NET-Associated Transcriptional Signatures and T-Cell Functional States During Allogeneic CAR-γδT-Cell Therapy: A Longitudinal Single-Cell Analysis

CAR T-cell therapy has transformed the treatment of hematologic malignancies, although conventional autologous αβ CAR-T products remain limited by complex manufacturing, variable persistence, and relapse risk. γδT cells provide a promising donor source for next-generation off-the-shelf CAR-T therapies due to their MHC-independent tumor recognition, potent cytotoxicity, and minimal risk of graft-versus-host disease; however, the determinants of treatment durability following allogeneic CAR-γδT-cell therapy remain poorly defined. NETs have recently emerged as key immunoregulatory components of the TME, where excessive NET formation sustains inflammation, reshapes immune-stromal interactions, and impairs lymphocyte function, yet their impact on CAR-T-cell fate is largely unknown.

In this study, we performed longitudinal single-cell transcriptomic profiling of patients treated with allogeneic CAR-γδT cells and integrated ligand-receptor inference, pathway enrichment, and transcriptional regulatory analyses.

We identified a 69-gene NET-associated transcriptional signature in the neutrophil population captured in PBMC samples. The NET-associated transcriptional score increased longitudinally in the single patient who subsequently relapsed, whereas longitudinal patterns varied across individual patients. Exploratory analyses identified associations between NET-associated transcriptional activity and T-cell functional programs, including an inverse association with proliferation. NicheNet, pathway enrichment, and SCENIC analyses highlighted candidate inflammatory and regulatory programs involving IL-1-related signaling, adhesion pathways, SMAD3 regulon activity, and E2F-related proliferative programs. Analysis of an independent cohort provided supportive evidence for related inflammatory transcriptional patterns, although differences in therapeutic platform and cellular composition limited direct validation.

Together, these findings suggest that NET-associated inflammatory states may be linked to peripheral immune remodeling and altered circulating T-cell functional states during CAR-γδT-cell therapy. Larger longitudinal cohorts, more comprehensive clinical annotation, and orthogonal measurements of NET formation are needed to establish the clinical and mechanistic significance of these associations.