| Mycobacterium tuberculosis(M.tuberculosis)infection mainly activates CD4+T cells and CD8+T cells.CD4+T cells were induced to differentiate into Thl type cells,which produce cytokines such as IFN-?,TNF-?,and IL-2,and activate cell-mediated immunity,CD8+T cells are differentiated into CTL cells to directly kill target cells,which play protective roles in clearing M.tuberculosis.However,long-term chronic infection of M.tuberculosis in patients with severe tuberculosis such as drug-resistant tuberculosis and sputum positive cavitary tuberculosis could lead to low immune response.In our previous studies,applying persistent M.tuberculosis antigen stimulation to mimic the status of M.tuberculosis persistent infection,we found that persistent stimulation with M.tuberculosis antigen could lead to T cells exhaustion,in which the proliferative ability of T cells decreased,and the production of cytokines IFN-? and IL-2 also decreased.On the animal model of T cells exhaustion induced by persistent M.tuberculosis antigen stimulation,further therapeutic studies on T cells exhaustion had found that IL-2 supplementation could reverse the exhaustion.Based on the above research,we further want to know whether persistent antigen stimulation could lead to the changes of bone marrow hematopoiesis during T cell exhaustion.Maintaining the steady state of bone marrow hematopoietic stem cells(HSCs)and precursor cells are the basis for the establishment of an effective anti-tuberculosis immune response.The differentiation and proliferation of HSC in bone marrow are regulated by cytokines such as GM-CSF,IL-2 and IFN-y.It was reported that in the process of persistent viral and bacterial infections,such as lymphocytic choriomeningitis virus(LCMV)infection,cytokine IFN-? was continuously secreted,which might cause hematopoiesis dysfunction.There are few studies on immune exhaustion and bone marrow hematopoiesis dysfunction in severe tuberculosis(TB),and the relevant mechanisms are still unclear.Objective:We speculated that under the continuous stimulation with M.tuberculosis antigen,high levels of cytokines were induced,which would cause the abnormal function of bone marrow HSC and hematopoietic precursor cells.This experiment continued to use the animal model of T cell exhaustion induced by persistent M.tuberculosis antigen stimulation,which previously established in our laboratory,and study the effect of persistent M.tuberculosis antigen stimulation on bone marrow hematopoietic function.Methods:To study the effects of persistent M.tuberculosis antigen stimulation on bone marrow hematopoiesis and T cell immune function,M.tuberculosis antigen was used to persistently stimulate mice,in order to observe the changes in the proliferative capacity of bone marrow hematopoietic cells during persistent stimulation of M.tuberculosis antigen,and analyze the changes in the expression levels of transcription factors related to the differentiation of hematopoietic cells.The effect of immune response induced by M.tuberculosis antigen stimulation on hematopoietic function in bone marrow was evaluated.On this basis,the effect of IL2 on bone marrow hematopoietic function was further studied.(1)Antigen stimulation and IL-2 treatment procedure.Taking the factors such as biosafety into account,this study applied M.tuberculosis antigen persistent stimulation to mimic the effects of continuous M.tuberculosis infection in clinic on the immune system and bone marrow hematopoietic function.In our study,using Bacillus Calmette-Guérin(BCG)to immunize C57BL/6 mice for the first time,and repeated subcutaneous injection with M.tuberculosis antigen ESAT-6,CFP-10 combined with Mtb10.4-HspX(MH)and adjuvants DDA and Poly(I:C)weekly.After repeated antigen stimulation for 12 weeks,the proliferating ability of bone marrow hematopoietic cells(Lin-c-Kit+,LK cells)was decreased,and obvious T cell dysfunction was detected after antigen stimulation for 22 times.IL-2 was used to treat T cells and bone marrow hematopoiesis dysfunction mice to study the changes of bone marrow hematopoietic cell proliferation and differentiation after IL-2 treatment.In the study,boosting BCG with the antigens twice was used as a control of transient antigen stimulation.At the same time,an adjuvant control group was established to exclude the effects of adjuvant on T cell and hematopoiesis functions.(2)Analysis of T cell function.The level of IFN-? secreted by spleen CD4+T lymphocytes was detected by flow cytometry and cytokine intracellular staining.The number of TB10.4 4-12 specific memory CD8+T cells was detected by the technique of MHC class I peptide pentamer staining technique.