Pathogenesis And Treatment Strategies Of T Cell Acute Lymphoma Leukemia

BackgroundFor T cell acute lymphoma leukemia,the major treatments are chemotherapy and haematopoietic stem cell transplantation in clinical.By treatment of chemotherapy,about 25% children patients progress to refractory leukemia with a poor prognosis.Survival rate of three years of adult patients is about 60%-65%.Although there is less adverse effect in syngeneic HSCT than in allogeneic HSCT,there is more relapse rate in syngeneic HSCT than in allogeneic HSCT.And allogeneic HSCT can only be used to patients with high risk because its high occurrence of adverse effects.So it is necessary to research the pathogenesis and strategies of treatment in order to apply it to clinical patients to extend survival time.In the processes of lymphocytes mature,after Notch receptors interact with its ligands, differentiation of T cells can be enhanced.Andrew first found that activating mutations in NOTCH1 were found in more than 50%of human T-ALL samples in 2002.So Notch1 is well known as important oncogene in human T-ALL pathogenesis.It can instruct clinical treatment by establishing Notch1-transduced T cell acute lymphoma leukemia(TALL) model.Now it is popular to research the treatment of lymphoma leukemia by mouse lymphoma cell line EL4. But it is not enough to imitate the morbility and treatment of clinical lymphoma leukemia.So it will raise a question if Notch-T-ALL can be more similar to clinical T cell leukemia when it is compared with lymphoma cell line EL4.If the answer is yes,this reaserch can provide more direct experimental proofs for treatment of clinical lymphoma leukemia.So it is a question that deserved to research. MHCI moleculars are major ligands that ineract with self-inhibitory receptors expressed on the surface of NK cells.Expression of MHCI moleculars can inhibitate NK killing.So far, no research have reported that if Notch-T-ALL cells can express MHCI and if NK cells can kill Notch-T-ALL cells by the mechanism of low expression of MHCI.So it is necessary to research if syngeneic NK cells can kill Notch-T-ALL cells and if MHCI is involved in the killing mechanism.CD25 isαchain of IL-2 receptors and it can express on surface of many malignant haematogenesis cells.And it can be an independent prognosis index in adult acute lymphoma leukemia.Anti-CD25 monoclone antibody target to CD25 molecular can extend survival time of mice loaded leukemia cells.And anti-CD25 monoclone antibody has been used toⅡclinical research.But it is not enough to treat clinical leukemia.The possible reasons are that: first,there are some CD25- leukemia cells existing at the same time.Targeting CD25+ could be more effective when in some leukemia with high occurance of expression of CD25 positive.Secondly,CD25+ lymphoma leuckemia specifically distribute in different tissuses.Thirdly,anti-CD25 monoclone antibody can induce increase of CD56 bright NK cells to 4-20 folds,sequently,CD56 bright NK cells can negative regulate immune reaction of organism.So there are some question left to answer such as differetiatation relationships between CD25+ and CD25- leukemia cells,characteristic of tissue distribution and reaction to treatment.What's more,since human CD56 bright NK cells can kill syngeneic activated CD25+ T cells,it is yet not known if syngeneic CD25+ leukemia cells are sensitive to NK mediated killing.If it can be demonstrated that NK cells can kill Notch-T-ALL cells effectively,it will be a new field to clinical treatment for lymphoma leukemia.It is an effective way to treat many kinds of malignant haematogenesis tumors.So far, lymphoma cell lines are major used to research graft versus leukemia(GVL) of mice.But it is difficult to imitate clinical leukemia by these cell lines.It is first time to use primary leukemia cells in GVL model.If we had have already demonstrated Notch-T-ALL more clinical in first step,we would provide more direct datas to clinical HSCT.It is found that IFN-γcan decrease effect of GVHD mediated by activated T cells and enhance effect of GVL selectively.Both direct role and indirect role involved in killing of IFN-γto tumor.But so far no research have reported that direct role or indirect role involved in killing of IFN-γ,to Notch-T-ALL.So it is necessary to establish Notch1 transduced IFN-γR deficient leuckmia model to research which role can be involved in killing of IFN-γ,to Notch-T-ALL.Objective:To research relationship of T-ALL,CD25 expression,anti-CD25 monoclone antibody,NK cells,allogeneic HSCT and IFN-γ.Methods:(1) MSCV-ICN1-IRES-GFP/Notch1 vector is transduced to Sca1+Lin- bone marrow cells of wt C57BL6 or GRKO C57BL6.Then to culture them in vivo by series of passage BMT.So it is first time to establish Notch1 transduced wt or GRKO leukemia model.Then to see if they are more clinical after compared them to EL4 cell line from phenotypes,tissues distribution,function characteristic.(2) By applicating technology of MACS microbeads selection to separate Notch-T-ALL to CD25+ or CD25- leukemia cells.Then to research differences from aspects of inversion relation,tissue distribution and mortality between these 2 category cells in vivo of mice.