*Purpose: Lymphatic vessel (LV) infiltration and sprouting, termed lymphangiogenesis, is a feature of chronic rejection (CR), but its role remains unclear. Although LVs play a pivotal role by resolving inflammation within tissues, they may propagate alloimmune-mediated injury by enabling alloantigen delivery to draining lymph nodes. We therefore aimed to characterise the structural and molecular properties of lymphatics in chronic kidney transplant rejection.
*Methods: We used wholemount immunofluorescence, optical clearing and three-dimensional microscopy to study LV architecture and spatial relationships in explanted renal allografts with chronic mixed (T cell- and antibody-mediated) rejection, using deceased-donor kidneys (not utilised for transplantation) and allografts with non-alloimmune chronic dysfunction as control tissues. In parallel, we used single-cell RNA-sequencing (scRNA-seq) to profile the gene expression of LVs, before ligand-receptor pair analysis to assess molecular crosstalk between LVs and T cells.
*Results: Donor kidneys displayed a hierarchical arrangement of LVs. Cortical LVs expand seven-fold in density in CR and infiltrate the medulla to invade and interconnect tertiary lymphoid structures. As well as demonstrating expression of class II human leukocyte antigens by LVs, we found C4d deposition along LVs in CR. In contrast to controls, the spatial proximity of CD4⁺ T cells to LVs was perturbed in CR, with concurrent loss of chemokine sequestration by LVs. Ligand-receptor pair analysis of scRNA-seq data suggested enhanced co-inhibitory interactions between LVs and CD4⁺ T cells (Figure 1).
*Conclusions: Our data provides a new perspective on the participation of lymphatics in human alloimmunity. We suggest that intra-graft LVs act to limit local immune responses, but are structurally and functionally perturbed in the setting of CR. We propose that targeting lymphatic dysfunction may represent a promising therapeutic strategy for promoting long-term kidney allograft survival.

Daniyal Jafree

University College London

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