| Part 1:The effect of bone marrow TRM on megakaryocyte maturation in vitro in primary immune thrombocytopeniaBackground:Immune thrombocytopenia（ITP）is an autoimmune-mediated platelet deficiency syndrome characterized by increased platelet destruction or decreased generation,and abnormalities in megakaryocyte maturation or apoptosis.It is the most common hemorrhagic autoimmune disease in clinical practice.The clinical manifestations of ITP mainly include spontaneous skin and mucosal bleeding without obvious cause,and can also cause visceral bleeding and even intracranial hemorrhage.The main pathological features of ITP are increased platelet destruction and/or decreased platelet generation,among which megakaryocyte maturation disorders,antigen-specific autoantibody abnormalities,and T cell-mediated platelet destruction are important pathogenic mechanisms of ITP.T cell dysfunction participant in the process of megakaryocyte and platelet destruction in ITP.Among them,cytotoxic T cells can accumulate in the bone marrow to directly lyse platelets;helper T cells（Th）can assist B cells in producing antibodies to kill platelets,and exhibit a stronger Th1 phenotype,thereby enhancing cell-mediated killing.Bone marrow immune tolerance defect is one of the main reasons for megakaryocyte maturation disorders and decreased platelet generation in ITP.Previous studies have confirmed that CD8+T cells can cause decreased platelet production by inhibiting the maturation of bone marrow megakaryocytes.As an important immune organ,bone marrow is not only the site where platelets are destroyed,but also the site where various blood cells and immune cells such as megakaryocytes and monocytes/macrophages develop and mature and exert their functions.In recent years,it has been found that there are a large number of tissue-resident memory T cell（TRM）in the bone marrow,which can reside in the bone marrow for a long time and have important immune regulatory functions.The chronic and protracted course of ITP may be closely related to abnormal memory T cells in the body.The region-specific TRM in the bone marrow may form a pathological microenvironment by secreting inflammatory mediators,affecting the activity and function of megakaryocytes and B cells,participate in the occurrence and development of ITP,and may be an important pathogenic mechanism of chronic or refractory ITP.Objective:（1）To explore the differences in TRM cells in the bone marrow of ITP patients compared with healthy controls,and clarify the abnormal distribution of TRM in ITP patients.（2）To investigate the differences in the expression of inflammatory factors in bone marrow TRM between ITP patients and healthy controls.（3）To analyze the correlation between the proportion of TRM in the bone marrow of ITP patients and the number of bone marrow megakaryocytes,peripheral blood platelets,age,hormone sensitivity resistance,and recurrence and difficult-to-treat conditions,and further explore the potential association between TRM and ITP disease.（4）To explore the effects of bone marrow TRM on megakaryocyte formation and platelet production in ITP patients.Methods:（1）Cases collection:Bone marrow samples of 22 ITP patients and 8 healthy donors who visited the Department of Hematology,Qilu Hospital of Shandong University were collected,and the patients’ basic information,clinical characteristics,and diagnosis and treatment process were recorded.（2）Flow cytometry analysis:Separation of bone marrow plasma,separation of bone marrow mononuclear cells（BMMCs）using Ficoll density gradient centrifugation method,and detection of the proportion of memory T cells in the bone marrow.（3）TRM cell isolation:The separated BMMCs were labeled with flow cytometric antibodies,and TRM cells were sorted using a flow cytometer under sterile conditions.（4）RNA extraction:RNA was extracted from the sorted TRM cells using an RNA rapid extraction kit,and the RNA samples were quantified using a microspectrophotometer.（4）Real-time fluorescence quantitative PCR:The expression of inflammatory factors IFN-y,NLRP3,IL-1 β,TNF-α,and IL-10 in bone marrow TRM of ITP patients and healthy controls was detected.（5）Correlation analysis:Collect information on bone marrow puncture results and treatment prognosis of ITP patients,and perform correlation analysis with the proportion of TRM in patients.（6）CD34+stem cell enrichment:CD34+stem cells in the bone marrow were enriched using anti-human CD34+magnetic beads.