(3) To research if leukemia can be aggravated and relationship between CD25 phenotype and NK cells after NK cells depletion in vivo of C57BL6.(4) To explore if NK cells can kill Notch-T-ALL in vitro by culturing NK cells.(5) To analyze if different CD25 expression of tumor cells can transform in other cell line such as EL4,which can be regarded as an analogy analysis to further clarify phenotypes transform on Notch-T-ALL.(6) To explore GVL effect of allogeneic HSCT on Notch-T-ALL in GVHD model by takeing wt Balb.c as donors and C57BL/6 or CB6F1 as recipients.Then takeing wt Balb.c or GKO Balb.c as donors to research killing mechanism of IFN-γto Notch-T-ALL by directly or indirectly without interaction with its receptor.Results:It is first time to establish Notch1 transduced wt or GRKO leukemia model that are more imitate to clinical T-ALL model by series of passage BMT in vivo of mice.In the process of passage BMT,CD4 expressed increased and CD8 expressed decreased gradually.Phenotype can be steady until generation 4 and generation 5.And we first demonstrated that Notch leukemia expressed 80%Notch cells were TCRVB8.1/2 positive and low expressed MHCI. But EL4 expresses increased MHCI when compared to normal mononuclear cell.Some leukemia cells are both CD4 and CD8 positive,but no FOXP3 expression.Notch-T-ALL can secrete IFN-γafter stimulation by ConA,but EL4 can not secrete IFN-γ.Notch-T-ALL can infiltrate all tissues including lymphoid tissues and non-lymphoid tissues extensively.But EL4 only infiltrate in non-lymphoid tissues.NK cells kill Notch-T-ALL in vivo demonstrated by shortened survival time in both normal and Rag1KO mice after NK cells depletion.NK cells also can kill Notch-T-ALL in vitro.We can show that NK killing to Notch1-T-ALL not by mechanism of low or no expression of MHCI on Notch1-TALL.In vitro,CD25+ Notch1-TALL cells can transform to CD25- cells,and CD25- Notch1-TALL cells can also transform to CD25+ cells.But in existence of NK cells,percentage of CD25+ cells decrease in the whole cell population.In vivo,CD25+ leukemia cells are sensitive to NK killing.In vivo of normal mice,CD25+ leukemia cells can differentiate into both CD25+ and CD25- cells, and CD25- cells seldom differentiate into CD25+.CD25+ cells significantly infiltrate into spleen and lymphoid.CD25- cells have less percentage when infiltrating in spleen,lymphoid, bone marrow,lung and kidney.When compared with CD25+ leukemia cells,CD25- leukemia cells can induce more mortality in wt B6 mice.In vitro,rhIL-2 can keep Notch1-T-ALL survival and CD25- cells can differentiate into CD25+ cells.CD25- cells can differentiate into CD25+ cells in mice with NK depletion.CD25+ Notch1-T-ALL more quick mortality in both B6 and RAG1KO mice with NK depletion.Both CD25+ EL4 and CD25- EL4 exist when in culture,but only CD25- EL4 exist in vivo.What's more,all CD25+ EL4 differentiate into CD25- cells.Both CD25+ EL4 and CD25- EL4 are not sensitive to NK killing and they both can not change their CD25 expression when culture in vitro even existence of rhIL-2.When compared to syngeneic HSCT,allogeneic HSCT significantly extend survival time of mice loaded Notch1-T-ALL.In chemira established already,survival time of leukemia mice is related to leukemia cell numbers given on the first time.Although survival time extend significantly in allogeneic HSCT compared to syngeneic HSCT when dosage of cell numbers is 2×106.Some of mice died of leukemia.But when dosage of cell numbers decreased to 5×105,60%mice died of GVHD,and 40%mice may long survive.In model of allogeneic HSCT, mice that first given small dosage leukemia cells then given more dosage of leukemia cells can long survive because immune remembrance.GVL effects of IFN-γto Notch-T-ALL is indirect without interaction with its receptor.Conclusions1.Compared to lymphoma cell lines,Notch1-T-ALL is more imitate to clinical T-ALL from aspects of phenotypes and function.2.Notch1-T-ALL is sensitive to NK killing both in vivo and in vitro.3.Under certain condition,CD25+ leukemia cells can differentiate into CD25-cells and CD25- cells can also differentiate into CD25+ cells.4.Compared to CD25-leukemia cells,CD25+ leukemia cells are more sensitive to NK killing.5.Although Notch1-T-ALL express low MHCI,we can show that NK killing to Notch1-T-ALL not by mechanism of low or no expression of MHCI on Notch1-TALL.6.When compared to syngeneic HSCT,allogeneic HSCT significantly extend survival time of mice loaded Notch1-T-ALL.7.In model of allogeneic HSCT,mice that first given small dosage leukemia cells then given more dosage of leukemia cells can long survive because immune remembrance.8.GVL effects of IFN-γto Notch-T-ALL is indirect without interaction with its receptor.SignificanceBy many research aspects in the pathogenesis and treatment strategies of mice lymphoma leukemia model,so it can be applied to clinical patients to extend survival time and can provide direct experimental datas for treatment of human T-ALL.