The Clinical Expression And Significance Of Inhibitory Receptor TIGIT On Peripheral NK Cells In Systemic Lupus Erythematosus

Objective:This study designed to investigate the expression of T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains（TIGIT）on peripheral CD3^-CD56⁺NK cells from patients with systemic lupus erythematosus（SLE）and its significance,for clarifying its role in the development of SLE.Methods:Patients with 44 SLE patients,21 RA patients and 27 healthy controls were recruited from the First Affiliated Hospital of Nanchang University.The medical history,clinical manifestations,physical examination,laboratory measurements,ther-apeutic regimen and treatment response were recorded.The expression of TIGIT on CD3^-CD56⁺NK cells were determined by flow cytome try.The percentage and the absolute number of NK cells in peripheral blood of patients with SLE,RA and healthy controls and the levels of TIGIT on NK cells were compared.The frequencies of TIGIT-expressing NK cells in patients with SLE were further analyzed for their correlation with markers of inflammation,autoimmune response,treatment with corticosteroids and immunosuppressive drugs and SLEDAI.Moreover,the function of TIGIT on NK cells in SLE was investigated.The expression of NK cell activation marker CD69,degranulation marker CD107a on NK cells after LPS stimulation and the secretion of IFN-γon NK cells after IL12 stimulation for 24h were detected by flow cytometry.Meanwhile,functional anti-human TIGIT antibody or IgG_2B control were added to the culture medium for blocking TIGIT pathway to detect the levels of IFN-γon T cells.Statistical analysis and graphic presentation were carried out with GraphPad Prism version 5.0.Statistical analysis was performed by one-way analysis of variance（ANOVA）followed by the Student-Newman-Keuls post test.A t-test was used where the normality test passed.Otherwise,the nonparametric Mann-Whitney test was used to analyze the data.For evaluation of changes with treatment in the group of SLE patients and blocking the TIGIT pathway on NK cells,paired t tests or Wilcoxon matched pairs test was used.Likewise,the Pearson method or the nonparametric Spearman method was used for correlation analysis.Results:1.The percentage of total lymphocytes of peripheral blood NK cells in the group of healthy controls（n=27）,RA（n=21）,SLE（n=44）were respectively11.9±4.94%,7.85±6.41%and 4.93%±4.45%,and the absolute number of NK cells in peripheralbloodofpatientswere24.38±2.46（x10⁷/L）,12.91±4.35（x10⁷/L）,5.83±0.78（x10⁷/L）respectively.The percentage and the absolute number of NK cells in peripheral blood of patients with SLE were significantly lower than those in healthy controls（P<0.010;P<0.001）.The absolute number of NK cells in peripheral blood of patients with SLE was lower than that in RA（P<0.010）,but there was no significant difference with the percentage of NK cells（P>0.050）.The absolute number of NK cells in peripheral b lood of RA group was significantly lower than that in HC group（P<0.010）,but there was no significant difference between RA and HC group with the percentage of NK cells（P>0.050）.2.The median of the percentage of TIGIT on NK cells in HC group,RA group and SLE group were 40.8%,28.5%,18.25%respectively,and the mean fluorescence intensity was 8.71±2.77,6.51±1.98,7.50±2.19 respectively.The percentage and the mean fluorescence intensity of TIGIT on NK cells in SLE group was lower compared with HC group（P<0.050;P<0.050）,but no difference was found between SLE group and RA group（P>0.050）.The mean fluorescence intensity of TIGIT on NK cells in RA group was lower compared with HC group（P<0.010）,but no difference was found in the percentage of TIGIT between them（P>0.050）.In HC group,No obvious correlation was observed between the proportion and absolute number of NK cells and the proportion of TIGIT on NK cells（r²=0.104,P=0.101;r²=0.004,P=0.758）.In RA group,positive correlations between the percentage of circulating NK cells and the percentage of TIGIT on NK cells were found（r²=0.373,P=0.003）,and positive correlations