellular matrix remodeling. Four cell types identified by scRNA-Seq prominently contributed to cGVHD skin lesions: cytokine-producing Th and Tfh-like tissue-resident memory cells, Ccl5+ TEM/TEMRA CD8+ T cells, hypoxia-induced inflammatory macrophages, and fibroblasts with tissue-remodeling phenotypes.

Our study presents the potential of PI3Kδ inhibition as a novel therapeutic strategy for cutaneous cGVHD. We tested the PI3Kδ inhibitor (PI3Kδi, Parsaclisib) in the Scl-cGVHD model, the major MHC-mismatched model (C57BL/6→BALB/c) and the Xeno-GVHD model. The results showed PI3Kδ inhibition markedly reducing cGVHD skin scores, with prevention of hair loss and reduced cell infiltration, collagen deposition, and skin fibrosis, as well as improving overall survival. Mechanistically, inhibition of PI3Kδ effectively suppresses HIF1αhi IL-13+ CD4+ T cells, HIF1αhi CCL5+ CD8+ T cells and pro-fibrotic macrophages, preserved HIF1αlo NKT and collagen degradation TLF+ macrophages. More importantly, inhibition of PI3Kδ reduces chemokine signaling, and prevents TLS formation.

In conclusion, our findings highlight three critical regulatory processes for cutaneous cGVHD pathogenesis: (1) Cellular response to hypoxia and PI3K/AKT activation, (2) Chemokine signaling mediating cell migration and adhesion, and (3) ECM remodeling and Fibrosis. For the first time, we describe a hypoxia gene signature in the skin of cGvHD patients and confirm this pathophysiology in murine models. Remarkably, PI3Kδ inhibition reversed skin hypoxia, prevented the development of pathogenic TLS, and ameliorated cGVHD histopathology, suggesting PI3Kd inhibition emerges as a promising therapeutic approach, alleviating disease symptoms and addressing fundamental mechanisms of disease progression. These insights pave the way for innovative treatments targeting the hypoxic milieu in cutaneous cGVHD.