CSF-1R+ macrophages orchestrate human skin chronic graft-versus-host disease

Abstract

Abstract Skin is the most frequently involved organ in chronic graft-versus-host disease (cGVHD) and presents as superficial lichenoid lesions and deep, fibrotic sclerosis that may arise sequentially. Preclinical models have demonstrated cGVHD involves colony stimulating factor-1 receptor (CSF-1R)-dependent tissue macrophage infiltration. However, spatial and temporal analysis of immune responses at high resolution has not been possible in human tissue to date, a major limitation to our understanding of disease pathophysiology and responses to therapy. We undertook sub-cellular spatial transcriptomics with the 10x Xenium platform to dissect tissue immune networks involved in human patient skin, first from lichenoid cGVHD biopsies relatively early after HCT (n=8), then before and after treatment with the CSF-1R-blocking antibody axatilimab in later sclerotic phases in patients enrolled in the AGAVE-201 trial that led to FDA approval (Wolff D et al, NEJM 2024) (n=5 responders + 5 non-responders). Skin with confirmed lichenoid cGVHD histopathology segregated into high (HI) and low (LI) immune cell-infiltrated tissue that reflected macrophage and T cell abundance. Non-cGVHD biopsies collected before or after HCT served as controls. While CSF1Rhi macrophages were equally abundant irrespective of cGVHD, an alternatively activated CSF1RintLYZhi population was the predominant subset differentially infiltrating HI skin (p=0.0011) and resided closest to basal keratinocytes (BKs) by proximity analysis. CD8 T cells were the dominant infiltrating lymphocytes distinctive to HI skin (p=0.0016) and in close bidirectional proximity to CSF1RintLYZhi macrophages (p<0.0001). These immune cells were, in turn, located adjacently to BKs, forming a putative pathogenic triad. Highly aberrant spinous and basal keratinocyte (BK) differentiation was seen deep within cGVHD epidermis. Closer BK proximity of CSF1RintLYZhi macrophages and type-1 and type-17 T cells (expressing IFNG, TBX21 and IL17A, RORC respectively) was associated with increased BK transcripts associated with immune cell recruitment (GJB2) and antigen presentation (CD74), suggesting immunomodulatory functions of keratinocytes in cGVHD. These HI keratinocytes bore high TGFB1 and ZEB2 and low CDH1 expression, consistent with epithelial-mesenchymal transition. Within the dermis, major fibroblast subsets were ablated during cGVHD, including the papillary fraction abutting the epidermal-dermal border and the dominant reticular LGR5+-equivalent subset (p=0.0376), pivotal to tissue organization and homeostasis. A proportion (5-10%) of residual skin fibroblasts were donor origin and expressed features of macrophage-mesenchymal transition. We next analyzed pre- and post-treatment skin biopsies from patients with steroid-refractory sclerosis enrolled on the AGAVE-201 study. As opposed to lichenoid cGVHD, cellular infiltration, including CSF-1R+ macrophages, was relatively sparse in this late phase of disease. Nevertheless, overall response to axatilimab was associated with a significant depletion of macrophages, including CSF-1Rhi and CSF1RintLYZhisubsets and concurrent increases in FoxP3+ regulatory T cells in the skin (p<0.05), suggesting that a systemic regulatory response may mediate clinical response. These immune subsets were unchanged in non-responders. LGR5+ fibroblasts, the main reticular population, demonstrated increased baseline collagen, M2 and myofibroblast-polarizing and collagen-amplifying gene transcripts in axatilimab non-responders. Similarly, papillary fibroblasts from non-responders expressed high baseline levels of profibrogenic TGF-β target genes. In contrast, papillary fibroblasts were characterized by lower activation states in responders. These data highlight a specific, aberrant CSF1R+ tissue macrophage population in lichenoid skin cGVHD and its colocalization in a putative pathogenic triad with CD8 T cells at the BK interface culminating in profound, previously unrecognized abnormalities in keratinocyte and fibroblast differentiation and mesenchymal transition—potential harbingers of fibrosis. The deletion of CSF-1R+ macrophages with axatilimab in more sparsely infiltrated sclerotic phases of cGVHD was associated with Treg expansion and clinical response. These data confirm the central role of macrophages in human cGVHD, provide a putative mechanism of response to CSF-1R inhibition, and suggestion treatment at the lichenoid stage may represent an optimal therapeutic window.