Acute Myeloid Leukemia Exosomes Induce CD8~+T Cell Apoptosis And AMLCD8~+T Cell TCR Characteristics

Acute myeloid leukemia(Acute myeloid leukemia,AML,non-acute promyelocytic leukemia or non-APL)is a malignant clonal tumor originating from immature myeloid hematopoietic cells in the bone marrow.It progresses quickly and has a poor prognosis.More than 50% of the first complete Remission of AML patients is expected to relapse within 3 years,and only about 10% of patients achieve long-term survival.At the same time,AML accounts for 80% to 90% of acute leukemia in adults and 15% to 20% of acute leukemia in children.AML has become a disease that seriously endangers people's health,bringing a heavy burden and suffering to patients,their families and society.However,in the past 40 years,there has been no major progress in the treatment of AML.The induction therapy mainly takes cytarabine combined with anthracyclines,and the consolidation therapy takes high-dose cytarabine combined with hematopoietic stem cell transplantation.With the development of science and the accumulation of clinical data,some targeted drugs against common mutations in AML,Rydapt?(FLT3-IDT),Tibsovo?(IDH1),Idhifa?(IDH2),have been approved by the US FDA for marketing.However,AML has extremely complex heterogeneity with a variety of common mutations and abnormal karyotypes,so most patients cannot receive targeted therapy.calling for more advanced treatments to be used more and newer treatments to be used in the treatment of AML.At present,a variety of immunotherapies have achieved encouraging results in a variety of relapsed and refractory malignant tumors.All of the therapies are different from the cytotoxic mechanism of chemotherapy and the targeted mechanism of targeted therapy,the core idea of ??immunotherapy is to activate the body's own immune system to recognize and kill tumor cells,which may also have extremely far-reaching application prospects for AML with strong heterogeneity,limited treatment methods and high mortality.Therefore,the establishment of a research platform related to AML and immunity,an in-depth understanding of the molecular mechanism of the interaction between AML and the immune system,and a comprehensive clarification of the relationship between the two will have important guidance for further promotion of AML immunotherapy,as well as for the increased survival rate.CD8~+ T cells are the main effector cells in transplant rejection and anti-tumor aspects.CD8~+ T cells often kill tumor cells by secreting granzyme,perforin,cytotoxin,?-interferon and tumor necrosis factor,inhibiting the occurrence and development of tumors;they can also directly recognize virus-infected cells and tumor cells to play the role of killing,assuming the functions of immune defense and immune surveillance.CD8~+T cells are the co-receptors that T lymphocytes(CTL)T-cell receptor(TCR)recognizes major histocompatibility complex I(major histocompatibility complex I,MHC I)molecule-antigenic peptide complex,whose? chain V-like functional region can bind to the non-polymorphic ? region of MHC class I molecules.TCR ? and ? chains are encoded by a series of variable(V)and joining(J)genes,and TCR ? and ? chains are also encoded by diversity(D)genes.Complementarity determining region 3(CDR3),as the most diverse part of the variable region,is the main contributor to the specificity of T cell antigen recognition.Recent studies have shown that ?-CDR3 s in ?? T cells can predict the clinical response of AML patients.Although TCR may be a therapeutic target,the dynamic changes of CD8~+TCR and the mechanism of TCR regulation during chemotherapy are still unclear.??T cells are the main subgroup,accounting for about 90-95% of the T cells in the peripheral blood of healthy adults.Therefore,it is very important to explore the characteristics and clinical significance of CD8~+TCR spectrum in patients with acute myeloid leukemia.Exosomes derived from AML cells can enhance glycolysis and promote chemoresistance,inducing apoptosis of T-cell,regulating functional T cells(Treg),inducing the polarization of M2 macrophages,inhibiting antitumor