Sophie M. Bilik, Caroline Dodson, Katelyn Rivas, Nathan Balukoff, Anthony Griswold, Jamie L. Burgess, Andrew P. Sawaya, Irena Pastar, Natasa Strbo, Maria I. Morasso, Marjana Tomic-Canic, Rivka C. Stone

Diabetic foot ulcers (DFUs) are a common and debilitating complication of diabetes, and amputations from non-healing ulcers carry high morbidity and mortality. A critical need exists for biomarkers that can identify healing potential early and guide targeted interventions. To address this, we applied an integrated multi-omics approach across four patient cohorts comprising 51 DFUs (29 Healing, 22 Non-healing). Bulk RNA-sequencing revealed marked activation of Th1 and Th2 pathways (activation z-score +4.8, p = 3.8×10-15), and immune cell deconvolution predicted higher proportions of T cell populations in Healers. Spatial proteomics in a second cohort identified elevated CD3+ T cell density and selective enrichment of PD-1 and PD-L1 expression in vascular niches of the papillary dermis in Healers (p < 0.001). Flow cytometry in a third cohort further demonstrated higher proportions of CD3+PD-1+ and CD3+PD-L1+ T cells in Healers compared with Non-healers. Single-cell RNA-sequencing from a fourth cohort showed upregulation of PD-1 and PD-L1 within CD4+ T cells from Healers. Complementary immunofluorescence and serological profiling confirmed that both PD-1 and PD-L1 are elevated in tissue and circulating serum of healing DFUs, supporting their potential use as systemic biomarkers. Taken together, vascular-enriched PD-1/PD-L1 signaling and T cell activation were observed in association with healing DFUs, supporting PD-1/PD-L1 as candidate biomarkers in both tissue and blood with potential translational relevance for predicting DFU outcomes and informing precision therapies.