TNFR-CD3-engineered regulatory T cells attenuate neuroinflammation and neuropathic pain

Immunol Res. 2026 Mar 6;74(1):22. doi: 10.1007/s12026-026-09744-8.

Neuropathic pain is a debilitating condition characterized by neuronal hyperexcitability and neuroimmune dysregulation. Regulatory T cells (Tregs) are critical for immune homeostasis, but their survival and function are severely impaired in the inflammatory milieu, particularly under the influence of tumor necrosis factor-α (TNF-α). Objective This study aimed to determine whether engineering Tregs with a TNFR-CD3 fusion construct could enhance their stability, survival, and therapeutic efficacy in a rat model of chronic constriction injury (CCI).

Methods: CD4⁺CD25⁺ Tregs were isolated from rats, transduced with a TNFR-CD3ζ-IRES-GFP lentiviral construct, and characterized for proliferation, apoptosis, and chemotaxis in vitro. CCI was induced by loose ligation of the sciatic nerve. Rats received adoptive transfer of either unmodified Tregs or TNFR-CD3 Tregs. Behavioral assays assessed mechanical hypersensitivity, while flow cytometry and molecular analyses evaluated immune cell infiltration, phenotype, and signaling pathway activation in the spinal cord.

Results: Endogenous Tregs were insufficient to control CCI-induced inflammation: depletion exacerbated hypersensitivity, and adoptive transfer of unmodified Tregs failed to relieve pain due to apoptosis in the TNF-rich environment. In contrast, TNFR-CD3 Tregs retained TNF-induced chemotaxis, exhibited enhanced proliferation without increased apoptosis, and persisted in vivo. Adoptive transfer of TNFR-CD3 Tregs significantly improved paw withdrawal thresholds, reduced macrophage infiltration, and promoted an M2-like phenotype. Microglial activation and pro-inflammatory cytokine production were markedly suppressed, accompanied by decreased NF-κB/p65 and MAPK phosphorylation. Additionally, TNFR-CD3 Tregs limited neutrophil infiltration, reduced IL-1β, TNF, IL-6, and MMP secretion, and attenuated ROS and superoxide production. 

Conclusion: TNFR-CD3 Tregs effectively reprogram the neuroimmune microenvironment by modulating macrophages, microglia, and neutrophils, thereby alleviating neuropathic pain. These findings highlight engineered Treg therapy as a promising immunomodulatory strategy for neuroinflammatory diseases and support its potential translational application in neuropathic pain management.

PMID:41787027 | DOI:10.1007/s12026-026-09744-8