Study On The Expression Level,Function And Related Mechanism Of CD47 On T Cell Subsets In Patients With SLE

Objective:Systemic lupus erythematosus（SLE）is an autoimmune disease that can affect the whole body,which is currently considered to be the destruction of immune tolerance caused by a combination of genetic,environmental and stochastic factors,the majority of patients died mainly from kidney damage and infection.T cells,as an important part of the immune system,play an important regulatory role in SLE.Among them,the decrease in the number of CD4+T cells reflecting the immune status of the body aggravates the risk of infection and delays immune recovery in patients.At present,the decrease of CD4+T cells is mainly due to the increase of apoptosis level,which also aggravates the autoantigen load of SLE patients.In addition,CD8+T cells were found to have the ability to inhibit the proliferation of CD4+T cells in a tolerant lupus mouse model after p Cons peptide treatment.However,this phenomenon has not been reported in patients with SLE.Therefore,this study takes the decrease of CD4+T cells in patients as the starting point.It is known that the function of T cell subsets is regulated by surface receptors.Among them,inhibitory receptors have the potential to down-regulate cell function and inhibit inflammation,which have become a hot point in the treatment of autoimmune diseases.CD47,a glycoprotein of the Ig superfamily,has received widespread attention in recent years for its role in mediating tumor cell immune escape due to its high expression on tumor cells and as an inhibitory receptor with regulatory cell subset functions.However,the expression and mechanism of CD47 on the surface of T cell subsets in SLE patients are still unknown.Therefore,this study mainly wants to clarify the changed expression of CD47 on T cell subsets in patients and its relationship with the clinical outcome of the disease,and to explore the mechanism of CD47 may lead to the decrease of CD4+T cell number in patients,hoping to provide new ideas and basis for targeted therapy of SLE.Methods:1.Study ParticipantsA total of 183 individuals were selected,including 82 cases of healthy controls and 101cases of patients with SLE.The included healthy controls were healthy people without autoimmune diseases and recruited from the Key Laboratory of AIDS Immunology,the First Affiliated Hospital of China Medical University.The included disease group was diagnosed as SLE by the Department of Rheumatology and Immunology of the First Affiliated Hospital of China Medical University,including the first visit and treated patients.2.Detection of immune regulatory receptors expression in multiple cell subsetsThe PBMC of the subjects was added with Fc Receptor Blocking Solution to reduce non-specific staining,then added with fluorescent antibodies for surface staining.Finally,PBMC was washed with PBS,added with 7-AAD for living/deading staining and detected by flow cytometry.3.Detection of the ability of CD8+T cells to secret Granzyme B and PerforinThe PBMC of the subjects was stained for living/deading,then washed with PBS containing 2%FBS,added with fluorescent antibodies for surface staining.PBMC was washed again and added with membrane-breaking reagent for quieting 30 minutes,then washed with membrane-breaking lotion.Finally,adding fluorescent antibodies into sample for intracellular staining.Flow cytometry was performed after the cells were washed.4.Detection of the ability of CD8+T cells to secret IFN-γand TNF-αThe PBMC of the subjects was resuspended with R10 at a concentration of 0.5million/200μl.Transferring cells in incubator for 24 hours.The cells were stimulated with a mixture of PMA ionomycin and Golgistop at the last 5 hours of cultivation.Then,the cells were stained for living/deading after being washed.Next,the cells were added with fluorescent antibodies for surface staining after being washed again.Then,adding membrane-breaking reagent to cells for quieting 30 minutes and washing cells with membrane-breaking lotion.Finally,adding fluorescent antibodies into sample for intracellular staining.Flow cytometry was performed after the cells were washed.5.Detection of mitochondrial phenotype and mt ROS content of CD4+T cellsThe PBMC of the subjects was added with Mito Tracker（?）Orange,Mito Tracker（?）Green FM and Mito SOX（?）Deep red for detecting mitochondrial phenotype and