（7）TRM function experiment:The sorted TRM cells and CD34+stem cells were placed in SFEM medium containing rhTPO,rhSCF,and rhIL-3,and cultured for 14 days with semi-quantitative exchange.Flow cytometry was used to detect the proportion of megakaryocytes,megakaryocyte ploidy,megakaryocyte apoptosis,and reticulated platelet production,and the morphology of megakaryocytes was observed under a microscope.Results:（1）Increased proportion of TRM cells in bone marrow of ITP patients:Bone marrow mononuclear cells were collected for flow cytometry analysis,and it was found that the proportion of CD8+TRM cells in ITP patients’ bone marrow was significantly higher than that in healthy controls,while the proportion of CD4+TRM cells had no significant difference.The proportion of CD8+TCm cells was lower in ITP patients than in healthy controls,while the proportion of CD8+TEM cells was higher in ITP patients,though there was no significant difference between the two groups.（2）High expression of pro-inflammatory factors in bone marrow TRM cells of ITP patients:RT-PCR detection showed that the expression levels of IFN-γ,NLRP3,TNF-α,and IL-1β in bone marrow TRM cells of ITP patients were higher than those in healthy controls,while the expression level of IL-10 was lower in ITP patients.（3）More TRM cells in bone marrow of steroid-sensitive ITP patients:Correlation analysis between the level of TRM cells in bone marrow and the results of bone marrow puncture and treatment prognosis information of ITP patients found that the level of TRM cells in ITP patients had no significant correlation with the number of megakaryocytes,age,antiplatelet antibodies,and refractory recurrence.However,the proportion of TRM cells was higher in steroid-sensitive patients than in non-sensitive patients,indicating that steroids may play a therapeutic role by correcting the function of TRM cells in ITP patients.Although the level of TRM cells in ITP patients had no significant correlation with the number of bone marrow megakaryocytes,it showed a negative correlation trend with peripheral platelet counts.（4）TRM cells inhibit megakaryocyte apoptosis and reduce reticulated platelet formation,and steroids can correct the function of TRM cells in ITP patients to play a therapeutic role.After co-culturing TRM cells with CD34+stem cells for 14 days,the cells in culture system were collected to analyse the proportion of polyploid cells.The results showed no significant difference in the proportion of CD41+megakaryocytes among the groups,but the detection of megakaryocyte polyploidy showed that the proportion of ≥4N polyploid megakaryocytes was increased in the TRM cells+CD34+ cells co-cultured group compared to the CD34+stem cells alone culture group,and dexamethasone inhibited the effect of TRM cells on promoting megakaryocyte polyploidization.Platelet-rich plasma was obtained by low-speed centrifugation,and thiazole orange was used to label reticulated platelets.It was found that the co-culture of CD34+cells and TRM cells significantly reduced reticulated platelet production,and dexamethasone-treated TRM cells could promote reticulated platelet production.Megakaryocyte apoptosis experiments showed that TRM cells could inhibit early-stage megakaryocyte apoptosis and platelet production inhibition,and dexamethasone could reverse the inhibitory effect of TRM cells.Cell suspension（100ul）was taken and made into a slide,which was stained with Wright’s stain and observed under a 100-fold oil microscope.Platelet-producing megakaryocytes with cytoplasmic platelets and even detached platelets could be seen in the CD34+stem cells alone culture group and the CD34+stem cells+dexamethasone-treated TRM cells group.In contrast,most of the cells in the CD34+cell+TRM cell co-culture group were granulocytic megakaryocytes,and platelet-producing megakaryocytes were rare.Conclusions:（1）The proportion of CD8+TRM cells in bone marrow of ITP patients is significantly higher than that in healthy controls,indicating that ITP patients have an abnormal increase in the number of bone marrow memory T cells,and immune homeostasis is disturbed.（2）TRM cells in bone marrow of ITP patients abnormally express pro-inflammatory factors such as IFN-γ,TNF-α,IL-1 β,and NLRP3,while low expression of the anti-inflammatory factor IL-10,suggesting that TRM cells play a role in activating local immunity in ITP patients.（3）There are more TRM cells in the bone marrow of steroid-sensitive ITP patients,and a higher proportion of TRM cells may indicate a better therapeutic effect of glucocorticoids.