Clinical Significance Of Tim-3 And PD-1 Expression On Immune Cells In Patients With Nasopharyngeal Carcinoma

Part 1 Tim-3 regulates cellular immune function and polarization of TAMs in NPC patientsObjects: 1.To determine the expression of Tim-3 on immune cells and to evaluate the function of immune cells in patients with NPC;2.To elucidate the relationship between the Tim-3 expression on macrophages and the polarization of macrophages.Methods: A total of 30 Patients with pathologically confirmed nasopharyngeal carcinoma were recruited from the Affiliated Hospital of Guilin Medical University between August 2019 and January 2020,22 healthy volunteers matched to the patients on age and sex were obtained from Medical Examination Center.Percentage of monocytes,CD4+ T lymphocytes and CD8+ T lymphocytes in peripheral blood and the expression levels of Tim-3 on immune cells were analyzed by Flow cytometry.Concentrations of cytokines(IFN-?,TNF-?,IL-10 and IL-12)in serum and in PBMCs cultures were detected by ELISA.The formalin-fixed paraffin-embedded NPC tissues and non-tumor nasopharyngeal epithelium(NPE)tissues were obtained from the Laboratory of Clinical Pathology.The expression levels of Tim-3,CD68 and CD163 in NPC tissues and NPE tissues were detected by immunofluorescence staining.Result:1.The percentage of CD14+CD64+monocytes in NPC patients was significantly up-regulated than that in control groups(3.00% ± 0.14% vs.2.24% ± 0.23%,P < 0.05).The ratio of CD4+/CD8+ was significantly declined in NPC patients(1.422% ± 0.13 vs.1.852% ± 0.1763,P < 0.05).The percentage of CD4+ T lymphocytes in NPC patients with stage IVA-B was significantly lower than that in patients with stage I-III(3.42% ± 0.32% vs.5.07% ±0.49%,P < 0.05).2.The concentration of IFN-?(pg/m L)in the peripheral blood of patients with NPC was significantly lower than that in control groups(3.19 ± 0.37 vs.8.81 ± 0.66,P < 0.05).Concentration of IFN-? in PBMCs cultures stimulated by phytohemagglutinin(PHA)was declined in NPC patients(70.67 ± 3.37 vs.87.30 ± 6.64,P < 0.05).There were no significant differences in the concentration of TNF-?(P > 0.05).3.Percentage of Tim-3+CD14+CD64+monocytes in NPC patients was significantly higher than that in normal controls(14.99% ± 2.13% vs.8.46% ± 1.63%,P < 0.05).Similarly,percentage of Tim-3+CD3+CD4+T cells(1.03% ± 0.09% vs.0.72% ± 0.09%,P < 0.05)was significantly upregulated.The expression of Tim-3 on monocytes and T cells was correlated with tumor stage.Percentage of Tim-3+CD14+CD64+monocytes(22.72% ± 3.63% vs.10.66% ± 1.95%,P < 0.05)and percentage of Tim-3+CD3+CD4+T cells(1.27% ± 0.16% vs.0.84% ± 0.09%,P < 0.05)in NPC patients with stage IVA-B were significantly higher than those with stage I-III.The percentage of Tim-3+CD14+CD64+monocytes was negatively correlated with the ratio of CD4+/CD8+(r =-0.430,P < 0.05).It was suggested that the higher the expression of Tim-3 on monocytes and T cells,the more serious the damage of immune function and the more serious the tumor.4.The expression levels of Tim-3,CD163 and CD163+Tim-3+TAMs in tumor tissues of NPC were higher than those in NPE tissues(P < 0.05).Tim-3 expression was positively correlated with tumor stage(r= 0.496,P < 0.05)and CD163 expression(r=0.685,P < 0.05).5.The concentration of M1 marker IFN-? decreased(3.19 ± 0.37 vs.8.81 ± 0.66,P<0.05),IL-12 increased(28.25 ± 2.35 vs.14.72 ± 1.66,P < 0.05)and M2 marker IL-10 increased(16.63 ± 1.10 vs.12.37 ± 1.29,P < 0.05)in patients with NPC than that of controls.There were no significant differences in the concentration of TNF-?(P > 0.05).After stimulating by LPS,PBMCs from NPC patients secreted more M1 marker IL-12(111.06 ± 2.07 vs.33.04 ± 5.28,P < 0.05)and M2 marker IL-10(39.54 ± 7.65 vs.12.93 ± 1.99,P < 0.05);PBMCs from controls secreted more M1 marker IL-12(83.66 ± 8.01 vs.15.99 ± 2.11,P < 0.05),However,there was no changes of M2 marker IL-10(19.72 ± 2.82 vs.14.43 ± 1.79,P > 0.05).Conclusion: Tim-3 was involved in nasopharyngeal carcinoma progression by inhibiting cellular immune function and regulating the polarization of macrophages to M2-like phenotype:Part 2 Expression of PD-1 on peripheral blood immune cells in NPC patients and its clinical significanceObjective: We detected he expression levels of programmed cell death protein 1(PD-1)on monocytes and T lymphocytes to explore its roles in the development of nasopharyngeal carcinoma.Method: A total of 30 Patients with pathologically confirmed nasopharyngeal carcinoma and 22 healthy volunteers were recruited from the Affiliated Hospital of Guilin Medical University.PD-1 expression on peripheral blood immune cells and changes of PD-1 expression before and after treatment were detected by flow cytometry.Result: 1.There was no significant difference in the expression of PD-1 on monocytes(21.88% ± 2.29% vs.16.34% ± 2.07%,P > 0.05),CD4+T cells(30.46% ± 1.53% vs.28.94% ± 1.40%,P > 0.05)and CD8+T cells(33.6% ± 2.02% vs.29.56% ± 2.37%,P > 0.05)between NPC patients and normal controls.There was no significant difference in the expression of PD-1 on monocytes and T lymphocytes in NPC patients with different tumor stages.2.There was a positive correlation between the expression of PD-1 on monocytes and expression of Tim-3 on monocytes(r =0.529,P < 0.05).3.After treatments,the expression of PD-1 on monocytes was significantly upregulated(20.16% ± 1.19% vs.33.15% ± 2.233%,P < 0.05)in NPC patients.Conclusion: Tim-3 may be a key negative regulator on immune cells leading to impaired function,and participating in the pathogenesis of NPC rather than PD-1.There may be a synergistic relationship between Tim-3 and PD-1,so the combination of blocking may be the best choice for immunotherapy of NPC.