| Objective:To investigate the effects of culture supernatant of esophageal squamous cell carcinoma(ESCC)cell on the subtype,surface receptor and effector molecule of CD3~+CD56~+NKT cells in healthy volunteers.Methods:In the first part,the peripheral blood mononuclear cells were isolated from 30 healthy volunteers by lymphocyte separating solution.The culture supernatants of esophageal squamous cell carcinoma cell lines Eca9706 and Kyse150 were mixed with RPMI1640 medium containing 10%fetal bovine serum at 1:1 to prepare conditioned medium.Peripheral blood monocytes were divided into control group,Eca9706 group and Kyse150 group.The control group cells were cultured in RPMI1640 medium containing IL-2 and 10%fetal bovine serum,and the Eca9706 and Kyse150 cells were cultured in conditioned medium(Eca9706 cell culture supernatant and Kyse150 cell culture supernatant)after 24 hours of IL-2 stimulation.The subsets of CD3~+CD56~+NKT cells were analyzed by flow cytometry.The expression of surface receptors(NKG2D,NKG2A,NKP30,NKP44,CD158b,CD271)and effector molecules(Perforin,GranzymeB Ki-67)in CD3~+CD56~+NKT cells were detected.The second part:Esophageal squamous cell carcinoma tissue was taken for primary culture.After primary culture was successful,the supernatant was collected and centrifuged.The supernatant was mixed with RPMI1640 medium containing 10%fetal bovine serum in 1:1 to prepare conditioned medium.Peripheral blood mononuclear cells were isolated from healthy volunteers by routine lymphocyte separation solution.Peripheral blood monocytes were divided into control group and primary culture group.The control group was cultured in RPMI1640medium containing IL-2 and 10%fetal bovine serum,and the cells in primary culture group were stimulated by IL-2 for 24 hours,then cultured in conditioned medium(supernatant of primary culture group)for 72 hours.The subsets of CD3~+CD56~+NKT cells were analyzed by flow cytometry.The expression of surface receptors(NKG2D,NKG2A,NKP30,NKP44,CD158b,CD271)and effector molecules(perforin,granulase,Ki-67)in CD3~+CD56~+NKT cells were detected.Results:1.Comparison of the proportion of different subtypes of CD3~+CD56~+NKT cells in each group:(1)The supernatant of esophageal squamous cell line culture can induce subtype changes of CD3~+CD56~+NKT cells.The results of co-culture showed that compared with the NC group alone,the proportion of CD3~+CD56~+NKT cell subtype CD4~+CD8~-NKT cells in the co-culture group(Eca9706 and Kyse150 groups)was higher than that in the separate culture group(NC)(4.19%±0.68%,3.46%±0.27%vs2.56%±0.34%p=0.011)and the proportion of CD3~+CD56~+NKT cell subtype CD4~-CD8~-NKT cells decreased(7.72%±2.47%,9.37%±3.06%vs 15.68%±2.4%,p=0.025)and the proportion of CD3~+CD56~+NKT cell subtype CD4~-CD8~+NKT cells showed no significant change(75.68%±9.9%,74.97%±8.31%vs 73.07%±8.09%,p=0.931).There was no significant change between Eca9706group and Kyse150 group.(2)The supernatant of primary culture of esophageal squamous cell carcinoma cells can induce subtype changes of CD3~+CD56~+NKT cells:The results of co-culture experiment showed that the proportion of CD3~+CD56~+NKT cell subtype CD4~+CD8~-NKT cells in co-culture group was higher than that in single culture group(4.47%±0.38%vs 2.48%±0.27%p=0.013)and the proportion of CD3~+CD56~+NKT cell subtype CD4~-CD8~-NKT cells decreased(8.38%±0.87%vs 14.68%±1.52%p=0.023)and the proportion of CD3~+CD56~+NKT cell subtype CD4~-CD8~+NKT cells did not change significantly(73.05%±4.28%vs 78.41%±4.37%p=0.431).2.The expression rate of surface receptors of CD3~+CD56~+NKT cells in each group was compared:(1)The supernatant of esophageal squamous cell carcinoma cell line could induce the