| IntroductionT-cell lymphoblastic leukemia(T-ALL)is a group of heterogeneous malignant diseases with clonal proliferation of precursor T lymphocytes that cause normal hematopoietic inhibition.The clinical manifestations include increased white blood cell count,neutropenia and thrombocytopenia,hematopoietic failure,lymph node or central nervous system infiltration,and mediastinal mass may appear in some patients.T-ALL accounts for 10%-15%of childhood acute lymphoblastic leukemia,accounting for 25%of adult acute lymphoblastic leukemia,and the incidence rate of males is two times that of women.The clinical diagnosis of T-ALL mainly depends on immunological tests.According to the corresponding stages of intra thymus differentiation,the European Leukemia Immunoclassification Group proposed to divide T-ALL into:pro-T,pre-T,cortical-T and mature-T.pro-T(c CD3~+,s CD3~-,CD1a~-,CD2~+,CD5~-,CD7~+,CD34~-),pre-T/immature(c CD3~+,s CD3~-,CD1a~-,CD2~+,CD5~+,CD7~+,CD34~-),cortical-T(c CD3~+,s CD3~+/~-,CD1a~+,CD2~+,CD5~+,CD7~+,CD34~-)and mature-T(c CD3~+,s CD3~+,CD1a~-,CD2~+,CD5~+,CD7~+,CD34~-).Although great efforts have been made in recent decades to improve the treatment of T-ALL,multi drug combination and high-dose chemotherapy and the application and promotion of all kinds of hematopoietic stem cell transplantation,the treatment effect of drug-resistant or relapsed T-ALL patients is still poor,and the overall efficacy and prognosis of the disease are far less than that of common acute B-lymphocytic leukemia.Therefore,it is of great clinical significance to explore more specific therapeutic targets of T-ALL and explore the pathogenesis of T-ALL.Immunoglobulin D(Immunoglobulin D,IgD)was first discovered in 1965,including secreted IgD(secreted IgD,s IgD)and membrane-binding IgD(membrane IgD,m IgD).IgD it self can act as a surface receptor for B cells,and can also function in combination with membrane receptor immunoglobulin D receptor(Immunoglobulin D receptor,IgDR),where IgDR is found in both human exodus T cells and mouse T cells.The interaction between IgDR and IgD on T-cells can lead to an increase in antigen-specific T-cell cloning,while crosslinking between IgDR on T-cells and m IgD on B-cells inhibits T-cell apoptosis.Prior studies in this group found that s IgD showed significantly higher expression in the serum of patients with diffuse large B cell lymphoma(DLBCL),and that IgD was able to increase the expression of Burkitt lymphoma cell Daudi surface IgDR,which can significantly promote the proliferation of Daudi cells,reduce apoptosis,and accelerate the conversion process of cell cycles from G1 to S.Early in vitro experiments,IgDR was found in human peripheral blood CD4~+T cells and human T-ALL cell lines(Jurkat and MOLT-4 cells),IgD and IgD-Fc-Ig can bind to IgDR on CD4~+T,Jurkat and MOLT-4 cells.IgD-Fc-Ig inhibited the proliferation of Jurkat and MOLT-4 cells induced by IgD,and promoted the apoptosis of Jurkat and MOLT-4 cells inhibited by IgD.Western blot results showed that IgD-Fc-Ig inhibited the up-regulated mTOR,Rictor and p-Akt levels(Ser473).The results suggest that IgD/IgDR may be a new therapeutic target for T-ALL,and specific blocking of IgD/IgDR signal transduction may be a new immunotherapy for T-ALL by affecting mTORC2/Akt signaling pathway.The limitation of the earlier experiment was that the effect of IgD-Fc-Ig on T-ALL cell lines was only studied in vitro,and there was a lack of studies on clinical samples of T-ALL and whether the expression of IgDR differed between T-ALL patients and healthy controls.In addition,there was no study on the effect of in vivo administration of IgD-Fc-Ig on T-ALL.Targeting IgD-IgDR,this topic has successfully connected the human IgD-Fc segment with the human Ig G1-Fc segment and synthesized the fusion protein IgD-Fc-Ig(Idfigine),intending to specifically block the IgD-IgDR path,high selective inhibition of abnormal proliferation and resuspension of T cells,which has been granted the Chinese invention patent.In this study,peripheral blood mononuclear cells(PBMCs)of T-ALL patients,thymus T cells of C57BL/6 mice,EL-4 cells of mouse T-ALL cell line and T-ALL mouse model were used as the research objects,to observe the difference of IgDR on peripheral blood T cells between T-ALL patients and healthy controls,detect the effect of IgD on T-cell function and the effect of