Objective To optimize in vitro amplification of human γδ T cells with cytokines for tumor adoptive immunotherapy.Methods On the basis of the immobilized anti-TCR γδ antibody plus IL-2 system, other γ chain receptor family cytokines, including IL-7, IL-15 and IL-21, were tested to amplify human peripheral blood γδ T cells either alone or in diversity combination.The percentage of γδ T cells was measured by flow cytometry, and the proliferation efficiency of γδ T cells was calculated.The expression of proliferation-or cytotoxicity-related molecules on γδ T cells was examined by flow cytometry in order to explore the relevant mechanisms.The cytotoxicity of γδ T cells to Daudi cells was detected by lactate dehydrogenase.Results IL-15 alone but not IL-7 or IL-21 increases the γδ T cell purity, amplification efficiency and cytotoxicity to reach comparable levels to those of IL-2.IL-2 plus IL-15 up-regulates the expression of CD69 on γδ T cells and significantly increases their amplificationefficiency (P<0.05).IL-2 plus IL-21 enhanced the cytotoxicity of γδ T cells against Daudi cells by increasing the expression of granzyme A (P<0.001).The combination of IL-2, IL-15 and IL-21 significantly improves cytotoxicity of γδ T cells but reduces their amplification efficiency.In addition, when IL-21 was applied for a short time, it also enhanced the cytotoxicity of γδ T cells (P<0.05).Conclusions The combination of IL-2 and IL-15 as well as a short time addition of IL-21 is the best cytokine recipe to amplify human peripheral blood γδ T cells in vitro with immobilized anti-TCR γδ antibody, which can increase both the proliferation efficiency and the cytotoxicity to tumor cells of γδ T cells.

Objective To construct human lung cancer cell model with human MutS homologous protein 2 (hMSH2) overexpression for exploring the effect of hMSH2 molecule in the cytotoxicity of γδ T cell against lung cancer cells.Methods hMSH2 coding sequence was cloned by PCR for construction of recombinant vector which over expressed hMSH2-EGFP fusion protein using homologous recombination.The recombinant vector was transfected to lung cancer cell line NCI-H520 to construct human lung cancer cell model overexpressing hMSH2 molecule.The expression of hMSH2 molecule in NCI-H520 was detected by Western blot.The expression of hMSH2 on the cell membrane was measured by flow cytometry.Cytotoxicity of expanded γδ T cells from peripheral blood mononuclear cells against NCI-H520 cells was detected by LDH release assay in vitro.Results hMSH2 coding sequence (2805 bp) was cloned and the result of restriction endonuclease digestion of Fugw-hMSH2 recombinant vector was accordance with the anticipated objective strip size.Exogenous hMSH2-EGFP fusion protein was expressed in NCIH520 cells.The level of hMSH2 molecule on the surface of NCI-H520 cells with overexpression of hMSH2 was significantly increased (P＜0.001).Cytotoxicity of γδ T cells against NCI-H520 cells with overexpression of hMSH2 was significantly increased compared to the wild type NCI-H520 cells (P＜0.05).Conclusions Lung cancer cell model that overexpresses hMSH2 molecule is successfully constructed,hMSH2 molecule on the cell membrane is increased and the cytotoxicity of γδ T cells against lung cancer cells is enhanced.

Objective: To evaluate measurement acquisition, operability, and measurement agreement of the OA-2000 in cataract eyes with high myopia. Methods: A diagnosis test examined 1 030 eyes of 780 high-myopia cataract patients at the Wuhan Aier Eye Hospital from October 2017 to October 2018.The eyes were examined using the OA-2000, the IOLMaster 500 and the Lenstar-LS900 preoperatively.Measurement acquisition was recorded.The total ophthalmic exam, the duration of patient data entry, the actual measurement process, and the time from intraocular lens power calculation to printout were calculated.Ocular biometric parameters, including anterior chamber depth (ACD), axial length (AL), keratometry for the flattest meridian (Kf), and keratometry for the steepest meridian (Ks) were compared among the devices.This study complied with the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Wuhan Aier Eye Hospital (No.2017IRBKY07). Results: The OA-2000 was capable of obtaining an AL measurement in 94.95% of the eyes, which was significantly higher than the 83.98% and 83.01% measured by the IOLMaster 500 and Lenstar-LS900, respectively (χ²=38.171, 46.208; both at P<0.001). The total ophthalmic exam with the OA-2000 took a significantly shorter time than the Lenstar-LS900 ([109.34±2.22]s vs. [135.64±5.55]s) (P<0.05). Excellent correlations were found for the AL, ACD, Kf, and Ks measurements between the OA-2000 and the IOLMaster 500 (r=0.999, 0.937, 0.996, 0.996; all at P<0.001). Good correlations were also found between the OA-2000 and the Lenstar-LS900 (r=0.999, 0.965, 0.995, 0.996; all at P<0.001). The respective 95% limits of agreement of the OA-2000 and IOLMaster 500, and the OA-2000 and Lenstar-LS900 were -0.19 to 0.20 mm and -0.13 to 0.17 mm for AL; -0.23 to 0.23 mm and -0.18 to 0.16 mm for ACD; -0.48 to 0.47 D and -0.64 to 0.65 D for Kf; -0.49 to 0.47 D and -0.60 to 0.61 D for Ks. Conclusions In terms of AL, ACD, Kf and Ks in cataractous eyes with high myopia, the OA-2000, IOLMaster 500, and Lenstar-LS900 show excellent correlation and agreement.The OA-2000, a new swept-source OCT-based biometer, outperforms both the IOLMaster 500 and the Lenstar-LS900 in terms of AL measurement acquisition and operability.