(3)Functional analysis of bone marrow hematopoietic function.The bone marrow cells were aseptically separated,and the proliferating activity of bone marrow hematopoietic cells was detected by EdU proliferation assay.The secretion levels of IFN-? and IL-2 in the bone marrow hematopoietic inductive microenvironment was detected by ELISA.The hematopoietic cells were sorted by magnetic beads,and the expression levels of transcription factors Batf2,IRF8,GATA2 and NOTCH1 in hematopoietic cells were detected by Real-time PCR,to analyze the effect of antigen stimulation on the differentiation of hematopoietic cells.(4)Treatment effect and mechanism of IL-2 on bone marrow hematopoietic cells.After IL-2 intervention,the changes in the above indicators were tested to measure the therapeutic effect and mechanism of IL-2.Results:(1)Effect of persistent stimulation with M.tuberculosis antigen on T cell function.Applying M.tuberculosis protein antigen to persistently stimulate mice,the detection of cytokines indicated that compared with transient antigen stimulation group,when antigen persistently stimulated for 12 weeks,higher levels of IFN-? were secreted by antigen-specific spleen CD4+T cells(p<0.05),however,IFN-? decreased after antigen persistently stimulated for 22 weeks.MHC antigenic epitope pentamer detection showed that after persistent antigen stimulation from 12 to 22 weeks,the number of TB 10.4 4-12-specific memory CD8+T cells in the spleen significantly decreased(p<0.01).(2)Persistent stimulation with M.tuberculosis antigen does effects on hematopoiesis function.Applying M.tuberculosis protein antigen to persistently stimulate mice,when antigen persistently stimulated for 12 weeks,higher levels of IFN-? in the bone marrow hematopoietic inductive microenvironment were detected(p<0.05),but eventually showed a downward trend.EdU proliferation test showed that compared with transient antigen stimulation group,after persistent antigen stimulated for 12 and 22 weeks,the proliferating activity of LK cells decreased(p<0.05).The expression levels of transcription factors in c-Kit+ cells in bone marrow also changed:compared with the adjuvant group,the expressions of Batf2 and IRF8,which involved in myeloid differentiation,were up-regulated,while the expressions of NOTCH1,which governing lymphoid commitment,were down-regulated.(3)The therapeutic effect of IL-2.While persistently stimulated mice with M.tuberculosis protein antigen,IL-2 were used to treat mice.The results showed that,the level of IL-2 in the hematopoietic inductive microenvironment was increased after IL-2 intervention.Compared with antigen persistence group,IL-2 treatment could down-regulate the expression of Batf2 and IRF8,up-regulate the expression of GATA2 and NOTCH1,and restore the proliferating activity of LK cells(p<0.05).At the same time,T cell exhaustion was also reversed:the levels of IFN-? secreted by spleen CD4+T lymphocytes increased(p<0.05),the number of splenic TB10.4 4-12 specific memory CD8+T cells was significantly increased(p<0.05).(4)Study of JAK3-STAT5 signaling pathway.JAK3-STAT5 signal pathway regulates the differentiation and proliferation of hematopoietic cells.The expression levels of JAK3 and STAT5 mRNA in antigen persistence group were lower than the adjuvant group.And the expression levels of JAK3 and STAT5 were significantly upregulated after IL-2 treatment.Conclusion:M.tuberculosis antigen persistently stimulates a Thl-type immune response with IFN-? secretion.M.tuberculosis antigen persistent stimulation decreased the proliferating activity of LK cells,up-regulated the expression of transcription factors Batf2 and IRF8,which promoted myeloid differentiation as the consequence of elevated IFN-? production.IL-2 supplementation enhanced the proliferating activity of LK cells,up-regulated the expression of transcription factors GATA2 and NOTCH1 involved in lymphoid commitment,and contributed to maintaining the homeostasis of hematopoiesis.In addition,IL-2 could up-regulate the expression levels of JAK3 and STAT5,and presumably enhanced the proliferation and differentiation of hematopoietic cells by JAK3-STAT5 pathway.Thus,persistent stimulation with M.tuberculosis antigens affected hematopoiesis,resulting in decreasing of the proliferating activity of hematopoietic cells,upregulating the expression of transcription factors that promote myeloid differentiation.However,IL2 treatment maintained the homeostasis of hematopoiesis.This study is the first to analyze the effect of