toxicity of natural killer cells(NK)and inhibiting the differentiation of dendritic cells into mature DCs.Exosome expression TGF-?1 secreted by a subset of AML blasts can induce repression of NK cell-mediated antitumor responses and impair NKG2D-mediated cytotoxicity.The FMS-like tyrosine kinase 3(FLT3)gene is a member of the type III tyrosine kinase family.The FLT3 mutation is closely related to the occurrence of AML.which is the most common molecular abnormality in AML,and mutation occur in 25%-30%of AML patients.Among them,the FLT3-ITD mutation is the most common type of all mutations,accounting for about 20% of AML cases.At the same time,the FLT3-ITD mutation is also the most common molecular abnormality associated with poor prognosis in AML patients.A series of research results have suggested that the bone marrow tumor microenvironment plays a key role in leukemia cell formation in AML and drug resistance,and both innate immunity and adaptive immunity are severely affected.In the tumor microenvironment(TME),Dysfunction and failure of CD8~+ T are caused by immunosuppressive mechanisms.Tumor cell-derived exosomes play a key role in the regulation of CD8~+T cell toxicity.Tumor-derived exosomes participate in the proliferation and immune evasion of nasopharyngeal carcinoma cells by regulating exosomal mi RNAs.In a mouse model reported in an earlier study,exosomes from the glioblastoma cell line GL26 reduced the number and activity of CD8~+ T cells,and down-regulated the expression levels of IFN-? and granzyme B.Tumor-derived exosomes also mediate T cell apoptosis.Fas ligand membrane exosomes expressed in the serum of oral cancer patients induce apoptosis of activated CD8~+ T cells.However,the potential effect of AML primary cells,especially FLT3-ITD mutation-positive exosomes on CD8~+ T cells is not fully understood.This study will mainly focus on the analysis of exosomes derived from FLT3-ITD mutation-positive AML patients and the FLT3-ITD mutation-positive cell line MV-411 cells,and explore its effect on CD8~+T cell apoptosis and possible mechanisms.It is of great significance to analyze CD8~+ T cell clonal expansion and TCR diversity characteristics of AML patients after initial treatment and chemotherapy(including patients with complete remission of CR and patients with refractory relapse)and healthy controls by using single-cell sequence,to elaborate the immunosuppression mechanism of CD8~+ T cells influenced by exosomes derived from AML,to analyze the relationship between tumor antigens in AML cells and identification of TCR.Part I FLT3-ITD exosomes activate PI3K/AKT/mTOR to induce AML cell immune escapeObjective:To clarify the characteristics of the apoptosis effect of FLT3-ITD mutation positive exosomes on CD8~+ T cells;and to explore the possible mechanism of the immune escape of FLT3-ITD mutation-positive exosomes on CD8~+ T cells.Methods :From June 2018 to May 2020,4 cases of FLT3-ITD mutation-positive AML patients(non-APL patients)admitted to the Department of Hematology,Affiliated Hospital of Guizhou Medical University,and 1 healthy donor were collected.Peripheral blood(PB)and bone marrow(BM)were collected respectively.Purchase The FLT3-ITD positive human myeloid leukemia cell line MV411 was purchased and cultured for passage.The exosomes were extracted by density gradient centrifugation,and morphological characteristics were identified by transmission electron microscopy.At the same time,Zetaview analyzer was used to determine the size and concentration of exosomes by nanoparticle tracking analysis,and the BCA protein analysis kit was used for protein concentration determination.The protein levels of Alix,TSG101,CD63 and CD34,which are specific biomarkers of exosomes,were determined by blotting.According to the CD8~+ T cell isolation kit(Militenyi Biotec),CD8~+ T cells were extracted and purified from the peripheral blood of the above-mentioned patients and healthy donors and cultured.Apoptosis was detected by flow cytometry,mitochondrial membrane potential was ceding by TMRM method,and protein expression of AKT,p-AKT,m TOR,p-m TOR,Bcl-x L,Cyto-C,and Caspase3 were