（4）TRM cells inhibit megakaryocyte apoptosis and reduce reticulated platelet formation,and steroids can correct the function of TRM cells in ITP patients to play a therapeutic role.Part 2:Transcriptome sequencing of bone marrow TRM and their impact on humoral immunity in ITPBackground:The pathogenesis of ITP involves multiple factors and processes,with numerous immune cells participating in immune tolerance imbalance,including T cells,plasma cells,macrophages,etc.Among them,T cell-mediated immune abnormalities are considered an important link in the pathogenesis of ITP.RNA-Seq is a new high-throughput gene expression analysis technology that uses high-throughput sequencing to sequence all RNA reverse-transcribed into cDNA libraries in tissues or cells.By calculating biological methods,it is converted into quantitative gene expression data,and the expression levels of different RNAs are calculated by counting the relevant reads.It can be used to detect differences in gene expression between normal and tumor tissues,before and after drug treatment,and between different developmental stages and tissues.Among all the types of differences detected,the most common is the difference in the expression level of all mRNAs.B cells,cytotoxic T cells,and other cellular components participate in the abnormality and damage of ITP megakaryocytes/platelets.Some groups of cells in these cells also show bone marrow residency,and TRM has close interactions with these cells.TRM can regulate the number and function of megakaryocytes/platelets through intercellular interactions,participate in the formation of bone marrow pathological immune microenvironment,form a complex network relationship with various immune cells in the bone marrow.When the body experiences immune disorders,it participates in the formation of pathological immune microenvironment in the bone marrow.B cells,as the source of antibodies and lymphocytes that generate immune memory,play an important role in the development of many autoimmune diseases.B cells differentiate into plasma cells after losing tolerance to self-antigens,which can produce pathogenic autoantibodies.In the field of ITP,there are also many explorations of B cells.Lyu et al.found that active ITP patients have increased peripheral total B cells and memory B cells compared with normal controls and relieved patients,and upregulated expression of co-stimulatory receptor CD72,while initial B cells are reduced.Previous studies have confirmed that the bone marrow pathological microenvironment in ITP patients leads to the long-term survival and continuous secretion of antibodies by plasma cells,leading to continuous destruction of platelets in the body.The bone marrow is the site of B cell development,and TRM participates in the formation of the bone marrow regional immune microenvironment,regulates the immune response in the bone marrow,and may affect B cell development and differentiation,participating in the pathogenesis of ITP and causing chronic persistence.Objective:（1）Explore the differences in the TRM cell transcriptome between ITP patients and healthy controls in the bone marrow,and clarify the abnormal status of TRM cells in the bone marrow of ITP patients.（2）Investigate the effect of TRM cells in the bone marrow on B cell differentiation in ITP patients.Methods:（1）RNA extraction and transcriptome sequencing:RNA of TRM cells in the bone marrow was extracted using TRIZOL,isopropanol,anhydrous ethanol,and chloroform.The SYBR Green Pro TaqHS pre-mixed qPCR kit was used to reverse transcribe RNA,and sample concentration was detected using Agilent2100 bioanalyzer.Library construction,whole transcriptome sequencing,and result analysis were performed.（2）TRM cell isolation:BMMCs was labeled with flow cytometry antibodies,and sterile conditions were used to sort TRM cells using a flow cytometer.（2）CD 19+B cell enrichment:CD19+magnetic beads were used to enrich CD19+ B cells in the bone marrow.（3）Co-culture of TRM cells and B cells:The cells were co-cultured with a culture medium containing IgM cytokines for 3 days.Cells and supernatants were collected to detect the proportion of memory B cells and cytokine concentration in the supernatant.（4）Flow cytometry:The proportion of memory B cells was detected by flow cytometry,and LEGENDplex multi-factor online analysis software was used to analyze the concentrations of BAFF,APRIL,CD40L,IFN-γ cytokines.（5）Enzyme-Linked Immunosorbnent Assay（ELISA）:ELISA was used to detect IgG concentration in the culture supernatant.Results:（1）The TRM cells in the bone marrow of ITP patients show higher expression of immune activation-related genes:Transcriptome sequencing was performed on TRM cells from the bone marrow of ITP patients and healthy controls sorted by flow cytometry.A total of 1929 differentially expressed genes were identified,among which 897 genes were up-regulated and 1032 genes were down-regulated in ITP patients with adjusted P<0.05（Figure 3）.The up-regulated genes include IGLC2,ASAP2,HIST2H2AA4,IGLV2-11,C1QA,ATP5MC1,IL5RA,CYC1,RTKN2.GO/KEGG analysis of the top 500 up-regulated genes revealed that the genes highly expressed in TRM cells of ITP patients were enriched in lymphocyte and B cell-mediated immune pathways.