Clinical Significance And Immune Regulation Of Th9/Tc9 Cells In Chronic Lymphocytic Leukemia

Objective:Chronic lymphocytic leukemia(CLL)is a malignant tumor of the blood system that mainly occurs in the elderly,and its pathogenesis is still unclear.This study uses CLL as a disease model,analyzing differentially expressed genes(DEGs),related signal pathways,and immune cell infiltration in CLL samples by comprehensive bioinformatics to understand the molecular mechanisms involved in CLL.By studying the characteristics of Th9,Tc9 cells and their related molecules before and after CLL treatment,activation of TGF-β/Smad pathway in patients with CLL,the role of TGF-β and TNF-α in Th9/Tc9 cells inducing differentiation to explore the immunomodulatory role of Th9/Tc9 cells in CLL.By observing the levels of MDSCs and their cytokines Arg-1 and iNOS in CLL,we preliminarily explored the correlation between MDSCs and Th9/Tc9,and analyzed the clinical significance and prognostic value of MDSCs and Th9/Tc9 in CLL based on patient clinical data.Methods:Part I:Download the original data from the gene expression omnibus(GEO)database,screen the DEGs between CLL and the control,and conduct functional and pathway enrichment analysis.Identify the top 20genes with the highest connectivity in CLL through protein-protein interaction.Use Immune cell abundance identifier(ImmuCellAI)to evaluate the differences in immune cell infiltration between CLL and the controls,and analyze the correlation between signal pathways and immune infiltrating cells.Collected peripheral blood from 15 newly diagnosed CLL patients and 10healthy controls,and the mRNA expressions of TGF-β/Smad were detected by real-time fluorescent quantitative PCR(RT-qPCR)and the expressions of CD4+T lymphocytes as well as CD8+T lymphocytes were detected by flow cytometry.Part Ⅱ:Collected a total of 50 newly diagnosed patients with CLL as experimental group and 30healthy volunteers with physical examination as healthy control.The percentages of Th1,Th2,Th17,Treg,Th9,Tc9,granzyme B and perforin in peripheral blood of two groups were detected by flow cytometry.The protein and mRNA expressions of TGF-β,Smad3(p-Smad3),Smad7,PU.1 and IL-9 were detected by Western Blot(WB)and RT-qPCR.The level of IL-9,IFN-γ,IL-4,TNF-α,IL-17,IL-10 and TGF-β were detected by ELISA.Blood samples were collected from 20 patients with CLL who received treatment.The frequency of Th9,Tc9,granzyme B,and perforin in peripheral blood after treatment was detected by flow cytometry.The expression of PU.1 and IL-9 proteins and mRNA in T lymphocytes of CLL patients after treatment was detected by WB and RT-qPCR.The Changes of cytokine in serum of CLL patients after treatment was detected by ELISA.Sorted initial CD4+T cells and initial CD8+T cells from CLL patients were induced by TGF-β,IL-4 and TNF-α.Part Ⅲ:The frequency of MDSCs in peripheral blood was detected by flow cytometry,and the concentrations of Arg-1 and iNOS in serum were detected by ELISA.The differences in the expression of MDSCs and Th9/Tc9 in different clinical characteristics and laboratory indicators of CLL were analyzed.Pearson correlation analysis was performed to analyze the correlation between MDSCs and Th9,Tc9,Th9/Tc9 ratios;Arg-1 and IL-9,PU.1,IFN-γ;TGF-β and MDSCs,Th9,Th9/Tc9 ratios.Generate receiver operating characteristic curves for MDSCs and Th9/Tc9 ratios.Results:Part Ⅰ:There were 98 genes upregulated and 3002 genes downregulated in DEGs between CLL patients and control group.DEGs played a biological role in biological processes,molecular functions,and cellular components,while the above DEGs participate in TGF-β and multiple signal paths.Among the DEGs analyzed by PPI network,20genes such as SRC,SYK,and FYN have the highest node degree.There was a difference in the infiltration level of immune cells in the CLL group and the control group,and T cells in the CLL patient were significantly lower than those in the control group.The correlation between signal pathways and immune infiltrating cells shows that TGF-βpathway is positively correlated with multiple cells such as CD4+T cells and CD8+T cells.In the validation section,RT-qPCR detection showed