IL-6R-CD3-engineered regulatory T cells alleviate neuropathic pain by remodeling the neuroimmune microenvironment.

Chronic neurological condition Neuropathic pain is marked by sustained neuronal sensitization. Regulatory T cells are crucial for preserving immune homeostasis. however, their stability and suppressive activity are often compromised within inflammatory environments generated after nerve injury, limiting their ability to restrain neuroinflammation. This study sought to determine whether genetically modified regulatory T cells expressing an interleukin-6 receptor-CD3ζ fusion receptor could enhance Treg persistence, functional capacity, and therapeutic benefit in a rat chronic constriction injury (CCI) model. CD4⁺CD25⁺ regulatory T cells were isolated from rat spleens and then transduced with a lentiviral vector expressing IL-6R-CD3ζ-IRES-GFP. The modified cells were subsequently evaluated in vitro for their proliferative capacity, susceptibility to apoptosis, and migratory behavior under inflammatory cytokine stimulation. To establish neuropathic pain, a chronic constriction injury model was generated by placing four loose chromic gut ligatures around the sciatic nerve at approximately 1-mm intervals proximal to the trifurcation. The animals then underwent adoptive transfer of either conventional regulatory T cells or IL-6R-CD3ζ-engineered regulatory T cells. Mechanical hypersensitivity was measured by behavioral testing, whereas flow cytometry together with molecular assays was applied to assess immune cell infiltration, the expression of inflammatory mediators, and the activation of intracellular signaling pathways in spinal cord tissue. Native Tregs did not effectively suppress CCI-associated neuroinflammation. Depletion of these cells exacerbated mechanical hypersensitivity, while adoptive transfer of unmodified Tregs conferred only limited analgesic effects. By contrast, IL-6R-CD3ζ-engineered Tregs retained cytokine-responsive migratory ability, displayed greater proliferative activity, and persisted longer in vivo. Transfer of the engineered Tregs significantly increased paw withdrawal thresholds and alleviated neuropathic pain. Mechanistically, this treatment reduced macrophage infiltration and shifted macrophages toward a less proinflammatory phenotype. It also markedly inhibited microglial activation and inflammatory mediator production, together with suppression of nuclear factor kappa B and mitogen-activated protein kinase signaling. In addition, neutrophil recruitment and effector functions were significantly reduced, including diminished secretion of IL-1β, TNF, IL-6, and matrix metalloproteinases as well as decreased oxidative stress. IL-6R-CD3 engineered Tregs remodel the neuroimmune after nerve injury by regulating macrophage, microglial, and neutrophil responses.