Fig 2: Postoperative NK cells show increased TGF-β and POD1 plasma induces NK cell dysfunction in vitro. (A) Platelet-free plasma was isolated by centrifugation from whole blood from 25 baseline and POD1 patients undergoing cancer surgery. TGF-β was quantified by ELSA. Median ± IQR is shown (p = ns). (B) Single-cell RNA sequencing in six patients with colorectal cancer underwent TGF-β activity analysis. Median ± IQR is shown. Flow cytometry staining showing expression, (C) %, and (D) MFI of NK activating receptors, NKG2D, DNAM-1, CD212, CD132, and intracellular IFNγ in NK cells isolated from health donors incubated in the absence (green) or presence (purple) of recombinant TGF-β (10 ng/mL). Healthy donor isolated NK cells were also incubated with baseline (blue) or POD1 (red) plasma and expression, (E) %, and (F) MFI of the same targets as (C,D) were determined. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005.

Fig 3: Proposed mechanisms of TGF-β-mediated Natural Killer cell dysfunction. Upon binding to its receptor, TGF-β may signal through SMAD2/3 to directly inhibit the expression of the transcription factor T-bet (Tbx21) or to inhibit mTOR via unknown intermediary protein(s). Smad-independent mechanisms may include the production and activation of SHP-2 phosphatase, which may indirectly mediate mTOR activity. These changes in intracellular signaling may then contribute to downregulation or activating receptors (IL-2R, IL-12R, NKG2D, DNAM-1). Finally, these phenotypic changes result in reduced cytokine (IFNγ) secretion and suppressed NK cell function.

Fig 4: Postoperative NK cells have significantly reduced cytokine and activating receptor expression and reduced phosphorylation of downstream signaling proteins. Whole blood was collected from healthy donors (green, median ± IQR shown) or patients undergoing cancer surgery at baseline (blue) and POD1 (red). Matched baseline and POD1 samples are indicated. Expression (% and MFI) of IL-2R subunit CD132 and IL-12R subunit CD212 ((A) (%)/(B) (MFI)) and activating receptors NKG2D and DNAM-1 ((C) (%)/(D) (MFI)) is shown. The phosphorylation of all assessed downstream signaling proteins (pSTAT5, pSTAT4, pP38 MAPK, pS6) in response to stimulation with rIL-2/12 was significantly reduced in NK cells on POD1 (E). * p ≤ 0.05, ** p ≤ 0.005, **** p ≤ 0.00005.