expression change of surface receptors of CD3~+CD56~+NKT cells.The co-culture experiment showed that compared with the NC group:the expression percentage of NKG2A in the conditioned medium group(Eca9706and Kyse150 group)increased(35.33%±9.29%,32.82%±8.12%vs17.47%±9.51%p=0.042)and CD158b in the conditioned medium group(Eca9706 and Kyse150 group)increased(64.05%±5.28%,62.34%±4.89%vs52.32%±3.73%p=0.013)while the expression rates of NKP44(8.61%±1.65%,9.78%±1.42%vs 11.63%±1.40%p=0.017)and NKP30(25.68%±8.49%,25.96%±6.60%vs 40.63%±8.23%p=0.038)and CD271(24.82%±4.89%,26.69%±4.50%vs34.23%±3.25%p=0.028)were decreased in the conditioned medium group(Eca9706 and Kyse150 groups).The expression rates of NKD2D showed no significant change between the conditioned medium group(Eca9706 and Kyse150 group)and NC group(p>0.05).(2)The supernatant of primary culture of esophageal squamous cell carcinoma cells can induce changes in the expression of surface receptors of CD3~+CD56~+NKT cells:The co-culture experiment showed that compared with the single culture group(NC),the expression percentage of NKG2A the co-culture group(primary culture supernatant group)increased(29.14%±6.10%vs 14.02%±2.13%p=0.047)and CD158b in the co-culture group(primary culture supernatant group)increased(65.36%±5.41%vs 41.91%±6.42%p=0.024)while the expression rates of NKP44(10.93%±1.54%vs 25.57%±1.40%p=0.0003)and NKP30(23.98%±3.41%vs 40.4%±4.74%p=0.048)and CD271(18.11%±1.54%vs 41.67%±7.22%p=0.033)were decreased in the co-culture group(primary culture supernatant group).The expression rates of NKD2D showed no significant change between between the co-culture group and the NC group(75.53%±8.14%vs 82.56%±6.93%p=0.53).3.The expression of effector molecule in CD3~+CD56~+NKT cells of each group was compared:(1)The supernatant of esophageal squamous cell carcinoma cell line could induce the changes of biological function of CD3~+CD56~+NKT cells.The co-culture experiment showed that the expression of Perforin in the conditioned medium group(Eca9706 group)was lower than that in the single culture group(93.98%±1.74%vs 98.70%±0.96%p=0.001)while the expression of Granzyme B did not change significantly(p>0.05).The expression of Ki-67 in the conditioned medium group(Eca9706 group)was increased(75.69%±3.74%vs61.83%±6.12%p=0.033).(2)The primary culture supernatant of esophageal squamous cell carcinoma cells can induce the biological function changes of CD3~+CD56~+NKT cells.The results of co-culture experiment showed that the expression of Perforin in co-culture group(primary culture supernatant group)decreased(57.43%±4.74%vs78.70%±15.69%p=0.037)while the expression of Granzyme B did not change significantly(p>0.05).The expression of Ki-67 in co-culture group(primary culture supernatant group)decreased(30.23%±3.63%vs 54.89%±8.76%p=0.04).Conclusion:1.The culture supernatant of esophageal squamous cell carcinoma cells can change the subtype distribution of infiltrated CD3~+CD56~+NKT cells by up-regulating the proportion of CD4~+CD8~-NKT cells and down-regulating the proportion of CD4~-CD8~-NKT cells.2.The culture supernatant of esophageal squamous cell carcinoma cells can inhibit the expression of surface activated receptor on CD3~+CD56~+NKT cell and promote the expression of surface inhibitory receptor,so that the expression ratio of the two is unbalanced.3.The culture supernatant of esophageal squamous cell carcinoma cells can inhibit the killing activity of CD3~+CD56~+NKT cell by down-regulating the expression of perforin.4.The culture supernatant of esophageal squamous cell carcinoma cells can change the expression of CD3~+CD56~+NKT cell effector molecule ki-67. |
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