| BackgroundRheumatoid arthritis (RA) is a chronic, progressive and invasive disease that can cause the loss of joint function and major disabilities. Early detection and treatment is critical for better prognosis in RA. Earlier studies have shown that T cell dysfunction caused by dysregulation of the human immune system is one of the major causes of RA. Moreover, T cell dysfunction in different RA patients is closely linked with prognosis and disease turnover. The immune regulation of T cells during RA disease is still unclear.Many studies have shown that multiple immune regulatory molecules play a role in T cell dysregulation in disease. T cell immunoglobulin-and mucin-domain-containing molecule-3(Tim-3) and programmed death1(PD-1) are two important regulatory molecules expressed on T cells. Tim-3is a transmembrane protein discovered in2002, and is found to express specifically on the surface of terminally differentiated Thl cells. The interaction of Tim-3with its cognate ligand galactin-9can negatively regulate the production of IFN-γ, and affect T cell tolerance. PD-1preferentially expresses on the surface of activated T cells, B cells and monocytes. The signal transduction of PD-1leads to recruitment of SHP-2domain to phosphorylated cysteine, which then suppresses immune activation. Preliminary results have shown that Tim-3and PD-1have suppressive effects in mouse autoimmune disease, suggesting that Tim-3and PD-1may have important roles in regulating excessive T cell-mediated inflammation in RA and ameliorate RA symptoms. In this study, we will investigate the role of Tim-3and PD-1in the development and progression of RA using clinical studies and in vitro cell culture. Results will be used to extend our understanding of RA disease progression and the underlying mechanisms, and provide new targets for disease therapy.Methods1. Tim-3expression in RA patients and its correlation with RA progression1.1Tim-3expression in the peripheral blood T cell subsets of RA patients Collect the peripheral blood of RA patients and healthy control volunteers. Detect the expression of Tim-3on the surface of peripheral blood CD4+T cells and CD8+T cells using flow cytometry.1.2Tim-3expression in the synovial fluid T cell subsets of RA patientsCollect the synovial fluid of RA patients. Detect the expression of Tim-3on the surface of synovial fluid CD4+T cells and CD8+T cells using flow cytometry.1.3Correlation between Tim-3expression on various T cell subsets in the peripheral blood of RA patients and their DAS28scorePerform Pearson correlation analysis on the Tim-3expression of peripheral blood T cell subsets in RA and their DAS28score using Prism GraphPad software.1.4Correlation between Tim-3expression on various T cell subsets in the synovial fluid of RA patients and their DAS28scorePerform Pearson correlation analysis on the Tim-3expression of synovial fluid T cell subsets in RA and their DAS28score using Prism GraphPad software.2. Effect of Tim-3expression on T cell function and regulation in RA2.1Tim-3expression on T cell proliferation in RACollect the peripheral blood of RA patients and healthy control volunteers. Use magnetic negative selection kit to purify peripheral blood T cells. Add anti-Tim-3blocking antibody or isotype control antibody for6days. Use CFSE staining to examine the proliferation of CD4+T cells and CD8+T cells.2.2Tim-3expression on the cytokine production of T cells in RACollect the peripheral blood of RA patients and healthy control volunteers. Use magnetic negative selection kit to purify peripheral blood T cells. Add anti-Tim-3blocking antibody or isotype control antibody for6days. Use florescent antibodies to stain for the expression of IFN-y and TNF-a in CD4+T cells and CD8+T cells.3. PD-1expression in RA patients and its correlation with RA disease progression 3.1PD-1expression on different peripheral blood T cell subsets in RACollect fresh peripheral blood of RA patients and healthy volunteers. Examine PD-1expression on peripheral blood CD4+T cells and CD8+T cells using flow cytometry.3.2Correlation between PD-1expression and CRP in RA patientsAnalyze the correlation between PD-1expression on different T cell subsets and CRP in RA patients using Pearson correlation analysis with Prism GraphPad software.3.3Correlation between PD-1expression and DAS28scores in RA patientsAnalyze the correlation between PD-1expression on different T cell subsets and DAS28score in RA patients using Pearson correlation analysis with Prism GraphPad software.3.4PD-1expression on T cell proliferation and cytokine production in RACollect the peripheral blood of RA patients and healthy control volunteers. Use magnetic negative selection kit to purify peripheral blood T cells. Add anti-PD-1blocking antibody or isotype control antibody for6days. Use CFSE staining to examine the proliferation of CD4+T cells and CD8+T cells. Use florescent antibodies to stain for the expression of IFN-y and TNF-α in CD4+T cells and CD8+T cells.4. Statistical analysisStudy data is analyzed using SPSS13.0software. Various analyses, including Student’s t test, Mann-Whitney nonparametric test, and Pearson correlation test, were performed. p<0.05is considered statistically significant.Results1. Tim-3expression is elevated in RA patients compared to healthy controls, and is negatively correlated with disease progression 1.1Tim-3expression on the peripheral blood T cells of RA patients is elevated compared to healthy controlsAccording to flow