between the absolute number of circulating NK cells and the percentage of TIGIT on NK cells（r²=0.279,P=0.017）.Also positive correlations between the percentage of circulating NK cells and the percentage of TIGIT on NK cells in SLE group were found（r²=0.219,P=0.001）.There were also positive correlations between the absolute number of circulating NK cells and the percentage of TIGIT on NK cells in SLE group（r²=0.599,P<0.001）.3.The percentage of TIGIT NK cells in SLE patients with decreased C3 and C4group were decreased compared with normal C3 and C4 group（P=0.015;P=0.035）,but no significant difference was found between high IgG,ESR,WBC group and the respective normal group（P>0.050）.The mean fluorescence intensity of TIGIT on NK cells between SLE patients with low C3 low C4 group,high IgG,high ESR,high WBC group and respective normal group showed no significant difference（P>0.050）.The correlation of the percentage of TIGIT on NK cells and related indexes of these inflammatory in SLE patients was analysed,the results showed that the percentage of TIGIT on NK cells in SLE patients was correlated positively with C3（r²=0.101,P=0.038）,but not with C4（r²=0.007,P=0.583）.In addition,no obvious correlation was observed between the correlation of the mean fluorescence intensity of TIGIT on NK cells and ESR,IgG and WBC（P>0.050）.4.The percentage of TIGIT NK cells was significantly decreased in patients with positive anti-rRNP compared to patients with negative anti-r RNP（P=0.022）,but no significant difference was found between other antibodies such as anti-ds DNA,anti-SSA,anti-SSB,anti-Ro52,anti-Sm,anti-nRNP/Sm,anti-AnuA.There was also no significant difference in the mean fluorescence intensity of TIGIT on NK cells between these antibodies（P>0.050）.5.The percentage of TIGIT NK cells in patients with inactive patients was significantly higher than that in those with active patients（P=0.021）.Further correla-tion analysis showed that the percentage of TIGIT NK cells was negatively correlated with the SLEDAI（r²=0.090,P=0.048）.we performed a at least one week follow-up evaluation in 8 SLE patients received regular treatment with corticosteroids and immunosuppressive drugs.The percentage of TIGIT NK cells were significantly increased in SLE patients that received a one week regular treatment with corticosteroids and immunosuppressive drugs（P=0.046）.We also compared the percentage of TIGIT NK cells between new-onset and revisiting SLE patients.Data showed that the percentage of TIGIT on NK cells tends to be decreased in new-onset patients,but a significant difference was not reached（P=0.153）.The clinical features of patients with SLE including fever,cutaneo us manifestat–ions,oral ulcer,alopecia,arthritis,effusion,neuropathic lupus,24 hour proteinuria,hematuresis,pyuria,leucopenia,erythrocytopenia and thrombocytopenia were analyzed and correlated with the expression of TIGIT on NK cells,but no significant d ifference was found（P>0.050）.6.We further evaluated the relationship between TIGIT and the function of NK cells.After LPS stimulation,there was a significantly decreased expression of activat-ion markers CD69 and CD107a on TIGIT⁺NK cells from patients with SLE than that in TIGIT^-NK cells in patients with SLE（P=0.019;P=0.044）.Moreover,the percentage of CD69⁺NK cells was significantly inversely correlated with the percentage of TIGIT⁺NK cells among patients with SLE（r²=0.413,P=0.033）while no correlation was found with CD107a（P=0.367）.After IL-12 stimulation,the produ-ction of IFN-γin TIGIT⁺NK cells was significantly lower than that in TIGIT^-NK cells in patients with SLE（P=0.014）.Furthermore,the percentage of TIGIT⁺NK cells was significantly inversely correlated with their IFN-γ-producing capacity between different patients with SLE（r²=0.610,P=0.021）.In addition,functional anti-human TIGIT antibody or IgG_2B control were added to PBMC from 17 SLE patients.After IL-12 stimulation,we observed that the functional anti-TIGIT monoclonal antibody could increase the IL-12-stimulated IFN-γproduction by T lymphocytes in SLE patients（P=0.020）.