Preventing Surgery-Induced NK Cell Dysfunction Using Anti-TGF-β Immunotherapeutics
Marisa Market1,2, Gayashan Tennakoon1,2, Marlena Scaffidi1,2
1Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1H8L6, Canada.
Abstract:
Natural Killer (NK) cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed postoperatively. This dysfunction is associated with increased morbidity and cancer recurrence. NK activity depends on the integration of activating and inhibitory signals, which may be modulated by transforming growth factor-beta (TGF-β). We hypothesized that impaired postoperative NK cell IFNγ production is due to altered signaling pathways caused by postoperative TGF-β. NK cell receptor expression, downstream phosphorylated targets, and IFNγ production were assessed using peripheral blood mononuclear cells (PBMCs) from patients undergoing cancer surgery. Healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma and in the presence/absence of a TGF-β-blocking monoclonal antibody (mAb) or the small molecule inhibitor (smi) SB525334. Single-cell RNA sequencing (scRNA-seq) was performed on PBMCs from six patients with colorectal cancer having surgery at baseline/on POD1. Intracellular IFNγ, activating receptors (CD132, CD212, NKG2D, DNAM-1), and downstream target (STAT5, STAT4, p38 MAPK, S6) phosphorylation were significantly reduced on POD1. Furthermore, this dysfunction was phenocopied in healthy NK cells through incubation with rTGF-β1 or POD1 plasma and was prevented by the addition of anti-TGF-β immunotherapeutics (anti-TGF-β mAb or TGF-βR smi). Targeted gene analysis revealed significant decreases in S6 and FKBP12, an increase in Shp-2, and a reduction in NK metabolism-associated transcripts on POD1. pSmad2/3 was increased and pS6 was reduced in response to rTGF-β1 on POD1, changes that were prevented by anti-TGF-β immunotherapeutics. Together, these results suggest that both canonical and mTOR pathways downstream of TGF-β mediate phenotypic changes that result in postoperative NK cell dysfunction.
Insights
Postoperative transforming growth factor-beta (TGF-β) impairs Natural Killer (NK) cell function by altering signaling pathways. Blocking TGF-β with immunotherapeutics restores NK cell interferon-gamma (IFNγ) production and activity.
Area of Science:
- Immunology
- Cellular Biology
- Cancer Research
Background:
- Postoperative Natural Killer (NK) cell dysfunction, characterized by suppressed cytotoxicity and interferon-gamma (IFNγ) production, is linked to increased patient morbidity and cancer recurrence.
- Transforming growth factor-beta (TGF-β) is a key regulator of immune cell signaling and may influence NK cell activity following surgery.
Purpose of the Study:
- To investigate the hypothesis that elevated postoperative TGF-β levels disrupt NK cell signaling pathways, leading to impaired IFNγ production.
- To assess the potential of TGF-β blocking agents to restore NK cell function in the postoperative setting.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) from cancer surgery patients were analyzed for NK cell receptor expression, downstream signaling, and IFNγ production.
- In vitro experiments involved incubating healthy NK cells with postoperative plasma or recombinant TGF-β1, with or without TGF-β blocking agents (monoclonal antibody or small molecule inhibitor).
- Single-cell RNA sequencing was performed on patient PBMCs to analyze gene expression changes.
Main Results:
- Postoperative day 1 (POD1) showed significantly reduced intracellular IFNγ, activating receptor phosphorylation, and downstream signaling targets (STAT5, STAT4, p38 MAPK, S6) in NK cells.
- Incubation with recombinant TGF-β1 or POD1 plasma mimicked this NK cell dysfunction, which was reversed by anti-TGF-β immunotherapeutics.
- Gene expression analysis revealed altered levels of key signaling molecules (S6, FKBP12, Shp-2) and reduced NK cell metabolism transcripts on POD1.
- Increased pSmad2/3 and decreased pS6 phosphorylation in response to TGF-β1 were observed, and these changes were abrogated by TGF-β blockade.
Conclusions:
- Postoperative TGF-β signaling, acting through both canonical and mTOR pathways, mediates phenotypic changes that impair NK cell function.
- Targeting TGF-β represents a promising therapeutic strategy to restore NK cell immunity in the postoperative period, potentially reducing cancer recurrence and improving patient outcomes.