IgD-Fc-Ig in T-ALL patients,observe the effects of IgD and IgD-Fc-Ig on the proliferation,apoptosis,and related proteins of C57BL/6 mouse thymus T cells and EL-4 cell lines.Besides,through the establishment of the T-ALL mouse model,the therapeutic effect of IgD-Fc-Ig on high-level T-ALL mice was observed at the overall level,to providethe experimental basis for the development of IgD-Fc-Ig as an innovative drug for the highly selective treatment of T-ALL.Objective:To clarify the difference in the expression of IgDR on the characteristic marker T cell subsets in peripheral blood between T-ALL patients and healthy controls,to clarify the effect of IgD on the characteristic marker T cell subsets in T-ALL patients and the effect of IgD-Fc-Ig,to clarify the effect of IgD on the proliferation of thymus T cells in C57BL/6mice and EL-4 cells and the effect of IgD-Fc-Ig,and to study the relevant mechanisms to reveal the therapeutic effect and partial mechanism of IgD-Fc-Ig on T-ALL mice to elucidate the role and mechanism of IgD/IgDR signal transduction in the pathogenesis of T-ALL by regulating mTORC2/Akt signaling pathway,and to provide the experimental basis for using IgD-Fc-Ig as an innovative drug for the treatment of T-ALL.Methods:Collecting T-ALL patients and healthy human extensor blood,centrifugal collection of plasma,PBMCs for culture testing by Ficoll density gradient centrifugal method,flow cytometers and laser cofocus detection of IgDR expression on the characteristic marker T cell subsets,flow cytometry was used to detect the effect of IgD-Fc-Ig on the characteristic marker T cell viability in patients with IgD-induced T-ALL.CCK-8 assay was used to detect the effect of IgD on T cell activity in the thymus of C57BL/6mice.CCK-8 method was used to detect the effect of IgD-Fc-Ig on the activity of IgD induced EL-4 cells,and flow cytometry was used to detect the effect of IgD-Fc-Ig on the apoptosis of IgD inhibited EL-4 cells,and Western blot method detects the expression of mTOR,Rictor,Akt,p-Akt(Ser473)protein in IgD induced EL-4 cells;The model of T-ALL mice was established,and BALB/c-nu naked mice were randomly divided into 7 groups,the normal group,the model group,the three groups of IgD-Fc-Ig(1.625,3,25,6.5 mg/kg),the positive control group admin(Adriamycin:3 mg/kg),and the negative control group Ig G-Fc(6.5 mg/kg),the normal group had 8 mice,the remaining group had 9 mice.ELISA tested plasma IgD levels in mice in each group;Reich-Gimsa staining method detected the effects of IgD-Fc-Ig on Jurkat cells in exosome blood and bone marrow in mice in each group;flow cytometry was used to detect the effect of IgD-Fc-Ig on Jurkat cells in bone marrow and spleen of T-ALL mice,and the effect on the apoptosis of IgDR,IgD plasma cells and apoptosis of peripheral blood lymphocytes;to investigate the effects of IgD-Fc-Ig on the overall indexes and pathological changes of liver and spleen in T-ALL mice;and Western blot method detection of expression of mTOR,Rictor,Akt,p-Akt(Ser473)proteins in the spleen of T-ALL mice.Result:1.Difference in IgDR expression of characteristic marker T cell subsets in peripheral blood between T-ALL patients and healthy controls8 T-ALL and physical examination were collected,patients’age range is 15～69 years old,male to female ratio of 1:1,the lymphocyte percentage and white blood cell count in7 patients were higher than normal.Flow cytometry detection results show that the characteristic marker T cell IgDR in T-ALL patients was significantly higher than that in healthy controls.The T-ALL patients and healthy controls already had IgDR on the surface of cells at the pro-T stage(CD7~+CD5~-CD3~-CD4~-),and the level of IgDR on T cells at all stages of T-ALL patients was higher than that of healthy controls,and the difference was statistically significant.2.The difference in expression of CD7~+T cells and IgDR in peripheral blood between T-ALL and healthy controlsThe laser confocal results show that:the expression of IgDR on CD7~+T cells was verified again.The expression of IgDR on CD7~+T cells of some T-ALL patients was higher than that of healthy controls,which was consistent with the results of flow cytometry.3.The effect of IgD on Tcell subsets in patients with T-ALLIgD(1,3,10μg/ml)stimulated PBMCs in T-ALL patients in vitro,flow cytometry results showed that IgD concentration group(3,10μg/ml)could