The Study Of T-cell Receptor Repertoire And Functional Phenotypes Of Cardiac T Cells In The Patients With Ischemic Heart Failure

Aim To probe the pathological local infiltration of T cells in human ischemic failing myocardium after myocardial infarction and uncover the functional and phenotypic characteristics.Methods 1)Collected the left ventricular myocardial samples from post-STEMI ischemic heart failure(IHF)patients who received heart transplantation(n=5).Peripheral venous blood samples(around 5ml)were also collected from the same individuals;The control myocardium samples in the same myocardial location were collected.2)Routine Hematoxylin-eosin(HE)staining was used for the evaluation of basic histopathological features.And immunofluorescent(IF)staining assays were used to visualize the infiltration of cardiac CD3⁺,CD4⁺and CD8⁺T cells and colocalization of CD3 with the myocardial fibrotic marker(Collagen-I),proliferative marker ki67,BDCA3⁺dendritic cells marker(C-type lectin domain containing 9A,CLEC9A),macrophage markers(HLA-DR and CD68);3)The myocardial tissues were enzymatically digested by Liberase TH.And the peripheral blood mononuclear cells(PBMCs)and cardiac mononuclear cells(buffy coat)were isolated.Flow cytometry assays(FCAs)were used to detect the number and frequency of cardiac CD4⁺and CD8⁺??T cells in the myocardial samples.Frequency of CD45RA⁺CCR7⁺naive T cells(T_naive),CD45RA^-CCR7⁺central memory T cells(T_CM),CD45RA^-CCR7^-effector memory T cells(T_EM)and CD45RA⁺CCR7^-terminally-differentiated effector memory T cells(T_EMRA)in the cardiac or circulating CD4⁺and CD8⁺T cells were analyzed in the FCAs.The frequency of IFN-?⁺Th1,IL-4⁺Th2,IL-17⁺Th17 and Foxp3⁺Treg in the cardiac and circulating CD4⁺T cells and the frequency of IFN-?/GZMB/PRF1-producing CD8⁺T cells were also analyzed in the FCAs.Real-time polymerase chain reaction(RT-PCR)was used to detect the expression of IFN-?,IL-4,IL-17,FOXP3,GZMB,PRF1,IL-6 and HLA-DR in the control and diseased myocardial samples.Results 1)HE staining showed that amounts of inflammatory cells gather in the myocardial fibrotic areas;2)IF assays illustrated that significantly increased infiltration of CD4⁺and CD8⁺T cells in the myocardial samples from IHF patients compared to controls.The geographical distribution of CD3⁺T cells was correlated with the fibrotic areas in the myocardial samples.Ki67⁺CD3⁺T cells were found and co-localizations of HLA-DR^highCD68⁺macrophages and CLEC9A⁺DCs with CD3+T cells were also observed in the diseased hearts;3)Significantly increased number of cardiac CD4⁺and CD8⁺T cells isolated from diseased myocardium per gram compared to control myocardium.The number of cardiac CD4 and CD8⁺T cells was nearly equal but the ratio of CD4 to CD8 in hearts was significantly decreased compared to blood from the same individuals;4)Compared to blood,the frequency of T_CMwas significantly lower but T_EMRAwas significantly higher in the cardiac CD4⁺T cells.The frequencies of Th1 was significantly higher but Th17 and Treg were significantly lower in the cardiac CD4⁺T cells compared to blood.Compared to blood,the frequencies of TEM and TEMRA were significantly higher but T_naiveand T_CMwere significantly lower in the cardiac CD8+T cells.The higher frequencies of IFN-?/PRF1/GZMB-producing in the cardiac cytotoxic CD8⁺T cells compared to blood were also observed;5)RT-PCR revealed that the higher expression of IFN-?,GZMB,PRF1 and HLA-DR in the diseased myocardium compared to controls.Conclusion 1)In the myocardium from IHF patients,pathological infiltration of CD3+T cells is observed in the fibrotic areas;2)The activation and clonal proliferation of local T cells in the ischemic failing myocardium might be controlled by the cardiac-resident antigen presentation cells(APCs);3)The cardiac T cells in the IHF patients might be able to proliferate and self renew;4)Cardiac T cells in IHF patients might be mainly composed of TEM and TEMRA,characterized by predominant CD4⁺Th1 and cytotoxic T cells producing IFN-?,GZMB,and PRF1 abundantly,which play a pro-inflammatory and cytotoxic role in failing myocardium.Aim The high throughput sequencing of the TCR? CDR3 region was helpful to uncover the global characterization of the TCR repertoire.In this section,the aim is to demonstrate the clonality and antigen specificity of cardiac T cells in the IHF patients according to the high throughput sequencing of the TCR? CDR3 region.Methods 1)Collected the myocardial and blood samples from 14 IHF patients(including 5IHF patients mentioned above)and 6 donors(including 5 control samples mentioned above).The peripheral venous blood samples were also obtained immediately from 14 age and sex-paired non-IHF controls;2)The genomic DNA molecules were isolated from myocardium or blood samples.Multiplex PCR was used for the amplification of the TCR?CDR3 region.The PCR products were purified and used for library construction.The high-throughput deep sequencing of the TCR? CDR3 region was performed in the Illumina Hiseq2000 platform.Then the raw sequencing data were aligned according to international IMGT database;3)According to all CDR3 sequences and frequency distribution in each sample,4 indexes were calculated to illustrate the clonal expansion,including the total frequency of high expanded clones(HECs),total/unique clonotypes,inverse Simpson's index,and Shannon H's index.The information about clonality was reflected based on 4indexes mentioned above.Principal component analysis was used to discriminate the difference in clonality of IHF-hearts from the other 3 groups;4)The differences in the usage profile of 624 TRBV-TRBJ rearrangements were analyzed among 4 groups.Principal component analysis was