MHC class I polypeptide-related sequence B shedding modulates pancreatic tumor immunity via the activation of NKG2DLow T cells

Abstract

Natural killer group 2 member D ligands (NKG2DLs) are expressed as stress response proteins in cancer cells. NKG2DLs induce immune cell activation or tumor escape responses, depending on their expression. Human pancreatic cancer cells, PANC-1, express membrane MHC class I polypeptide-related sequence A/B (mMICA/B), whereas soluble MICB (sMICB) is detected in the culture supernatant. We hypothesized that sMICB saturates NKG2D in NKG2D^Low T cells and inhibits the activation signal from mMICB to NKG2D. Knockdown of MICB by siRNA reduced sMICB level, downregulated mMICB expression, maintained NKG2D^Low T cell activation, and inhibited NKG2D^High T cell activation. To maintain mMICB expression and downregulate sMICB expression, we inhibited a disintegrin and metalloproteinase (ADAM), a metalloproteinase that sheds MICB. Subsequently, the shedding of MICB was prevented using ADAM17 inhibitors, and the activation of NKG2D^Low T cells was maintained. In vivo xenograft model revealed that NKG2D^High T cells have superior anti-tumor activity. These results elucidate the mechanism of immune escape via sMICB and show potential for the activation of NKG2D^Low T cells within the tumor microenvironment.

Keywords: ADAM; Immunity; MICB; NKG2D; NKG2DL; Pancreatic tumor; T cell.

Fig. 1

NKG2D^High CD8 T cells are more activated than NKG2D^Lowcells are. (a) A representative histogram showing NKG2D expression before (left) and after (right) sorting. (b) IFNG mRNA expression of NKG2D^High or NKG2D^Low CD8 T cells cocultured with PANC-1 cells for 72 h. Data are represented as fold-changes relative to the NKG2D^High T cells and are means with S.E.M. from four experiments using T cells from two different donors. Each pair of connected dots represents data from a single experiment. (c) Expression of surface activation markers of NKG2D^High or NKG2D^Low CD8 T cells cocultured with PANC-1 cells. Data are mean with SEM from four independent experiments using T cells from three different donors. Each pair of connected dots represents data from a single experiment.

Fig. 2

Anti-MICA/MICB antibody 6D4 blocks activation of T cells by coculture. (a) IFNG mRNA expression of NKG2D^High (left) and NKG2D^Low (right) CD8 T cells cocultured with PANC-1 cells with or without 6D4 antibody at 100 nM. Data are mean with SEM from three independent experiments. (b) Apoptotic PANC-1 cells after coculture with NKG2D^High (left) or NKG2D^Low (right) CD8 T cells with or without 6D4 antibody at 100 nM. Data are mean with SEM from three independent experiments.

Fig. 3

Differential responses of NKG2D^High and NKG2D^Low CD8 T cells cocultured with PANC-1 cells treated withMICB siRNA. (a) MICB mRNA expression of PANC-1 cells treated with control (−) or MICB (+) siRNA. Data are mean with SEM from three independent experiments. (b) IFNG mRNA expression of NKG2D^High (left) and NKG2D^Low (right) CD8 T cells cocultured with PANC-1 cells treated with control (−) or MICB (+) siRNA. Data are mean with SEM from three independent experiments. (c) Expression of membrane MICB on PANC-1 cells treated with control (−) or MICB (+) siRNA. Data are mean with SEM from three independent experiments. (d) Concentration of soluble MICB in culture supernatant of PANC-1 cells treated with control (−) or MICB (+) siRNA. Data are mean with SEM from three independent experiments. (e) Cytotoxicity of NKG2D^High (open) and NKG2D^Low (closed) CD8 T cells against PANC-1 cells treated with control (−) or MICB (+) siRNA. Data are expressed as ratio of PANC-1/T cell number and are mean with SEM from three independent experiments. ns, not significant.

Fig. 4

Soluble MICB regulates the activation of T cells cocultured with PANC-1. (a) IFNG mRNA expression of NKG2D^High (left) and NKG2D^Low (right) CD8 T cells cocultured with PANC-1 cells with or without ADAM inhibitor GW280264x at 10 µM. Data are mean with SEM from three independent experiments. (b) Apoptotic PANC-1 cells after coculture with NKG2D^High (left) and NKG2D^Low (right) CD8 T cells with or without ADAM inhibitor GW280264x at 10 µM. Data are mean with SEM from three independent experiments. (c) Apoptotic PANC-1 cells after coculture with NKG2D^Low CD8 T cells with or without MICB-Fc fusion protein at 200 ng/mL. Data are mean with SEM from two independent experiments.

Fig. 5

Superior anti-tumor activity of NKG2D^High CD8 T cells in PANC-1 xenograft model. (a) Experimental schedule of PANC-1 xenograft model. SCID beige mice were subcutaneously transplanted with PANC-1 cells (3 × 10⁶) with Matrigel in the right flank at day − 12. At day 0, NKG2D^Low or NKG2D^High CD8 T cells (1 × 10⁶) were intravenously injected. (b) Tumor growth curves of individual mice injected with NKG2D^Low (left) and NKG2D^High (right) CD8 T cells. (c) Tumor burden was expressed as area under the curve (AUC) over baseline (tumor size at day 0) of individual tumor growth curve.