Single-cell spatial mapping of immune microenvironments across the spectrum of skin rejection in vascularized composite allotransplantation

Introduction: Rejection of the skin component remains an important challenge in vascularised composite allotransplantation. Despite this, detailed molecular characterisation of allogeneic skin rejection remains limited due to sample scarcity and the resolution of prior transcriptomic studies. A deeper understanding of the cellular and immune coordination underlying skin rejection may refine histopathological assessment and support emerging strategies that utilise vascularised skin for monitoring of solid organ transplant status. To investigate rejection-associated changes within the cutaneous microenvironment, a cohort of allogeneic skin biopsies spanning the spectrum of rejection severity was analysed using spatial single-cell transcriptomics.
Methods: Thirteen skin biopsies from eight skin allograft recipients were profiled on the Xenium platform (10x Genomics) using a 420-gene panel comprising a pre-designed 370 gene multi-tissue panel, supplemented with 50 custom genes selected from a literature review and preliminary whole-transcriptome analysis. Following quality control and removal of low-quality regions, 217,860 cells were retained for downstream analyses. Data were normalised and integrated prior to automated cell annotation augmented with manual curation, using an inflammatory skin reference atlas. Cell composition, cellular neighbourhoods, and receptor-ligand interaction analyses were performed to investigate rejection-associated spatial and cellular changes.
Results: Distinct changes in the cutaneous immune microenvironment were observed across rejection grades. Spatial analyses demonstrated clustering of inflammatory and regulatory immune infiltrates around vascular structures, consistent with the perivascular organisation of cutaneous rejection. A trend towards reduced Langerhans cell abundance and shifts in macrophage subset frequencies were amongst the observed changes with increasing rejection severity. Differential gene expression analyses between populations within tissue niches, and receptor-ligand analyses, demonstrated spatial changes in cell phenotypes and associations; fibroblast associated Tregs, as an example, exhibited upregulation of canonical regulatory and tissue resident transcripts including FOXP3, IL2RA and CXCR4 whilst Tregs in immune infiltrated areas exhibited signatures suggestive of increased dendritic cell subset interactions.
Conclusion: In summary, these findings provide a high-resolution, descriptive analysis of cutaneous alloimmune responses across the spectrum of rejection severity.