（2）TRM cells in the bone marrow of ITP patients promote B cell differentiation towards memory B cells:In co-culture of TRM cells and naive B cells from ITP patients,there was a decrease in the proportion of naive B cells and an increase in the proportion of memory B cells.A similar trend was observed in the co-culture of TRM cells and na?ve B cells from healthy controls.Additionally,when comparing TRM cells from ITP patients with healthy controls,there was no significant difference in the proportions of naive B,but more memory B cells were formed in the TRM group of ITP patients.（3）TRM cells in the bone marrow of ITP patients promote the generation of cytokines involved in B cell differentiation:The levels of B cell growth-dependent cytokines such as BAFF,APRIL,and CD40L were significantly increased in the supernatant of the co-culture of TRM cells and B cells compared to that of memory T cells.Further analysis revealed that BAFF,APRIL,and sCD40L levels were higher in the TRM group of ITP patients.（4）TRM cells in the bone marrow of ITP patients promote antibody production by B cells:ELISA detected an increase in IgG concentration in the supernatant of the co-culture of TRM cells and B cells from ITP patients（CD 19++TM co-culture group 238.2±115.3 ng/mL vs.CD19++TRM co-culture group 293.7±113.9 ng/mL,P<0.05）.A similar trend was observed in the co-culture of TRM cells and B cells from healthy controls.Additionally,there was a higher production of IgG in the TRM group of ITP patients compared to healthy controls.（5）TRM cells in the bone marrow of ITP patients produce more IFN-γ:There was a higher concentration of IFN-γ in the supernatant of the co-culture of TRM cells and B cells compared to that of memory T cells.Additionally,there was greater production of IFN-y in the TRM group of ITP patients compared to healthy controls.Conclusion:（1）TRM cells in the bone marrow of ITP patients show higher expression of immune activation-related genes,which are enriched in lymphocyte and B cell-mediated immune pathways.（2）TRM cells in the bone marrow of ITP patients promote B cell differentiation towards memory B and.（3）TRM cells in the bone marrow of ITP patients promote the generation of cytokines involved in B cell differentiation.（4）TRM cells in the bone marrow of ITP patients promote antibody production by B cells.（5）TRM cells in the bone marrow of ITP patients produce more IFN-γ,promoting the generation of B cell differentiation-related cytokines such as BAFF,APRIL,and CD40L.Part 3:The role of ICOS gene frameshift mutation in autoimmune diseasesBackground:Inducible T-cell co-stimulator（ICOS）is a member of the CD28 co-stimulatory molecule family and plays an important role in the activation and proliferation of T cells.ICOS ligand（ICOSL）is widely expressed on antigen-presenting cells such as B cells,macrophages,and dendritic cells.The activation of these cells further upregulates ICOS,and the connection between ICOS and ICOSL initiates the interaction between T cells and B cells,which is crucial for the generation and maintenance of germinal centers and the formation of memory B cells and antibody responses.The co-stimulatory signal transmitted by ICOS plays an important role in the development and maintenance of helper T cells（including Th1,Th2,Th17）and regulatory T cells（Treg）.Abnormalities in the ICOS signaling pathway can lead to various immune-related diseases,such as autoimmune diseases,immunodeficiency diseases,and allergies.Therefore,studying the relationship between ICOS gene mutations and immune-related diseases is of great significance for elucidating the molecular mechanisms of the ICOS signaling pathway and the pathogenesis of immune-related diseases.ICOS gene mutations are also associated with other autoimmune diseases such as rheumatoid arthritis,systemic lupus erythematosus,and Sj?gren’s syndrome.A study of ICOS mutations found that these mutations can lead to reduced T cell activation and proliferation,promote the proliferation and hyperfunction of immunosuppressive cells,and thus result in the loss of immune tolerance and the occurrence of autoimmune diseases.This study identified a novel disease-causing mutation,ICOS p.L96Sfs,which is associated with monogenic CVID.This mutation was validated by Sanger sequencing,and the clinical manifestations and laboratory findings of this mutation were further explored to investigate its significance in immune-related diseases.Objectives:（1）To explore the protein structural changes caused by the newly discovered ICOS mutation and clarify the structural functional domains affected by this mutation.