Vascularised composite allotransplantation (VCA) can offer functional and aesthetic restoration after significant tissue loss. Despite its potential, VCA recipients face significantly heightened rates of acute rejection compared to solid organ transplant recipients. The immunogenicity of skin, a common component of VCAs, is theorised to contribute to this apparent vulnerability to rejection. Due to small patient cohorts and limited clinical data, it remains unclear whether the skin component increases sensitisation or alters rejection mechanisms in this setting. To address this gap, we investigated the tissue transcriptome of a cohort of biopsies from patients undergoing intestinal transplantation alone or in combination with an abdominal wall VCA, assessing the allograft immune niche during stable function and active rejection.

FFPE biopsies from combined abdominal wall/small bowel VCA (n=11) and intestinal transplant (ITx) alone (n=6) recipients were evaluated via whole transcriptome (~18,000 genes) spatial profiling (Nanostring GeoMx), augmented with a custom T-cell receptor spike-in for in situ TCR diversity assessment. Within the biopsies, specific regions of interest were selected and analysed based on current Banff rejection criteria. A serial section was processed via iterative rounds of multiplex immunofluorescence (~21 markers) to characterise the allograft immune niche and tissue architecture. Data analysis was conducted using R, with DESeq and Clusterprofiler used for differential gene expression (DGE) and GSEA respectively. Differentially expressed genes were defined as log fold change > 0.5 and adjusted p value < 0.05.

Within the rejecting intestinal component of the VCAs, crypt regions demonstrated pronounced induction of apoptotic signalling and myeloid cell differentiation compared to the ITx cohort. DGE analysis revealed minimal overlap in upregulated genes between the VCA and ITx cohorts, although both showed upregulation of the anti-angiogenic gene SERPINF1 and apoptotic marker CLU. Notably, 48 genes were specifically upregulated in the VCA group, including CXCL14, implicated in dendritic cell maturation and MHC-I upregulation, as well as CD44 and CD55. DGE analysis of graft infiltrate regions between groups revealed a significant upregulation of neutrophil chemoattractants CXCL1, CXCL5 and CXCL6 in the rejecting VCA cohort. Multiplex immunofluorescence provided further insight into the composition of infiltrates within the biopsies and will be correlated with transcriptomic findings.

The distinct rejection signatures observed in the VCA cohort compared to ITx alone group suggests the addition of a skin component to visceral transplant may have unique immunological consequences with potential clinical significance. Ongoing analysis will focus on the rejection signatures between tissues, with the aim of determining any tissue specific pathways that may be relevant to rejection processes.