used to discriminate the differences in the profile of TRBV-TRBJ rearrangements of ICM-hearts from the other 3 groups.Results 1)The frequencies of HECs were significantly higher in IHF-heart and Con-heart compared to IHF-blood and Con-blood respectively.The ratio of total/unique clonotypes in the IHF-heart group was significantly higher than the other 3 groups,including Con-heart,IHF-blood,and Con-blood.The Shannon's H index and inverse Simpson's index were significantly lower in ICM-heart and Con-heart compared to IHF-blood and Con-blood respectively.An obvious geographical difference in the loading plot between the IHF-heart group and the other three groups in regards to clonality was observed according to PCA,suggesting the difference in clonality between IHF-heart groups and other groups;2)The similarity in the usage profile in TRBV-TRBJ rearrangements between IHF-heart and Con-heart groups was more obvious than between IHF-heart and IHF-blood groups.The same geographical location in loading plot between IHF-heart and Con-heart groups in regards to usage profile of TRBV-TRBJ rearrangements was observed according to PCA,which could be distinguished from the sites of IHF-blood and Con-blood groups.Conclusion Compared to from controls,it is probable that more obvious clonality but similar usage profile of TRBV-TRBJ rearrangements in cardiac T cells from IHF patients was observed,suggesting theoretically the similar antigen specificity between cardiac T cells from diseased and healthy myocardium.Compared to circulating T cells from IHF patients,it is probable that not only more obvious clonality,more reduced diversity but also distinct usage profile of TRBV-TRBJ rearrangements were observed in the cardiac T cells from IHF patients,suggesting that the cardiac T cells from IHF patients were characterized by obvious clonal expansion and antigen specificity.Aim In this section,the aim is to explore the shareability of TCR signature among different IHF patients and reveal the interrelationship between the shareability of TCR signature and HLA genotype among different IHF patients.Methods 1)Collected the myocardial and blood samples from 14 IHF patients(including 5 IHF patients mentioned above)and 6 donors(including 5 control samples mentioned above).The peripheral venous blood samples were also obtained immediately from 14 age and sex-paired non-IHF controls;2)The genomic DNA molecules were isolated from myocardium or blood samples.Multiplex PCR was used for the amplification of the TCR?CDR3 region.The PCR products were purified and used for library construction.The high-throughput deep sequencing of the TCR? CDR3 region was performed in the Illumina Hiseq2000 platform.Then the raw sequencing data were aligned according to international IMGT database;3)According to top 1000 CDR3 amino acid sequences listed by frequency from each sample,pairwise overlapping rates of TCR clonotypes between random two samples were calculated,including intragroup and intergroup overlapping rate among 4groups;2)Defined the IHF-associated clonotypes according to the share TCR clonotypes in the IHF-heart group,calculate the total frequency of these IHF-associated clonotypes between circulating T cells from ICM patients and controls;3)Established 3retrospectively cohorts,including controls(n=60),stable IHF patients(n=52)and severe IHF patients(n=40).Sequencing-based HLA typing was used for the detection of HLA genotype of 4 gene locus including HLA-A,HLA-B,HLA-DRB1 and HLA-DQB1 in all subjects;4)Chi-square test was used for comparison of differences in the carrier rate of specific HLA alleles among 3 cohorts.Analyzed the correlation between the risk of severe IHF and HLA alleles carrier.Calculated the odds ratio and 95% confidence intervals.Results 1)The intragroup pairwise overlapping rates of TCR clonotypes among samples of IHF-heart group was significantly higher than samples of IHF-blood group;2)The intergroup pairwise overlapping rates between IHF-heart and Con-heart groups and IHF-heart and paired IHF-blood groups were significantly higher than the intergroup overlapping rate between IHF-heart and Con-blood groups;the intergroup overlapping rate between IHF-heart and unpaired IHF-blood groups was slightly higher than between IHF-heart and Con-blood groups;3)The frequency of shared clonotypes in all clonotypes was the highest in the IHF-Heart samples.These clonotypes shared in IHF-heart samples were defined as IHF-associated clonotypes.The frequency of IHF-associated clonotypes was significantly higher in the circulating T cells from IHF patients than controls;4)The patients with shared and high-expanded TCR clonotypes carry generally one or several common HLA alleles;5)There was no difference in the frequency of HLA-A*11:01 and HLA-B*46:01 between control and stable IHF patients.However,significantly higher frequency of HLA-A*11:01 and HLA-B*46:01 in severe IHF patients compared to controls;frequency of HLA-B*46:01 in the severe IHF patients was also higher compared to stable IHF patients,but for HLA-A*11:01,there was a tendency towards an increase in severe IHF patients compared to stable patients.Conclusion The shareability of TCR clonotypes of T cells in the heart among individuals might be significantly higher than ones in blood,especially among cardiac T cells of IHF patients,and have a similar tissue-specific expression and TRBV gene segments,suggesting theoretically that the cardiac T cells from control or diseased hearts might recognise common cardiac self-antigen.The HLA alleles might be associated with the frequency spectrum of TCR clonotypes.HLA-A*11:01 and HLA-B*46:01 might be considered as risk factors for the prediction of severe IHF after MI.