（2）To investigate the differences in peripheral total B cells,naive B cells,and memory B cells between patients with this ICOS mutation and healthy controls,and to clarify the effects of this mutation on B cell differentiation,development,and function.（3）To investigate the effects of this ICOS mutation on T cell subset distribution,T cell activation,secretion of T cell inflammatory cytokines,and T cell STAT phosphorylation,and to refine the mechanism of disease onset caused by this mutation.Methods:（1）Sample collection:The peripheral blood of one patient with ICOS mutation and six healthy controls were included in this study.Plasma was separated,and PBMCs were isolated using Ficoll density gradient centrifugation.CD4+T cells were isolated using CD4 magnetic beads.（2）Sanger sequencing:DNA was extracted from PBMCs of healthy controls,patients,and their family members,and first-generation sequencing was performed to verify the ICOS mutation status.（3）Prediction of mutant gene nucleotides and protein structures:Transcripts mRNA were obtained from NCBI,and the coding sequences for amino acids in the protein（CDS）were identified.The mutant nucleotide sequence was predicted,and Swissmodel was used to predict the tertiary structure of the mutant protein.PyMol was used to construct the mutant protein model.（4）Flow cytometry:The expression of ICOS on T cells was detected,and the proportions of total B cells,naive B cells,memory B cells,total T cells,memory T cells,and Treg cells were measured.（5）In vitro functional assays of T cells:CD4+T cells were activated with CD3/CD28 antibodies,and the expression of Th cell subsets,activated T cells,cytokines IL-2,IL-4,TNFα,IFN-γ,IL17,and the phosphorylation levels of STAT1 and STAT4 were detected.Results:（1）Based on the revised ESID（2014）CVID diagnostic criteria and clinical manifestations,the patient was diagnosed with Common Variable Immunodeficiency（CVID）and received immunoglobulin replacement therapy.（2）Sanger sequencing showed that the pedigree analysis of the patient and his parents did not conform to Mendelian inheritance law.Only one of the parents carried a heterozygous mutation while the other was wild-type.Based on this result,it was initially suspected that the proband may have had a maternal lineage mutation in one allele and a de novo mutation in the same gene on the other allele.（3）ICOS mutant verification:This study predicted the structural model of the protein:homozygous ICOS mutation（c.279dupT,p.L96Sfs,NCBI NM012092）,which led to early truncation of the ICOS protein at residue 114 in the extracellular domain.Part of the extracellular domain,the entire transmembrane domain,and the cytoplasmic tail were all missing.（4）Abnormal distribution and impaired function of B cells in ICOS-deficient patients:The CD19+B cells in the patient’s peripheral blood were reduced,with a significant increase in the proportion of immature B cells and a decrease in memory B cells.The levels of IgG and IgA in the serum were significantly reduced,while the low level of IgM indicated impaired B cell function.（5）Abnormal distribution and impaired function of T cell subsets in ICOS-deficient patients:The levels of CD3+,CD4+,and CD8+T cells in the patient were extremely low.The number of central memory T cells（TCM）and effector memory T cells（TEM）decreased.There was a decrease in Th1 cells and an increase in Treg cells（Figure 3B,C）.The Th1/Th2 and Th17/Treg cell ratios were reduced.The expression of CD69 and CD25 on CD4+T cells was reduced after stimulation with anti-CD3/anti-CD28 antibodies.The levels of IL-2,TNF-α,and IFN-γ cytokines in the culture supernatant were down-regulated.This indicates that this ICOS mutation not only causes abnormal distribution of helper T cells and memory T cell subsets but also impairs T cell function.Conclusions:（1）The homozygous ICOS mutation（c.279dupT,p.L96Sfs）leads to structural changes in the ICOS protein,causing the loss of part of the extracellular domain,the entire transmembrane domain,and the cytoplasmic tail,which affects the expression and function of the ICOS molecule.（2）This ICOS mutation causes abnormal distribution and impaired function of B cells.（3）This ICOS mutation causes abnormal distribution of T cell subsets,inhibition of T cell activation,decreased secretion of T cell inflammatory cytokines,and abnormal T cell STAT phosphorylation,resulting in abnormal distribution of helper T cell and memory T cell subsets and impaired T cell function. |
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