M.tuberculosis antigen stimulation on the development and differentiation of bone marrow hematopoietic cells.Our study contributes to reveal the mechanism of immune dysfunction and the changes of bone marrow hematopoietic function in patients with tuberculosis under M.tuberculosis continuous infection,which provides a way to further study the precise diagnosis and treatment of severe pulmonary tuberculosis.Miliary tuberculosis is caused by a large amount of M.tuberculosis entering into blood circulation in a short period of time.Its onset is acute,the condition is dangerous,and the mortality rate is high.The immune responses in miliary tuberculosis patients declined due to long-term chronic infection with M.tuberculosis.The relevant mechanisms are still unclear.Objective:We hypothesized that miliary tuberculosis,which is caused by a large number of tuberculosis entering into blood,might induce excessive immune response of T cells at early stage,and the produced cytokines act on bone marrow hematopoietic cells,affect the development and differentiation of immune cells,at last the development of lymphocytes would be reduced,and ultimately caused immune dysfunction.In order to verify the above hypothesis,in this study,BCG entering into blood infection was used to mimic the immunopathological changes of clinical miliary tuberculosis infection,and its effects on the bone marrow hematopoietic system and immune system were investigated.Methods:First,through investigating the data of clinical cases with miliary tuberculosis,the changes of peripheral blood cells count in miliary tuberculosis were analyzed to reflect the changes of bone marrow hematopoiesis function.Then,different doses of BCG were applied via intravenous injection or intranasal route to infect mice.The changes of T cell function and bone marrow hematopoietic function were analyzed.Meanwhile,we analyzed the changes of expression levels of transcription factors related to the differentiation of hematopoietic cells,and evaluated the effect of BCG infection on bone marrow hematopoietic function.After infecting mice with different doses of BCG via different routes,the immune response characteristics and the effects on the function of bone marrow hematopoietic cells were compared.By this way we used BCG infection to establish an animal model to mimic the impaired hematopoiesis of miliary tuberculosis.(1)Analysis of clinical case data.We analyzed 28,047 cases of tuberculosis from Lanzhou Pulmonary Hospital during 2015 to 2019,including 118 cases of miliary tuberculosis.Through retrospective analysis,the number of peripheral blood cells in clinical cases of miliary tuberculosis was investigated.(2)BCG infection and IL-2 treatment program.According to literature reports and taking the factors such as biosafety into account,we applied high-dose BCG replacing M.tuberculosis to infect mice to mimic the infection of clinical miliary tuberculosis.In this study,mice were first infected with high-dose BCG(5 x 107 CFU/ml)via intravenous injection or intranasal route,peripheral blood cells count,T cell function and the proliferation and differentiation characteristics of bone marrow hematopoietic cells(LK cells)were detected respectively at 3 weeks,5 weeks and 8 weeks after infection.The mice infected by a low-dose of BCG(5 x 105 CFU/ml)via intranasal route were established as a control,and the PBS control group was designed to exclude the influence of external factors and age on the function of T cells and bone marrow hematopoietic cells.(3)Changes of peripheral blood cells counts,the levels of IFN-? in serum and organ index.Adding the fresh blood from mice into the anti-coagulation tube,and the changes of blood cells count fromdifferent groups were measured by an animal blood analyzer.Peripheral blood without anti-coagulation was centrifuged,and the level of IFN-? in serum was measured by ELISA.The effects of BCG infection on spleen index,liver index and lung index were observed by weighing the spleen,liver,lung and mouse.(4)Analysis of T cell function.The levels of IFN-? and IL-2 secreted by spleen lymphocytes after stimulated with BCG purified protein derivative(PPD)were detected by ELISA.The expression levels of inhibitory receptor PD-1 on CD4+and CD8+T lymphocytes were detected by flow cytometry.The proliferative capacity of CD4+and CD8+T lymphocytes after PPD stimulation were detected by EdU proliferation assay.