Multi-Omics Profiling of Skin Biopsies of Patients with Sclerodermatous Graft-Vs-Host Disease Suggests Therapeutic Potential of Targeting Don't Eat Me Signals

Introduction: Chronic graft-vs-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HCT). This debilitating, chronic condition is characterized by inflammation, cell-mediated and humoral immunity, and ultimately tissue fibrosis. There is currently little or no understanding of the molecular pathogenesis of chronic cGVHD resulting in poor effective treatment strategies. Sclerodermatous GVHD (sclGVHD) is a highly morbid form of cGVHD associated with poor prognosis and low sensitivity to immune suppressive therapy. Understanding the underlying pathophysiology of sclGVHD and identifying the keyfibroticpathways involved iscriticalfor translation into targeted therapies that improve patient care.

Methods: To further our understanding of the underlying pathophysiology of sclGVHD we used single cell RNA sequencing analyses on 5 fresh patient biopsy specimens and healthy controls. Human studies were carried out using: i) Patient skin biopsy samples (n =45) and matching them with 5 normal matched skin biopsy controls. These patient tissue samples were used to perform spatial gene expression analyses (10X Vizium) and identify the unique molecular profiles in each SclGVHD sample. ii) ATAC-sequencing analyses was performed on primary dermal fibroblasts from patients with SclGVHD to assess chromatin accessibility patterns genome wide in SclGVHD iii) TMA and tissues from patients were used to validate targets using immunofluorescent staining. iv) Animal studies were conducted to evaluate the therapeutic potential of an immune therapy-based treatment approach with a combined anti-fibrotic/anti-inflammatory strategy, and effectiveness of the treatment was determined with CyTOF studies.

Results: To investigate cGVHD immunopathogenesis, normal-appearing skin, lesioned skin, and circulating immune cells from chronic patients were analyzed via single-cell RNA sequencing (figure A). A distinct transcriptional signature, the upregulation of clusters 11, 3, 1 and 17 in Scl cGVHD and the downregulation of clusters 6, 5, 9, 8 was observed. To validate these results in the context of tissue architecture we integrated spatial RNA sequencing on a tissue microarray assembled with 45 skin biopsies of SclGVHD patients to demonstrate a tight correlation between JUN activation and immune checkpoint upregulation (figure B).Hierarchical clustering of ATAC-seq signals revealed that the chromatin landscape was greatly remodeled after JUN deletion (figure C).We blocked CD47 and IL6 in the SclGVHD mouse model to evaluate therapeutic efficacy. We found a significant decrease of dermal thickness and collagen deposition in SclGVHD mice treated with blocking antibodies against IL6 and CD47 but not in untreated mice (figure D). We also detected a noticeable decrease in the expression of CD47, pJUN and IL6 in the treated group (figure E). Analyses of skin tissues from sclerotic lesions in mice using CyTOF revealed different cell populations in the skin clustered in spatially distinct groups. Finally, an increase in total leukocyte as well as a significant decrease in the fibroblast populations was observed (figure F).

Conclusion: Our findings are significant because we profile skin biopsies matched with peripheral blood from patients with Scl GVHD with a multiomics approach and identify a unique molecular signature in patients with SclGVHD thus providing a rationale for subsequent combined interventions targeting innate immunity.

Arai:Kadmon: Membership on an entity's Board of Directors or advisory committees. Shizuru:Jasper Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; rBio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Shoreline BioSciences: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Müller:Novartis: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.