determined by western blotting.Results:(1)In the process of exosome identification,primary leukemia blasts and cell line that express FLT3-ITD mutations were cultivated and purified exosomes were identified for size and surface biomarkers.The exosomes showed a spherical structure on transmission electron micrographs,and had an average diameter of around 100 nm as determined by NTA.Western blot analysis verified the expression of exosomal protein markers CD63,Alix and TSG101,as well as CD34,frequently expressed on leukemic blasts.An extended duration of incubation of 48 h demonstrated a higher exosome isolation efficacy relative to the 12 h and 24 h.A similar trend was observed within 72 h.Therefore,primary leukemic blasts and MV-411 cells were cultured for48 h before isolating exosomes for downstream analysis.(2)We assessed the effects of apoptosis of CD8~+ T cells in vitro with various concentrations(0,50,100,200 ?g/m L)of exosomes derived from MV-411 cells and different culture durations(24,48,and 72 h).found that the apoptosis of CD8~ + T cells changed in a dose-dependent manner after 24 hours of treatment.Treatment with100 or 200 ?g/m L exosomes for 48 h and 72 h increased the apoptotic effect to a marked degree.The difference was statistically significant,p<0.05.Thus,we hypothesized that apoptotic effect was more obvious at a lower dose of 100 ?g/m L and for shorter exposure duration of 48 h,so this dose and duration were used for subsequent analysis.(3)To determine whether exosomes derived from FLT3-ITD positive AML patients and from MV-411 cells have comparable effect on CD8~+ T cell apoptosis,CD8~+ T cells were treated with 100 ?g/m L exosomes from four FLT3-ITD positive AML patients and MV-411 cells for 48 h.Flow cytometry analysis demonstrated similar trends in the apoptotic rate of CD8~+ T cells in different patients and from MV-411 cells.TMRM staining was used to monitor the state of mitochondrial membranes of CD8~+ T cells.With the fluorescent dye accumulating in the inner mitochondrial membrane,control cells displayed greater fluorescence intensity.Loss of fluorescence emission intensity was observed in the cells treated with exosomes.The results suggested that exosomes caused dissipation of MMP in the CD8~+ T cells.In addition,after treatment,the pro-apoptotic protein cleaved Caspase-3 was upregulated.Altogether,the results suggested the pivotal role of mitochondria in mediating apoptosis of CD8~+ T cells treated with exosomes derived from leukemic blasts.The difference was statistically significant,p<0.05.(4)The effect of exosomes on the apoptosis of CD8~+ T cells could be reversed by PI3 K agonist 740 Y-P.Exosome treatment downregulated the phosphorylation levels of AKT(p-AKT)and m TOR(p-m TOR),the effects of which were reversed by 740Y-P.In line with these results,the expression level of the anti-apoptotic protein Bcl-x L was attenuated whereas the expression levels of the pro-apoptotic proteins Cytochrome C and cleaved Caspase-3 were increased upon exosome treatment,the effects of which could be reversed by 740 Y-P.Moreover,we investigated whether m TOR is a downstream component of the PI3 K signaling pathway in the apoptosis of CD8~+ T cells induced by exosomes,and rapamycin was found to have reversed the anti-apoptotic effect of 740 Y-P.Similarly,the phosphorylation level of m TOR(p-m TOR)upregulated by 740 Y-P was reversed by rapamycin.The difference was statistically significant,p<0.05.Taken together,these findings support the hypothesis that the PI3K/AKT/m TOR pathway could be inhibited by exosomes in the apoptotic process.The difference was statistically significant,p<0.05.