Redirecting IL-8 Signaling Through CXCR1-CD3ζ–Engineered Regulatory T Cells Enhances Treg Persistence and Alleviates Neuropathic Pain

BACKGROUND: Neuropathic pain is driven by persistent neuroimmune activation. Regulatory T cells (Tregs) can suppress inflammation, but their therapeutic efficacy is limited by poor survival and persistence in inflammatory tissues. We investigated whether engineering Tregs to redirect interleukin-8 (IL-8) signaling could improve their therapeutic activity. METHODS: A rat chronic constriction injury (CCI) model was used to evaluate CXCL1 signaling and engineered Treg therapy. Tregs expressing a CXCL1R-CD3ζ-IRES-GFP construct (CXCL1R-CD3 Tregs) were assessed for migration, proliferation, apoptosis, tissue persistence, and analgesic efficacy. Neuroimmune responses were evaluated by flow cytometry and inflammatory marker analysis. RESULTS: CXCL1 was markedly increased after CCI. Although CXCL1 promoted conventional Treg migration, it also increased apoptosis, and unmodified Tregs failed to improve mechanical hypersensitivity. In contrast, CXCL1R-CD3 Tregs retained CXCL1-directed migration, showed enhanced proliferation without increased apoptosis, persisted in the spinal cord and spleen, and significantly improved paw withdrawal thresholds. Engineered Tregs reduced macrophage accumulation and iNOS expression while increasing Arg1 expression. They also suppressed microglial activation, decreased TNF, IL-1β, IL-6, and iNOS expression, attenuated p65/MAPK signaling, reduced neutrophil infiltration and MMP expression, and diminished oxidative activity. CONCLUSION: CXCL1R-CD3ζ engineering converts inflammatory CXCL1 signaling into a proliferative and survival-supporting cue for Tregs, enabling sustained immunoregulation and alleviation of neuropathic pain.