significantly promote the expression of pro-T(CD7~+CD5~-CD3~-CD4~-T)and mature(CD7~+CD5~+CD3~+CD4~-T,CD7~+CD5~+CD3~+CD4~+T)cells in T-ALL patients after in vitro stimulation for 24h.4.The effect of IgD-Fc-Ig on T cell subsets in patients with T-ALL induced by IgD Flow cytometry results showed that IgD(3,10μg/ml)group significantly promoted the expression of pro-T(CD7~+CD5~-CD3~-CD4~-T)and mature(CD7~+CD5~+CD3~+CD4~-T,CD7~+CD5~+CD3~+CD4~+T)cells in T-ALL patients,and IgD-Fg-Ig(3,10μg/ml)group could down-regulate the expression of CD7~+CD5~-CD3~-CD4~-T,CD7~+CD5~+CD3~+CD4~-T and CD7~+CD5~+CD3~+CD4~+T.5.The effect of IgD on T cell viability in the thymus of C57BL/6 miceCCK8 results showed that human IgD could also stimulate mouse T cells,and IgD concentration groups(3,10μg/ml)could up-regulate the activity of mouse T cells.6.The effect of IgD on the activity of T-ALL mouse EL-4 cell lineCCK8 results showed that IgD concentration groups(3,10μg/ml)could promote the activity of mouse T-ALL cell line EL-4 cells.7.The effects of IgD-Fc-Ig on the viability of IgD-induced EL-4 cell linesCCK8 results showed that IgD-Fc-Ig(3,10μg/ml)could significantly inhibit the proliferation of IgD-induced EL-4 cells,but Ig G-Fc protein(10μg/ml)had no significant effect on the effect of IgD.8.The effects of IgD-Fc-Ig on apoptosis of EL-4 cell line induced by IgDFlow cytometry showed that the IgD-Fc-Ig(3,10μg/ml)could significantly up-regulate the apoptosis of EL-4 cells inhibited by IgD,but Ig G-Fc(10μg/ml)had no significant effect on the effect of IgD.9.Theeffect of IgD-Fc-Ig on expression of mTOR、Rictor、Aktand p-Akt(Ser473)protein in EL-4 cell line induced by IgDThe results of immunoprecipitation showed that IgD(3μg/ml)significantly up-regulated the protein expressions of mTOR,Rictor,and p-Akt(Ser473)in EL-4 cells,and IgD-Fc-Ig(3,10μg/ml)down-regulated the protein expressions of mTOR,Rictor,and p-Akt(Ser473)induced by IgD.10.The establishment and evaluation of T-ALL mouse modelIn this study,the T-ALL model was established by caudal vein transplantation,the result shows:T-ALL mice showed no significant changes on the 7th day,while began to show drowsiness and poor diet on the 14st day.On the 21st day,the caudal vein transplantation group showed arched back,emaciation,decreased vitality and obvious listlessness,etc.On the 28th day,some nude mice showed near-death state with spinal curvature.Nude mice in normal control group grew normally,were active,had good appetite and spirit,and had no pathological signs.11.Jurkat cells of model mice were infiltrated in peripheral bloodThe results of Ray-Gimsa staining showed that:Jurkat cells were mainly erythrocytes and nucleated cells were rare in peripheral blood of normal control group.In the cudal vein transplantation group,a small number of Jurkat cells were found in peripheral blood at d7,but nucleated cells increased significantly at d10,and the number of Jurkat cells was also higher than d7.Flow cytometry results showed:the results of Jurkat cells in peripheral blood at d14 were significantly different from those at d7.Combined with the above results,it was suggested that the administration of Jurkat cells was started at d10.12.The effect of IgD-Fc-Ig on serum IgD level in T-ALL miceThe serum IgD level of T-ALL mice was significantly higher than that of normal mice.13.The Swiss-Gimsa stain of peripheral blood and bone marrow of T-ALL miceMice were sacrificed after blood was taken from the eyeball and peripheral blood and bone marrow cells were taken fora smear.The number of nucleated cells and Jurkat were recorded by randomly selecting 3 fields,and the results showed that the percentage of Jurkat cells in the peripheral blood of the model group mice was the highest,which could be as high as 11.21%,the IgD-Fc-Ig(3.25,6.5 mg/kg)and amycin(3 mg/kg)groups reduced the percentage of Jurkat cells in the peripheral blood,which was statistically different from the model group.The results in the bone marrow are consistent with the peripheral blood.14.The effects of IgD-Fc-Ig on the survival of Jurkat cells in bone marrow and spleen of T-All miceJurkat cell surface markers were CD3,and fluid cytometers detected the expression of CD3 in the spleen and bone marrow of different groups of mice,and the results showed that T-ALL mice had the highest percentage of Jurkat cells in the spleen and bone