(5)To determine whether exosomes derived from leukemic blasts have impacts on the cytotoxic ability of CD8~+ T cells against myeloid leukemia cells,CD8~+ T cells isolated from untreated AML patients and normal donors were cocultured with MV-411 cells,and a cytotoxicity assay was performed to determine the extent of lysis with the administration of different concentrations of exosomes.25 ?g/m L exosomes significantly suppressed the cytotoxic activity of CD8~+ T cells against MV-411 cells.Exosomes derived from four AML patients harboring FLT3-ITD showed similar inhibitory effects on cytotoxicity of CD8~+ T cells.To further determine the proteins that were regulated by the exosomes in the inhibition process,perforin,granzyme B and tumor-necrosis factor-alpha(TNF-?)were measured in the supernatants of CD8~+T cells cultured with exosomes derived from MV-411 cell line or AML patients.Expression levels of perforin and TNF-? did not vary in the presence or absence of exosomes.Granzyme B production from the control group was found to be remarkably higher found to be in the treatment group.Conclusions:(1)Exosomes derived from FLT3-ITD+AML cells are closely related to the cytotoxicity and apoptosis of CD8~+T cells.(2)The mitochondrial pathway plays a key role in the induction of CD8~+ T cell apoptosis by exosomes produced by FLT3-ITD AML cells.(3)FLT3-ITD exosomes maybe activate PI3K/AKT/m TOR as to induce immune escape of AML cells.Part II Clonal expansion of bone marrow CD8~+ T cells in acute myeloid leukemia patients at new diagnosis and after chemotherapyObjective:The differences of the clonal expansion of CD8~+T cell populations and the differences in TCR?V-J gene rearrangements between healthy blood donors and AML patients were compared;the differences in CD8~+T cell receptor TCR expression diversity between newly-treated and relapsed AML patients and normal controls.Methods :Collected 31 AML patients(non-APL patients)admitted to the Department of Hematology,from June 2018 to May 2020,and 10 healthy blood donors during the same period.Peripheral blood(PB)and bone marrow(Bone marrow,BM)were collected,and peripheral blood mononuclear cells(PBMC)and bone marrow mononuclear cells(BMNC)were extracted.CD8~ + T cells were extracted and purified according to the CD8~ + T cell isolation kit.According to Thermofisher single-cell sequencing instructions,total RNA was extracted,and c DNA was synthesized using total RNA as a template.The template was prepared and sequenced using Ion Chef?and Ion Gene Studio? S5 series systems.View the analysis results in the Ion Reporter? software.Framework 3 and joining gene targeted primers were used to analyze the human T cell receptor ? chain amplified from g DNA or cf DNA.Short amplicons(with an average length of 80 bp)provide coverage of the CDR3 region,allowing clones to track and measure the T cell diversity of degraded materials.The frequency and sequence characteristics of clones were reported,and the library characteristics were analyzed again.Ion Reporter? Uploader was used to upload sequence files,Oncomine? TCRBeta determination results are shown graphically,and the selected VJ? Gene use thermgram.When sequencing with long amplicons covering all three CDR domains,it ensures the most accurate identification of variable genes and alleles.Results:(1)The distribution plot of the top 100 TCR clonotypes in BM and PB from one AML patient and one healthy donor.The graph demonstrates increased clonal expansion in the BM of the AML patient compared to the other groups.The total/unique clonotype ratios in BM and PB of AML patients was significantly higher than that of healthy donors.A markedly higher frequency of highly expanded clones(HECs)was noted in the BM and PB of AML patients than in those of healthy donors.In addition,Which showed the Shannon index and Gini index were used to evaluate the TCR repertoire diversity,the Shannon index for the BM of AML patients was significantly higher than that for the PB of AML patients and the BM and PB of healthy donors;in contrast,the Gini index for the BM of AML patients showed a pronounced reduction compared with that for the other groups.Collectively,these findings indicated that in CD8~+ T cells from the BM of AML patients,a decline in T cell repertoire diversity is closely associated with clonotypic expansion.The difference was statistically significant,p<0.05.