(5)Analysis of bone marrow hematopoietic function.The bone marrow cells were aseptically separated and subjected to magnetic bead sorting to obtain LK cells.The proliferating activity of bone marrow hematopoietic cells was detected by EdU proliferation assay.The secretion levels of IFN-?,IL-2,TNF-?,IL-6 and GM-CSF in the bone marrow hematopoietic inductive microenvironment was detected by ELISA.The RNA was extraction from LK cells and the expression levels of transcription factors Batf2,IRF8,GATA2 and NOTCH1 in LK cells were detected by Real-time PCR,to analyze the effect of BCG infection on the differentiation of hematopoietic cells.(6)Analysis of IL-2 treatment.After high-dose BCG entering into blood infection,IL-2 was used to treat mice with T cells exhaustion and abnormal proliferation and differentiation of bone marrow hematopoietic cells.After IL-2 intervention,the changes in the above indicators were tested to measure the therapeutic effect and mechanism of IL-2.Results:(1)Analysis of clinical case data.According to the test results of peripheral blood cells from patients,the changes in many kinds of blood cells count were analyzed.It was found that including 118 cases of miliary tuberculosis,89%of them had lymphopenia(105 cases),23%of cases had leucopenia(27 cases),8.5%of cases had neutropenia(10 cases),49.2%of cases had hemoglobin reduction(58 cases),11%of cases had erythrocyte reduction(13 cases),8.5%of cases had thrombocytopenia(10 cases),and 4.2%of cases had pancytopenia.The above data indicated that miliary tuberculosis often had lymphopenia first,followed by various types of cytopenia such as leukopenia,hemoglobin reduction,and thrombocytopenia.(2)The effect of BCG infection on peripheral blood cells count and organ index.5 weeks after BCG infection,compared with PBS group,leukopenia,lymphopenia,and thrombocytopenia occurred in peripheral blood,and the spleen index,liver index,and lung index of mice were significantly increased in high BCG i.v.group,which indicated that the spleen,liver,and lung enlarged.(3)The effect of BCG infection on T cell function.At 3 weeks,5 weeks and 8 weeks after BCG infection,spleen CD8+T cells in high BCG i.v.group showed high expression level of inhibitory receptors PD-1 compared with PBS group,low BCG i.n.group and high BCG i.n.group(p<0.05).EdU proliferation test showed that the proliferation of CD8+T cells in low-dose BCG i.n.group was significantly stronger than other groups.Compared with PBS group,low BCG i.n.group and high BCG i.n.group,the proliferation of CD8+T cells in high BCG i.v.group was significantly decreased(p<0.05).The levels of IFN-? secreted by spleen lymphocytes stimulated by specific antigen PPD were detected by ELISA.The results showed that at 3-5 weeks,the levels of IFN-? in high BCG i.v.group were higher than that in low BCG i.n.group and PBS group(p<0.05),after 8 weeks,the ability of producing IFN-? was significantly reduced;IL-2 levels were higher in low BCG i.n.group at 3-5 weeks than that in PBS group and high BCG i.v.group(p<0.05).(4)The effect of BCG infection on the function of bone marrow hematopoietic cells.At 3 weeks after BCG infection,the proliferative capacity of LK cells in high BCG i.v.group was significantly higher than that in PBS group(p<0.01),and then gradually decrease.The level of cytokines in bone marrow supernatant were as follow:At 5 weeks after BCG infection,high BCG i.v.group induced higher levels of IFN-?and TNF-? than that in PBS group(p<0.05),while the levels of IL-2,IL-6 and GMCSF were not significantly different from those in PBS group,but the levels of IL-2,IL-6 and GM-CSF significantly higher than those in PBS group(p<0.05).The expression level of transcription factors in LK cells from high BCG i.v.group also changed significantly compared with PBS control group:the expression levels of transcription factors Batf2 and IRF8 involved in myeloid differentiation,were upregulated,while the expression levels of GATA2 and NOTCH1,which governing lymphoid commitment,were down-regulated.(5)Therapeutic effects of IL-2.After high-dose BCG entering into blood infected mice,IL-2 was used to treat mice.The results showed that the T cell exhaustion was reversed.Conclusion:Retrospective analysis of cases from patients with clinical miliary tuberculosis showed that impaired hematopoiesis function such as lymphopenia,thrombocytopenia and pancytopenia were found in the patients with miliary tuberculosis,being consistent with the literature reports.Using mouse model,we found that high-dose BCG entering into blood infection could cause peripheral lymphopenia and thrombocytopenia,and induce the increasing expression of cytokines IFN-? and TNF-?,which reduce the proliferation activity of LK cells,and up-regulate