marrow,while IgD-Fc-Ig(3.25,6.5 mg/kg)dose group and amycin(3 mg/kg)significantly reduced the immersion rate of Jurkat cells in the spleen and bone marrow of T-ALL mice.15.The effect of IgD-Fc-Ig on IgDR of Jurkat cells in T-ALL mice spleenFlow cytometry was used to detect the expression of IgDR in the spleen of Jurkat cells of T-ALL mice.The results showed that the expression of IgDR in the spleen of Jurkat cells ofthe model group was higher than that ofthe normal group.IgD-Fc-Ig(3.25,6.5mg/kg)and doxorubicin in the administration group could decrease the expression of IgDR in spleen cells,and the difference was statistically significant.16.The effect of IgD-Fc-Ig on IgD plasma cells in spleen of T-ALL miceCD19~-CD138~+IgD~+cells were detected by flow cytometry with spleen cells obtained from T-ALL mice,the results showed that the expression of CD19~-CD138~+IgD~+cells in the model group was higher than that in the normal group,and the difference was statistically significant.The expression of CD19~-CD138~+IgD~+was decreased by IgD-Fc-Ig(6.5 mg/kg)and positive doxorubicin in the administration group,and the difference was statistically significant compared with that in the model group.17.Theeffect of IgD-Fc-Ig on apoptosis of peripheral blood lymphocytes in T-ALL miceThe apoptosis of peripheral blood lymphocytes in T-ALL mice was detected by flow cytometry.The results showed that IgD-Fc-Ig(3.25,6.5 mg/kg)and positive drug groups could increase the apoptosis of peripheral blood lymphocytes in T-ALL mice.18.The effects of IgD-Fc-Ig on spleen pathology and liver pathology in T-ALL mice Put to death in mice,the spleen and liver,HE staining,observation of histopathological changes,the results found that in normal mice spleen visible red pulp and neat white pulp distribution,the white pulp is composed of the lymphatic sheath surrounding the central artery and artery,the red pulp is composed of splenic sinus and spleen of mice model slicing visible red pulp and white pulp line disappear,white pulp severe contraction,Jurkat tumor lymphatic cells stay or scattered,dosing group IgD-Fc-Ig(3.25,6.5 mg/kg)and positive medicine group can improve the pathological changes.The results of HE staining of liver tissue showed that the liver tissue of nude mice in the normal group was complete,and the liver cells were radially arranged from the center of the central vein.The liver tissue sections of nude mice in the model group without histopathological changes showed Jurkat tumor cells infiltration,partial liver tissue necrosis,and no radially arranged normal tissue structure.19.The effects of IgD-Fc-Igon p-Akt(Ser473),Akt,mTOR and Rictor protein expression in spleen of T-ALL miceWestern blot results showed that the total protein expression of Akt was not significantly different between the normal group and the model group,but the protein expression of p-Akt(Ser473),mTOR and Rictor in the model group was significantly increased.IgD-Fc-Ig(3.25,6.5 mg/kg)could down-regulate the protein expression of p-Akt(Ser473),mTOR,and Rictorin T-ALL mice.Conclusion1.The expression of IgDR on T cells in peripheral blood of some T-ALL patients was higher than that of healthy controls.IgD increase the percentage of pro-T(CD7~+CD5~-CD3~-CD4~-T)and mature-T(CD7~+CD5~+CD3~+CD4~-T,CD7~+CD5~+CD3~+CD4~+T)cells in T-ALL patients,IgD-Fc-Ig decreased the percentage of pro-T and mature-T cells in IgD-induced T-ALL patients2.IgD can stimulate the proliferation of thymus T cells in C57BL/6 mice and EL-4 T cells,and inhibit the apoptosis of EL-4 cells,IgD-Fc-Ig can down-regulate IgD-induced cell proliferation and promote the apoptosis of EL-4 cells.IgD-Fc-Ig inhibited the proliferation of IgD-induced EL-4 cells,promoted the apoptosis of IgD-inhibited EL-4cells,and down-regulated the expression of mTOR,Rictor and p-Akt(Ser473)proteins in IgD-induced EL-4 cells.3.The level of serum IgD in T-ALL mice was higher than that in normal mice.IgD-Fc-Ig improved the overall indexes of T-ALL mice,reduced the infiltration rate of Jurkat cells in peripheral blood spleen and bone marrow,and reduced the pathological degree of liver and spleen tissue.4.IgD-IgDR may be a new target for T-ALL therapy by regulating mTORC2/Akt signaling pathway,and IgD-Fc-Ig may have an important prospect for the treatment of T-ALL with high IgD level or high IgDR activity. |
PDF Full Text Request