TGF-β mRNA levels in T lymphocytes of CLL group were significantly increased,and flow cytometry showed a significant decrease in the percentages of CD4+T cells and CD8+T cells in the peripheral blood of the CLL group.Part Ⅱ:Flow cytometry showed a significant increase in Treg cells and Treg/Th17 ratio in the peripheral blood of CLL patients,with no significant difference in Th1,Th2,and Th17.Th9 cells were significantly increased in high-risk CLL patients,Tc9 cells were slightly increased in low-medium risk CLL patients,but decreased in high-risk CLL patients,and the Th9/Tc9 ratio was significantly increased.The increase of TGF-β,PU.1,IL-9 proteins and mRNA,as well as p-Smad3 protein in T lymphocytes of CLL patients is particularly significant in high-risk CLL patients,while the Smad7 protein and mRNA decreased.The frequency of granzyme B and perforin in the peripheral blood of CLL patients decreased,and the decrease was more significant in the high-risk group of CLL patients.The concentrations of IL-9,IL-10,and TGF-β in serum increased,with the former two increasing more significantly in high-risk CLL patients.The concentrations of IFN-γ and TNF-α decreased while the IL-4 and IL-17 did not significantly change in the CLL group.There were 20 CLL patients received treatment.In these patients the frequency of Th9 cells in the peripheral blood decreased,Tc9 cells,granzyme B and perforin increased,and the Th9/Tc9 ratio decreased.The protein and mRNA of PU.1 and IL-9 in T lymphocytes were downregulated,the concentration of IL-9,IL-10,and TGF-β decreased,while IFN-γ and TNF-α increased in these 20 CLL patients received treatment.The results of in vitro experiments showed that the levels of IL-9 and PU.1 in the culture system of initial CD4+T cells from CLL patients increased after adding TGF-β and IL-4,but decreased after adding TNF-α.The levels of IL-10,IFN-γ,IL-17,and granzyme B did not significantly change.After adding TGF-β and IL-4 to the initial CD8+T cells of CLL patients,the levels of IL-9,PU.1,IFN-γ and granzyme B in the culture system increased,but the increase in IFN-γ and granzyme B was greater,and the increase was more significant after adding TNF-α.There was no significant change in the levels of IL-10 and IL-17.Part Ⅲ:The frequency of MDSCs in the peripheral blood of CLL patients increased significantly,especially in high-risk CLL patients.The concentrations of Arg-1 and iNOS in the serum increased,with Arg-1 being significantly higher in high-risk CLL patients than in low-medium risk CLL patients.MDSCs were positively correlated with Arg-1 and iNOS,respectively.MDSCs and the ratio of Th9/Tc9 increased in patients with Binet C,β2-microglobulin>3.5(mg/L)group and IGHV non mutation group,while the frequency of MDSCs increased in the TP53 abnormal group.MDSCs are positively correlated with the frequency of Th9 and the ratio of Th9/Tc9,Arg-1 is positively correlated with IL-9 and PU.1,while there was no correlation between other indicators.Conclusion:Part I:Through comprehensive bioinformatics analysis of CLL samples in public databases and found that TGF-β pathway may be involved in the molecular imbalance mechanism of CLL,and immune cell infiltration is abnormal in CLL patients.The results of RT-qPCR validation suggested that TGF-β pathway may be activated in T cells of CLL patients,and flow cytometry shows that T cells in CLL patients were unbalanced.Part Ⅱ:Th9/Tc9 cells and related molecules,TGF-β/Smad pathways were involved in the occurrence and development of CLL.There was an imbalance between Th9 and Tc9 cells in CLL patients.TGF-β can induce the differentiation of Th9 and Tc9 cells in vitro.TNF-α inhibits the differentiation of Th9 cells but promotes the differentiation of Tc9 cells.Newly discovered Th9 and Tc9 cells may become targets for immunotherapy in CLL patients.Part III:Upregulated MDSCs and their cytokines may be related to the progression of CLL.Th9/Tc9 ratio and MDSCs may be used as prognostic indicators and biomarkers in patients with CLL.In addition,MDSCs and Th9 cells were positively correlated and they may have synergistic effects,which together participate in the occurrence and development of CLL.