Objective This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.
Humans ; Mice ; Animals ; Receptors, Chimeric Antigen/genetics* ; Interleukin-15 ; NK Cell Lectin-Like Receptor Subfamily K/metabolism* ; Granzymes ; Cell Line, Tumor ; Multiple Myeloma/therapy* ; Perforin

Objective To develop a novel transcatheter tricuspid valve replacement device and test its performance. Methods The transcatheter tricuspid valve stent consisted of double-layer self-expanding nitinol stent, biotissue-derived bovine pericardial leaflets, and PTFE woven. The delivery system, mainly consisting of a handle control unit and a delivery sheath, was sent to the correct position via right atrium or jugular vein. The sheath had a visualization feature, and the handle control unit could realize the functions of stable release and partial recovery of the interventional valve. In addition, this study performed animal survival experiments on the basis of in vitro experiments. A large-white pig was used as the experimental animal. Cardiopulmonary bypass was established through median thoracotomy, then the right atrium was opened, and the interventional valve was released under direct vision without cardiac arrest. Approximately 1 month after interventional valve implantation, the maneuverability and stability of the interventional tricuspid device were evaluated by autopsy. Results Through the animal experiment, the interventional valve was successfully released, and the anchoring was satisfactory. Postoperative transthoracic echocardiography showed that the interventional valve opened and closed well, the flow rate of tricuspid valve was 0.6 m/s, and there was no obvious tricuspid regurgitation. One month after the operation, we dissected the large-white pig and found the interventional valve was not deformed or displaced, the leaflets were well aligned, and there was thrombus attachment in the groove between the inner and outer layers of the interventional valve. Conclusion Animal experiment shows that the novel device can stably and firmly attach to the tricuspid annulus, with good anchoring effect, and effectively reduce paravalvular leakage.