mt ROS content.Next,the cells were stained for living/deading after being washed,and then added with fluorescent antibodies for surface staining after being washed again.Finally,flow cytometry was performed after the cells were washed.6.Detection of CD4+T cell death levelThe PBMC was resuspended with R10 at a concentration of 0.5 million/200μl.Using Dynabeads（?）Human T-Activator CD3/CD28 to stimulate cells for 48 hours.Then,the cells were added with fluorescent antibodies for surface staining after being washed.Next,the cells were added with PE-Annexin-V and 7-AAD for death detection after being washed with 1x Annexin V binding buffer and flow cytometry was performed.7.Detection of the effect of H₂O₂on CD4+T cell death levelThe PBMC of the subjects was resuspended with R10 at a concentration of 0.5million/200μl.Then,dividing the cells into two groups.The cells were added with H₂O₂and PBS respectively.After stimulation with Dynabeads（?）Human T-Activator CD3/CD28,cells were incubated in an incubator for 48 hours.Finally,the cells were collected to measure CD4+T cell death level,the next experimental operations are the same as experiment 6.8.The killing effect of CD8+T cells on autologous CD4+T cellsNegatively selected CD8+T cells and CD4+T cells from PBMC of the subjects were resuspended with R10 at a concentration of 1.0 million/ml.Then,CD8+T and CD4+T were co-cultured at a ratio of 2:1,and CD4+T cells that cultured alonely were used as control group.Adding Immuno Cult（?）Human CD3/CD28 to stimulate the cells respectively and incubating cells in an incubator for 24 hours.Next,the cells were added with fluorescent antibodies for surface staining after being washed with PBS containing2%FBS.Finally,the cells were washed again,added with 7-AAD for living/deading staining and detected by flow cytometry.9.CD47 blocking experiments of CD8+T and CD4+T cellsThe PBMC of the subjects was resuspended with R10 at a concentration of 0.5million/200μl.Then dividing cells into two groups and adding anti-CD47 antibody and Isotype-Ig G respectively;（1）The cells were incubated in incubator for 18 hours,washed with PBS containing 2%FBS and stained with fluorescent antibodies for surface receptors,the next experimental operations are the same as experiment 2.（2）The cells were incubated in incubator for 18 hours,and collected to measure the secretion of Granzyme B by CD8+T cells.The next experimental operations are the same as experiment 3.（3）The cells were incubated in incubator for 24 hours,and were stimulated with a mixture of PMA ionomycin and Golgistop at the last 5 hours of cultivation,and collected to measure the secretion of IFN-γand TNF-αby CD8+T cells.The next experimental operations are the same as experiment 4.（4）The cells were incubated in incubator for 18 hours,and collected to measure mitochondrial phenotype and mt ROS content of CD4+T cells.The next experimental operations are the same as experiment 5.（5）The cells were incubated in incubator for 2 hours.Then,adding Dynabeads（?）Human T-Activator CD3/CD28 into cells until 48 hours.Finally,the cells were collected to measure CD4+T cell death level,and the next experimental operations are the same as experiment 6.10.Statistical analysisFlow Jo v10.6.2 and Graphpad Prism 8.0 software were used to analyze and map the results of flow experiments.The independent data of the two groups were compared by non-parametric Mann-Whitney U test,and Spearman test was used for correlation analysis.Wilcoxon paired test was used to compare the two groups of data that met the paired data.P<0.05 was considered statistically significant.Results:（一）Five inhibitory receptors expression of CD4+T（CD3+CD8-T）,CD8+T and NK cells in SLE patients,and their relationship with disease progression and the number of CD4+T cells1.Five inhibitory receptors expression on the surface of CD4+T（CD3+CD8-T）,CD8+T and NK cells was changed to varying degrees in SLE patientsFive inhibitory receptors expression on the surface of CD4+T（CD3+CD8-T）,CD8+T and NK cells in SLE patients was detected by multi-color flow cytometry.It was observed that decreased expression of CD47,CD52 and CD161,and enchanced expression of CD200 on the surface of CD4+T cells（CD3+CD8-T）.Decreased expression of CD47 and CD161,and enchanced expression of CD200 on the surface of CD8+T cells.The expression of CD200 on NK cells was increased.There was no significant change in the