Fig 2: TGF-β1 mediates decreased NK cell cytotoxicity. (A) NK cells purified from PBMCs of JORRP patients were preincubated with LY364947 for 3 days in the presence of 100 U/ml IL-2, then stimulated with K562 target cells for an additional 6 h at the ratio = 1:1. (A, B) Representative paragraph and percentage analysis of K562 cell apoptosis (the percentage of Annexin V on K562 cells) was determined by flow cytometry. Blank, K562 cells alone; NC, NK cells only; DMSO, NK cells + DMSO; LY364947, NK cells + LY364947. (C, D) Representative histograms and percentage analysis of CD107a on NK cells. (E) NKp30, NKG2D, and NKp46 expression on NK cells. MFI, mean fluorescent intensity. (F, G) PBMCs from HCs, PBMCs and TILs were isolated from JORRP patients. Representative paragraph and percentage analysis of CXCR6 gated on CD56+CD3- cells. TIL, tumor-infiltrating lymphocyte. Data are presented as median with interquartile range. Each dot represents a single individual. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig 3: Comparison of NK cell phenotypes between fresh and cryopreserved NK cells.NK cell phenotypes between freshly isolated and cryopreserved thawed PBMC samples were compared. NK cells were either activated or non-activated with cytokines (IL-2+IL-15) and target cells (A431) alone or targets coated with cetuximab (CET). Expression levels of NKG2A (A), NKG2C (B), NKG2D (C), KIR2D (D) were compared for the following conditions: i) NK only ii) NK+A431 and iii) NK+A431+CET conditions. NK only conditions are depicted as open rectangles, followed by NK+A431 with grey shades and NK+A431+CET conditions represented as black rectangles. Columns represent data from12 donors, from each donor the mean of triplicate values was used; with bars showing SEM. Data are from independent experiments performed in triplicates from 12 PBMC donors (6 donors: BD LSRFortessa +6 donors: MACSQuant). Statistical analysis was performed using the Wilcoxon test.

Fig 4: The protein expression level of IL-2 and IL-15 costimulation culture 0 and 7 days. The FACS analysis NKG2D expression percentage of NK cells (a). Western blot analysis of p-PI3K/PI3K, MEK, and p-ERK/ERK protein expression levels of the uncultured NK cells compared with the IL-2 and IL-15 costimulation culture 7 days NK cells. For comparison, Student's t-test was performed. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Fig 5: UMCD6 induces upregulation of NKG2D on NK and CD8+ T cells.(A) NK-92 cells were incubated with 10 μg/mL of IgG or UMCD6 and harvested after 4 hours. Real-time PCR revealed significantly higher levels of mRNA for the activating receptor NKG2D, as well as perforin and granzyme B upon incubation with UMCD6, while mRNA for the inhibitory receptor NKG2A was downregulated. Data are expressed as mean ± SD; *P < 0.05. (B and C) Representative flow cytometry plots showing NKG2D-positive NK and CD8+ T cells. PBMCs from 6 donors (n = 6) were isolated as described in Methods and subsequently cultured with 10 μg/mL IgG or UMCD6. Increase of cell surface expression of NKG2D was evident 72 hours after treatment with UMCD6 on NK cells (gated on CD3–CD56+ cells) (B) and CD8+ T cells (gated on CD3+CD8+ cells) (C).