NKG2D receptor ligands are cell surface biomarkers for injured murine and human nociceptive sensory neurons

Nociceptors are primary afferent neurons that sense noxious stimuli. They can be activated by tissue injury as well as the accompanying local immune response. We have shown that following nerve injury in mice cytotoxic Natural Killer (NK) cells infiltrate the peripheral nerve and interact with stress-induced ligands of the activating receptor NKG2D (Klrk1). However, the diversity and specificity of NKG2D receptor ligands among sensory neuron subtypes, and translation of this mechanism to human cells, remains unknown. We used dorsal root ganglion (DRG) neurons cultured from C57BL/6J mice of both sexes with fluorescently labelled sensory neuron lines (Scn10a, Mrgprd, Calca, Trpv1, Th, Thy1), as well as human induced pluripotent stem cell-derived (hiPSCd)-sensory neurons after laser ablation, as in vitro models of axonal injury. We assessed the expression of NKG2D ligands by quantitative polymerase chain reaction (PCR) corroborated by publicly available RNA sequencing datasets and validated with single-cell PCR. Recombinant NKG2D receptor proteins in live cell-based assays were used to reveal the subcellular membrane localisation of NKG2D ligands with quantification by semi-automated image analysis. Functional interactions between human NK cells and sensory neurons were confirmed with co-cultures in microfluidic devices. We show that NKG2D ligands are expressed exclusively in unmyelinated DRG neurons after injury. Soluble mouse NKG2D receptors bound to puncta along distal neurites of injured axons enriched predominantly in Mrgprd-expressing non-peptidergic nociceptors. We observed low-level binding of soluble human NKG2D receptors to neurites of hiPSCd sensory neurons that increased after axonal laser ablation. Degeneration of hiPSCd sensory neurites by interleukin (IL-2) primed human NK cells was prevented by an NKG2D blocking antibody. The induction and enrichment of functional NKG2D receptor ligands selectively on pathological nerve fibres could aid the diagnosis of peripheral neuropathy in chronic pain conditions, and sheds new light on the potential role of nociceptive neurons in regulating the local tissue immune microenvironment.