cytometry analysis, Tim-3expression on the peripheral blood CD4+T cells and CD8+T cells from RA patients are significantly elevated than that of healthy controls (p<0.01), showing that the expression of Tim-3in RA disease is elevated.1.2Tim-3expression on the synovial fluid T cells of RA patients is further elevated compared to healthy controlsIn order to study the expression of Tim-3in RA disease, we collected synovial fluid samples of RA patients. Results show that the expression of Tim-3on synovial fluid CD4+T cells is not only significantly higher than that of healthy volunteers (p<0.001), but also significantly higher than that of peripheral blood CD4+T cells in RA patients (p<0.01). Similarly, Tim-3expression on synovial fluid CD8+T cells is not only significantly higher than that on healthy subjects (p<0.001), but also significantly higher than that on peripheral blood CD8+T cells from RA patients. Results further show that Tim-3expression is elevated in RA disease, and suggest that it has a role in disease development and progression.1.3Tim-3expression in RA patients is negatively correlated with DAS28scoreStatistical analyses show that the Tim-3expression son peripheral blood CD4+T cells, peripheral blood CD8+T cells, synovial fluid CD4+cells and synovial fluid CD8+T cells, respectively, are negatively correlated (p=0.009,0<0.001, p=0.002, and p=0.006, respectively), suggesting that Tim-3has suppressive function in the CD4+T cells and CD8+T cells in RA patients. Moreover, Tim-3expression may ameliorate RA disease progression by suppressing T cell function.2. Tim-3can suppress T cell function in RA2.1Tim-3expression can suppress T cell proliferationResults from CFSE staining and flow cytometry have shown that the proliferation of CD4+T cells from RA patients co-incubated for6days in the presence of anti-Tim-3blocking antibody is significantly higher than CD4+T cells co-incubated with isotype control antibody (p<0.05). Similarly, the proliferation of CD8+T cells from RA patients co-incubated for6days in the presence of anti-Tim-3blocking antibody is significantly higher than CD8+T cells co-incubated with isotype control antibody (p<0.05). These results suggest that Tim-3expression can suppress T cell proliferation in RA.2.2Tim-3expression can suppress T cell proinflammatory cytokine expressionResults from flow cytokinehave shown that the IFN-y expression and TNF-a expression of CD4+T cells from RA patients coincubated for6days in the presence of anti-Tim-3blocking antibody is significantly higher than CD4+T cells co-incubated with isotype control antibody (p<0.05and p<0.01, respectively). Similarly, the IFN-y expression and TNF-a expression of CD8+T cells from RA patients co-incubated for6days in the presence of anti-Tim-3blocking antibody is significantly higher than CD8+T cells co-incubated with isotype control antibody (p<0.05and p<0.05, respectively). These results suggest that Tim-3expression can suppress T cell proinflammatory cytokine expression in RA.3. PD-1expression is decreased in RA patients, and is negatively correlated with disease progression3.1PD-1expression is reduced on the peripheral blood T cells of RA patients and serumFlow cytometry analyses show that PD-1expression on peripheral blood CD4+T cells and CD8+T cells in RA is significantly lowered than that in healthy volunteers (p=0.002and p<0.001, respectively). Moreover, PD-1concentration in RA patient serum is significantly lower in RA patients compared to that in healthy controls (p<0.05).3.2PD-1expression is negatively correlated with the CRP result of RA patientsStatistical analyses have shown that PD-1expression on CD4+T cells of CRP-positive subjects is significantly lower than that on CRP-negative subjects (p<0.001). Similarly, PD-1expression on CD8+T cells of CRP-positive subjects is significantly lower than that on CRP-negative subjects (p<0.001). These results suggest that PD-1 expression is negatively correlated with CRP result. Furthermore, PD-1expression may be used as an diagnostic indicator for RA disease progression.3.3PD-1expression in RA patients is negatively correlated with DAS28scoreStatistical analyses have shown that PD-1expressions on peripheral blood Cd4+T cells and CD8+T cells in RA patients are negatively correlated with DAS28score (p<0.001and p=0.001, respectively), suggesting that PD-1may be correlated with RA disease progression, and may be used as a descriptive indicator of RA disease progression.3.4anti-PD-1blocking antibody has no effects on T cell proliferation and cytokine productionResults from CFSE staining and flow cytometry have shown that the proliferation of CD4+T cells or CD8+T cells from RA patients co-incubated for6days in the presence of anti-PD-1blocking antibody is not significantly changed. Similarly, the IFN-y expression and TNF-a expression of from RA patients co-incubated for6days in the presence of anti-PD-1blocking antibody is also not significantly altered. These results suggest that anti-PD-1blocking antibody may not have effects on T cell proliferation and cytokine production in RA.Conclusion(1) Tim-3expression is elevated in RA patient T cells, and is negatively correlated with disease progression.(2) Tim-3can negatively regulate proliferation and proinflammatory cytokine production of CD4+T cells and CD8+T cells of RA patients.(3) PD-1expression is decreased in RA patient T cells, and is correlated with disease progression. |
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