expression of LAG3 in the three subgroups.2.CD47 on the surface of T cell subsets is the immune regulatory receptor most closely related to disease outcomeFurther analysis of the relationship between the five immune regulatory receptors expression and the C3 or C4 levels that reflecting disease progression,and the relationship between the expression of five immune regulatory receptors and the absolute number of CD4+T cells that reflecting immune status in SLE patients.It was observed that only CD47 expression of CD4+T cells and CD8+T cells was correlated with the absolute number of CD4+T cells,patients with lower CD47 expression had lower absolute number of CD4+T cells.And in patients with SLEDAI>9,CD47 expression of CD4+T cells and CD8+T cells was positively correlated with C3 levels.Therefore,CD47 molecules on T cell subsets may be the most closely related inhibitory receptors to the disease outcome of SLE patients.（二）Study on the mechanism of CD47 leading to the reduction of CD4+T cell number in patients1.The decreased expression of CD47 on CD4+T cells in SLE patients induces cell death by promoting oxidative stressWe found that CD47 expression of CD4+T cells was negatively correlated with the level of cell death and mt ROS in SLE.And the previous results showed that the number of CD4+T cells in the CD47^LOWexpression group was significantly decreased compared with the CD47^HIGHexpression group.We further explored whether anti-CD47 antibody could block CD47 and promote the oxidative stress of CD4+T cells leading to death.We found that after blocking CD47 with anti-CD47 antibody,the levels of MMP,mt ROS and the percent of death cells increased,and the death of CD4+T cells increased after exogenous addition of ROS agonist H₂O₂.The results showed that decreased CD47expression induced CD4+T cell death in SLE patients by promoting oxidative stress.2.The decreased expression of CD47 on CD8+T cells in SLE patients promotes CD8+T killing autologous CD4+T cellsCD8+T cells in SLE patients secreted more Granzyme B which was negatively correlated with CD47 expression.And the previous results showed that the number of CD4+T cells in the CD47^LOWexpression group was significantly decreased compared with the CD47^HIGHexpression group.We further found that CD8+T cells can kill autologous CD4+T cells in patients with SLE.To further explore whether anti-CD47antibody can block CD47 and promote the killing function of CD8+T cells.The results confirmed that the secretion of Granzyme B by CD8+T cells increased after blocking CD47 with anti-CD47 antibody.The above results indicate that the decreased expression of CD47 on CD8+T cells in SLE patients promotes CD8+T killing autologous CD4+T cells.（三）Effect of CD47 on the secretion of proinflammatory cytokines by CD8+T cells1.Blocking CD47 reversed the inhibitory effect of CD47 signal on the secretion of IFN-γby CD8+T cellsThe systemic inflammatory symptoms of SLE disease are related to the level of IFN-γand TNF-αin vivo.We further explored the effect of CD47 expression level on the secretion of IFN-γand TNF-αby CD8+T cells.After blocking CD47 with anti-CD47antibody,it was found that the secretion of IFN-γby CD8+T cells increased and there was no significant difference in TNF-αlevel.Conclusion:The level of five inhibitory receptors on the surface of multiple immune cells in SLE patients changed.Through sorting,it was finally found that CD47 on the surface of T cell subsets in SLE patients was the most closely related inhibitory receptor to the clinical outcome of the disease.Among the five inhibitory receptors,CD47 was the only immune regulatory receptor related to the absolute count of CD4+T cells,and in active patients,the expression of CD47 is related to the level of serum complement C3.The decreased expression of CD47 on the surface of CD4+T cells in SLE patients can promote cell oxidative stress and eventually lead to cell death,and the decreased expression of CD47on the surface of CD8+T cells in SLE patients can also promote CD8+T cells to kill autologous CD4+T cells and secrete IFN-γ.These two mechanisms may eventually lead to a decrease in the number of CD4+T cells in patients,an increase in the risk of infection,a slower immune recovery,and a promotion of systemic inflammation.Therefore,CD47 is expected to become a promising target for immunological treatment and functional cure of SLE patients in the future.