the expression levels of transcription factors Batf2 and IRF8 invoved in myeloid differentiation,and down-regulate the expression levels of transcrip tion factors GATA2 and NOTCH1 involved in lymphoid commitment,eventually leading to T cells exhaustion.IL-2 supplementation could reverse T cells exhaustion.This study combines clinical case data with animal experiments,from the perspectives of the development and differentiation of bone marrow hematopoietic cells to reveal the mechanism of low immune function in miliary tuberculosis and the changes of bone marrow hematopoietic function,which provides a reference for the prevention and treatment of miliary tuberculosis.T cell development and differentiation processes require glycolysis and oxidative phosphorylation to provide energy,especially effector T cells with enhanced glycolysis processes.In the previous chapter,we found that high-dose BCG entering into blood infection could cause the over-activation of T cells,leading to the occurrence of T cell exhaustion.We hypothesized that inhibiting glycolysis and mitigating the activation of T cells,might block the immunopathological process of T cell exhaustion.Glycolysis is an important characteristic of tumor metabolism.In our laboratory,we found enolase 1(ENO1)highly expressed in cervical cancer tissue by proteomics.ENO1 is an enzyme in glycolysis process.Silencing ENO1 gene expression by ShRNA interference could significantly reduce the tumorigenic ability and invasion and metastasis ability of cervical cancer Hela and SiHa cells,indicating that inhibition of ENO1 expression could block glycolysis,and thereby play a role in treating tumor.Objective:Although the use of shRNA to interfere with ENO1 expression could inhibit tumor growth,the application of RNA interference technology in clinic is limited by viral vectors and transportation systems.In order to promote clinical applications,this study intends to prepare the monoclonal antibody of ENO1,to further evaluate the effect of the monoclonal antibody against tumor.In addition,the first two chapters found that both persistent M.tuberculosis antigen stimulation and large number of BCG entering into blood infection could lead to low immune response or even exhausted.How to reverse the exhaustion is of great significance for the prevention and treatment of tuberculosis.Changes in cellular metabolism can determine T cell function,and thus cellular metabolism considered to be a key regulator of T cell function and differentiation.This study intends to prepare ENO1 monoclonal antibodies for further study new methods and strategies for the treatment of T cell exhaustion by intervening T cell metabolism.Methods:This study intented to use the eukaryotic expression system to express and purify the ENO1 protein,and prepare ENO1 monoclonal antibodies.It was expected that ENO1 monoclonal antibodies could reduce the migration and invasion of tumor cell by interfering with ENO1 on the surface of tumor cells.For this reason,we screened the ENO1 monoclonal antibody with strong anti-cervical cancer SiHa cell migration and invasion ability.The gene encoding the ENO1 monoclonal antibody was sequenced,and the variable region gene sequence was recombined into an adeno-associated virus.In the follow-up study,we will identify the function of the protein polypeptide from antibody variable region expressed by adeno-associated virus,and conduct an in-depth study on the intervention of ENO1 monoclonal antibody on inhibition effect of glycolysis.(1)Cloning,expression and purification of ENO1Method one:Using pET30a-ENO1 plasmid as template,the ENO1 gene was cloned into the expression vector pcDNA3.1 to construct the recombinant plasmid pcDNA3.1-ENO1;the pcDNA3.1-ENO1 was successfully transfected into Human embryonic kidney cells(HEK-293T cells)by transient transfection;the target protein was expressed;and the target protein was purified by Ni-NTA column.Method two:The pcDNA3.1-ENO1 was successfully transfected into Chinese hamster ovary cells(CHO-K1 cells)in a stable transfection manner to express the target protein.Method three:The ENO1 gene was cloned into the baculovirus expression vector pFastBacl,transfected into insect cells Sf9,and the target protein was expressed and purified.(2)Preparation of ENO1 monoclonal antibody.BALB/c mice were repeatedly immunized with ENO1 protein,and spleen cells of mice with higher antibody titer were taken out and fused with mouse myeloma cells SP2/0.The hybridoma cells were repeatedly screened to obtain a hybridoma cell strain with high antibody titer.ENO1 monoclonal antibody was purified.