NKG2D interactions with NKG2DLs, such as MICB, play an important role in cancer immunity. However, their function has been primarily studied in the context of their anti-tumor activity on NK cells (21, 29). In our previous study (24), we observed that NKG2D + cells infiltrating tumor tissue were CD8 + T cells. Therefore, this study focused on CD8 + T cells. Recent studies have shown that CD8 + T cells exert anti-tumor effects on MHC-I-deficient cancer cells, which are less responsive to cancer immunotherapy via NKG2DL/NKG2D signaling (30). It has long been recognized that NKG2D-expressing CD8 + T cells are activated by membrane-type NKG2DLs (17, 25). However, it has not been fully elucidated whether different levels of NKG2D expression on CD8 + T cells alter their susceptibility to inhibition by soluble NKG2DLs. In this study, we demonstrate that the soluble form of one of the NKG2DLs, MICB, inhibits the activation of NKG2DLow T cells (Fig. 6B). Knockdown of MICB mRNA in cancer cells resulted in a decrease in the membrane form of MICB on the cell surface and a decrease in the soluble form of MICB in the culture supernatant. Consequently, the activation of NKG2DLow T cells co-cultured with these cancer cells was enhanced four-fold. This finding suggests that the de-inhibition due to the decrease in sMICB affects T cell activity more strongly than the decrease in activation caused by the reduction in membrane MICBs. Notably, the altered T cell activity observed in this co-culture system, even when T cells and cancer cells were not HLA matched, suggests signaling changes other than T cell receptor signals, pointing to NKG2D receptor activation by the NKG2DL. On the other hand, co-culture with cancer cells in which MICB mRNA was knocked down did not enhance the activation of NKG2DHigh T cells; rather, it suppressed it, indicating that NKG2DHigh T cells are less susceptible to the soluble form.
Soluble NKG2DLs are generated when membrane-type NKG2DLs are cleaved by enzymes such as ADAM. MICB is cleaved by ADAM10 and ADAM17 (14, 31), primarily expressed in PANC-1. In this study, we discovered that GW280264X (32), which selectively inhibits ADAM10 and ADAM17 (33, 34), reduces sMICB production. This reduction in sMICB is accompanied by enhanced activation of NKG2DLow T cells and increased apoptosis of cancer cells during co-culture. This suggests that the sMICB, which inhibits NKG2DLow T cell activation, is generated by ADAM-mediated cleavage. In contrast, for NKG2DHigh T cells, GW280264X did not alter apoptosis of cancer cells during co-culture, indicating that NKG2DHigh T cells are less susceptible to the soluble ligand, similar to the effect of siRNA-based knockdown.
This study suggests that regulating the cleavage of membrane-type NKG2DLs may reactivate NKG2DLow T cells that are inhibited by soluble NKG2DLs. As demonstrated in this study, ADAM inhibitors may increase membrane-type NKG2DLs by blocking NKG2DL cleavage. However, since ADAM is expressed outside of cancer cells, as shown in previous studies (35, 36, 37, 38), ADAM inhibitors may not be effective therapeutic agents. Future studies are needed to elucidate the regulatory mechanism of ADAM activation specific to cancer cells in order to control this mechanism.
This study also suggests that sMICB may serve as a prognostic marker for pancreatic cancer. Correlations between soluble NKG2DLs detected in the serum and prognosis have been reported in several cancer types (19, 39). Higher serum soluble ULBP2 levels have been associated with a worse prognosis in lung cancer (40). Additionally, it has been reported that soluble NKG2DL levels in the serum of melanoma patients inversely correlate with survival and serve as important biomarkers for anti-PD-1 therapy (41). Our results suggest that sMICB levels in the blood may be a new biomarker for pancreatic cancer immunotherapy.
We have demonstrated that CD8 T cells exhibit MICB-mediated anti-tumor activity in an in vitro system. In particular, the regulation of sMICB production enhances the anti-tumor activity of NKG2DLow T cells. However, it remains unclear whether this mechanism is significant in vivo. This issue needs to be addressed in the future, given the divergence of NKG2DLs in mouse and human cancer cells in our study (42) and the significant challenge of generating immunologically humanized mice transplanted with MICB-expressing pancreatic cancer cells.
In conclusion, our study reveals that MICB, the primary ligand of pancreatic cancer cells, inhibits the activation of NKG2DLow T cells by shedding through ADAM. This suggests that NKG2DLow T cells, which are suppressed by sMICB, can be reactivated, potentially enhancing the anti-tumor effect of NKG2D + CD8 + T cells. Although the development of patient-specific methods to stimulate NKG2DLow T cells is needed, our study suggests that the regulation of sMICB, a suppressor of NKG2DLow T cells, may be an effective therapeutic target.