(2)We identified a total of 60 distinguishable gene transcription segments from the TCR? V(TRBV)loci,2 from the TCR? D(TRBD)loci,13 from the TCR? J(TRBJ)loci,and 780 rearrangements in the TRBV-J region.Each rearrangement event in TRBV-J is denoted by a dot,with its size indicating the average frequency of the rearrangement in the sample group.Similar overall usage profiles of the rearranged TRBV/J segments were noted between BM and PB from healthy donors(81 differentially expressed rearrangements)and between PB from AML patients and PB from healthy donors(62 differentially expressed rearrangements);however,the differences were greater between BM and PB from AML patients(176 differentially expressed rearrangements)and between BM from AML patients and BM from healthy donors(202 differentially expressed rearrangements)(Figure 3B-E).Similarly,we found comparable usage patterns of TRBV gene segments between PB from healthy donors and BM from healthy donors and between PB from healthy donors and PB from AML patients but found relatively different in usage patterns between BM from healthy donors and BM from AML patients and between PB from AML patients and BM from AML patients.These data suggest that CD8~+ T cells in the BM of AML patients exhibit expression of specific TRBV-J rearrangements,indicating that they may recognize bone marrow-specific antigens.(3)We analyzed the CDR3 amino acid sequences of CD8~+ T cells in PB and BM from 31 AML patients and 10 healthy donors to identify the presence of shared clones(i.e.,shared CDR3 sequences)in the top 1,000 or 5,000 clones between any two sample pairs.Through intragroup analysis of the CDR3 sequences between different sample pairs,we found a higher ratio of identical T cell clones between sample pairs in the bone marrow of the AML patients(AML BM VS BM,n=465)when compared to sample pairs in other groups(AML PB VS PB,n=465 or healthy donor BM VS BM/PB VS PB,n=45).Intergroup analysis indicated that there was no difference in the percentage of identical T cell clones between sample pairs from different groups(AML patient BM vs.PB,n=930;healthy donor BM vs.PB,n=90;AML patient BM or PB vs.healthy donor BM or PB,n=310).By analyzing identical T cell clones in the PB and BM of AML patients or healthy donors(i.e.,comparison between BM and PB from the same individual,n=31 or n=10),we found that the percentage of identical T cell clones between PB and BM from the same individual was significantly higher than that between peripheral blood and bone marrow among different individuals.The difference was statistically significant,p<0.05.Moreover,the percentage of identical T cell clones between PB and paired BM from the healthy donors was significantly higher than that in PB and paired BM from the AML patients.These results suggested that the clonal expansion of BM CD8~+ T cells in AML patients was highly specific.The difference was statistically significant,p<0.05.(4)TCR? repertoire variety and stability among CR patients and relapsed patients show BM CD8~+ T cell clonal expansion in one relapsed patient and one patient with sustained CR at new diagnosis and after chemotherapy.After discontinuation of chemotherapy,markedly expanded T cell clones were noted in the BM from the relapsed patient but not that from the patient who achieved CR.Looking more deeply into the clonal diversity of the TCR? repertoires,we found considerably less diversity in the relapse post chemotherapy group than in the other groups.We further calculated the V gene usage of CD8~+ T cells in this relapse patient,and the results showed that compared to that at new diagnosis,the preferential usage pattern of some V gene segments changed dramatically after relapse,and the number of different clones with the same V gene usage was also remarkably different.The difference was statistically significant,p<0.05.We further analyzed the Shannon and Gini indexes of BM T cells in all 31 patients at new diagnosis and after chemotherapy.Compared to that at new diagnosis,the Shannon entropy of the relapsed patients showed a marked reduction,whereas the Gini index increased significantly.No difference was observed in the Shannon and Gini indexes in patients with CR.By assessing the ratio of identical clones among the top 1,000 T clones in the BM of patients who achieved CR or in patients who experienced relapse at new diagnosis and