(3)Evaluation of the inhibitory effect of ENO1 monoclonal antibody on migration and invasion of cervical cancer cells.The effect of five ENO1 monoclonal antibodies on the migration and invasion of cervical cancer SiHa cells was examined by cell scratch test and Transwell chamber assay,and the ENO1 monoclonal antibody with best effects was selected.(4)The variable region gene sequencing of ENO1 monoclonal antibody.RNA of five hybridoma cells were extracted separately,and the corresponding cDNA was obtained by RT-PCR.The 3'-end gene-specific primers were designed based on the known sequences of the heavy and light chain constant regions of antibody,and the universal primers of the 5'-end were used to PCR-amplify the cDNA gene.The amplified product was sequenced to obtain the variable region gene sequence encoding the heavy and light chain of ENO1 monoclonal antibody.(5)The variable region gene of ENO1 antibody packaged into the adenoassociated virus.The light chain and heavy chain variable region genes of ENO1 monoclonal antibody H1,which had a significant inhibitory effect on the migration and invasion of cervical cancer cells,were ligated with linker--(GGGGS)3 and cloned on pAAV-MCS vector,which synthesized by company,referred as rAAV-H1.The pHelper,pAAV-RC bacteria in AAV helper virus system and rAAV-H1 bacterium were activated,preserved and amplified,and the plasmid was extracted in large quantities,transfected into AAV-293 cells,the transfection efficiency was observed and the virus was collected.At the same time,pAAV-IRES-ZsGreenl with green fluorescent protein was transferred into AAV293 cells as a vector control(rAAV-GFP).Result:(1)The expression and purification of ENO1 protein.First,pcDNA3.1-ENO1 was successfully transfected into HEK-293T cells by transient transfection to express the target protein.However,the results showed that the protein yield was too low.Then,the pcDNA3.1-ENO1 was successfully transfected into CHO-K1 cells in a stable transfection manner.The protein yield was also very low.At last,the ENO1 gene was cloned into the baculovirus expression vector pFastBacl,and transfected into insect cell Sf9.The protein yield was very high.The ENO1 protein with high purity was successfully purified.(2)The preparation of ENO1 monoclonal antibody.After immunization of mice with ENO1 protein,5 positive hybridoma cell lines with higher titer and related ENO1 monoclonal antibodies were successfully screened by hybridoma technology.(3)The effect of ENO1 monoclonal antibody on the migration and invasion of cervical cancer cells.The anti-tumor migration and invasion effects of five monoclonal antibodies were initially evaluated using cell scratch test and Transwell chamber.The results showed that five ENO1 monoclonal antibodies could inhibit the migration and invasion of cervical cancer SiHa cells.Among them,H1 had the strongest inhibitory effect.(4)The variable region gene sequencing of ENO1 monoclonal antibody.On basis of the monoclonal antibody,the genes encoding heavy and light chain variable region of the antibody were extracted from the hybridoma cell RNA using universal primers.By gene sequencing,the variable region gene was successfully obtained.(5)The variable region gene of ENO1 antibody packaged into the adenoassociated virus.The obtained heavy and light chain variable region genes of ENO1 antibody were constructed on an adeno-associated virus vector to obtain rAAV-Hl.A large concentration of pHelper,pAAV-RC and rAAV-H1 plasmids were obtained and transfected into AAV293 cells.The results showed that the transfection was successful and the transfection efficiency reached 90%.Conclusion:ENO1 protein with high concentration and purity was successfully purified by baculovirus expression vector.Using ENO1 protein to immune mice,5 ENO1 monoclonal antibodies with higher titer were successfully prepared by hybridoma technology.By evaluation of inhibiting the migration and invasion of cervical cancer cells by ENO1 monoclonal antibody,we found that the ENO1 monoclonal antibody H1 had the significant inhibition effect.The variable region genes of ENO1 monoclonal antibodies were successfully obtained,then the variable region gene of ENO1 antibody was recombined into an adeno-associated virus vector to obtain rAAV-H1.The preparation and related research of ENO1 monoclonal antibody also lay the foundation for further research on the effect of ENO1 monoclonal antibody on anti-tumor and anti-T cell exhaustion through inhibiting glycolysis. |
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