after treatment,we found that the ratio of identical T clones in the relapsed patients before and after treatment was significantly lower than that in the CR group.However,when we increased the number of clones assessed to 5,000,no difference was observed between the relapse group and the CR group in terms of the ratio of identical T clones at new diagnosis and after chemotherapy.These results showed that compared to those of the AML patients who were in CR after chemotherapy,some of the CD8~+ T cells of the patients who relapsed after treatment exhibited massive clonal expansion and reduced clonal diversity,with a significant change in the T cell composition among the top 1,000 clones.The difference was statistically significant,p<0.05.(5)Four maturation states of CD8~+ T cells were distinguished using CD45 RA and CCR7.We found a significantly increased percentage of effector memory T cells(TEM,CD45RA-CCR7-),and CD45RA+ effector memory T cells(TEMRA,CD45RA+CCR7-)and a reduced percentage of naive T cells(T naive,CD45RA+CCR7+)and central memory T cells(TCM,CD45RA-CCR7+),in both peripheral blood and bone marrow of AML patients compared to those of HCs.Meanwhile,the percentage of TEM and TEMRA in bone marrow was higher than in peripheral blood of AML patients.The difference was statistically significant,p<0.05.Following chemotherapy,the percentages of the four subsets had no changes compared to pre-treatment levels.Several previous reports have suggested the functional involvement of co-inhibitors in AML progression.Compared to patients at the time of diagnosis,PD-1-,TIGIT-or TIM3-expressing CD8~+ T cells displayed a marked increase in BM of relapsed patients and a concomitant decrease in BM of patients who achieved CR.The difference was statistically significant,p<0.05.To determine whether CD8~+PD-1+ cells exhibited more clonal expansion,TCR? deep sequencing of CD8~+PD-1+ and CD8~+PD-1-T cells was performed.depicts the TCR repertoire distribution in 4 samples from one patient who relapsed and one patient who achieved sustained remission at new diagnosis and after chemotherapy.In all the samples analyzed,CD8~+ PD-1+ T cells were found to be more oligoclonal than CD8~+PD-1-T cells.The cumulative frequency of the top 50 clonotypes was 42.9%,35.9%,41.5%,and 50% of the total PD-1+ T cells at the time of CR diagnosis,CR post chemotherapy,relapse diagnosis,and relapse post chemotherapy,respectively,but only 18.2%,15.8%,20.4%,and 29.6% of the total PD-1-frequency at the time of CR diagnosis,CR post chemotherapy,relapse diagnosis,and relapse post chemotherapy.We further analyzed the distribution of the top 50 clonotypes among CD8~ + PD-1+cells and CD8~ + PD-1-T cells in bulk CD8~+ T cells based on previous results for these two patients.Both at diagnosis and after chemotherapy,the clones with a higher frequency and the overall frequency of the top 50 clonotypes in the CD8~+ PD-1+ group were higher than those in the CD8~+ PD-1-group.Moreover,the top 50 prevalent clonotypes in the CD8~+ PD-1+ group were far less frequent in the PD-1-group.Analysis of all samples from the 5 relapsed patients and 17 CR patients also revealed that the top 50 clonotypes in each CD8~+ population were more frequent in the CD8~+PD-1+ group than in the CD8~+ PD-1-group.There were significant reductions in the Shannon index in the CD8~+ PD-1+ group compared to the CD8~+ PD-1-group,indicating lower TCR repertoire diversity in the CD8~+ PD-1+ group.The difference was statistically significant,p<0.05.Conclusions:(1)In the bone marrow CD8~+ T cells of AML patients,T cell diversity is closely related to clonality.(2)CD8~+ T cell expression-specific TRBV-J rearrangement in the BM of AML patients may be one of the reasons for the recognition of AML-specific antigens.(3)The clonal expansion of bone marrow CD8~+ T cells in AML patients is highly specific.(4)The CD8~+ T cells of patients who relapsed after treatment showed a large number of clonal expansion and the most specific clonal diversity.(5)TCR spectrum distribution of CD8~+T cells based on PD-1 expression CD8~+PD-1+T cells are more oligoclonal than CD8~+PD-1-T cells.