Study Of The Effect Of WAS Protein Deficiency On CD4 Helper T Cells And Germinal Center B Cells Differentiation During Acute LCMV Infection

PART I STUDY OF THE EFFECT OF WAS PROTEIN DEFICIENCY ON CD4 HELPER T CELLS AND GERMINAL CENTER B CELLS DIFFERENTIATION DURING ACUTE LCMV INFECTIONResearch background and purpose: B cells responses against microbial infections such as bacterial and virus invasions to produce protective antibodies from the humoral immune system is one of the most important mechanisms for protective immunity.The production of memory B cells and long lived plasma cells is the core of the adaptive humoral immune response,and this process depends on the germinal center B cell immune response(Germinal Center B cell reaction),where activated B cells undergo clonal proliferative expansion.Ig somatic hypermutation,Ig class switch recombination and antibody affinity maturation.These processes in the germinal centers require a special kind of CD4 T helper cells named CD4 Follicular Helper T cells(Tfh).During acute LCMV virus infection,CD4 T helper cells sensate the complex of different signalings form TCR,cytokines,signal transduction molecules,which switch on the signal of nuclear transcription factors to control the differentiate into Tfh cells and Th1 cells,providing of help to B cells and CD8 T cells,respectively.Tfh cell differentiation is a multistep process,involving cell synapses and TCR-MHCII molecules interactions between DC cells and activated T cells,different cytokines and signal transduction molecules to regulate the master transcription factor BCL6 for the early differentiation fate of Tfh cell.Early lineage committed Tfh cells migrate to the germinal centers,where they interact with GCB cells through frequent cellular contacts,cytokines and co-stimulatory molecules for further development into GCTfh.The interactions between GCB cells and GCTfh cells not only promote GCB immune responses,but also promote and maintain the differentiation and function of GCTfh cells.Acute LCMV infection is an ideal model for the study of differentiation of CD4 T helper cells,Tfh cells development,germinal center B cells immune responses,antibody productions,Th1 cells function and the immunologic memory responses.Wiskott-Aldrich syndrome(WAS)is a rare x-linked latent genetic immunodeficiency disease,which is caused by the mutation of WAS gene encoding WAS protein(WASp).Patients with classical WAS are susceptible to various pathogens,including viruses,bacteria,fungi and so on.WASp is only expressed in hematopoietic system,it is the key molecule for actin polymerization and cytoskeleton remodeling.By cytoskeleton dependent and independent mechanisms,WASp plays an important role in hematopoietic cells differentiation,migration,immune synapse formation and signal transduction.WASp deficiency can lead to a variety of dysfunctions of immune cells,resulting in defective specific humurol and cellular immunologic responses against pathogens,while the detailed mechanisms have not been clarified clearly.In this study,WASKO mice undergone with acute LCMV infection was used as a model to investigate the effects of WAS protein deficiency on the differentiation of CD4 helper T cells and germinal center B cells and the possible molecular mechanisms.Methods: We detected some measurements by flow cytometry at different time points between WASKO and WT mice after acute LCMV infection,such as the proportion of spleen Th1 CD4 T helper cells and Tfh cells,mainly related co-stimulatory molecules,signal transduction molecules and master nuclear transcription factors,and the proportion of GCB cells,GCB subgroups,the main nuclear transcription factor BCL6.The ratio of antigen specific Th1 and Tfh cells was measured by tetramer technique.After immunization with NP-KLH,the proportion of memory B precursor cells in the spleen was tested to explore the differentiation of antigen specific GCB cells.Adoptive transfer of bone marrow chimeric mice were used to verify whether the specific T and B cells abnormalities were intrinsic or extrinsic during acute LCMV infection.Results: In WASKO mice,the influence of WASp deficiency on IL2-CD25-STAT5 signal transduction axis resulted in the phenotypes that Th1 cells development was impaired at the early stage of CD4 T helper cells differentiation,and in turn Tfh cells differentiation was dominant.However,WASp defects affected the further development of Tfh cells,leading to the dysplasia of GCTfh.The expression of BCL6,the main nuclear transcription factor of GCB cells was impaired in WASKO mice,the dark zone of germinal centers was arrested.Abnormal differentiation of GCB cells showed dominant differentiation into polyclonal memory B precursor cells with arrested further development and maturation into memory B cells during acute LCMV infection.The percentage of antigen-specific GCB cells differentiated into memory B cell progenitors increased,but the differentiation of antigen specific memory B cells was hindered after NP-KLH immunization.The expression of intracellular WASp in GCB was heterogeneous and positively with BCL6 in WT mice.Conclusion: As a regulatory factor of actin cytoskeleton protein,WASp involved in the early differentiation of CD4 helper T cells,development of Tfh cells and immune response of GCB cells and formation of memory B cells during acute LCMV infection.PART ? STUDY OF THE INFLUENCE OF EXCESSIVE ACTIVATION OF PI3 KDELTA ON THE DEVELOPMENT OF SPLEEN B CELLS PRECURSORSResearch background and purpose: Activated phosphoinositide3-kinase delta syndrome(APDS)is an autosomal dominant primary immunodeficiency disease caused by gain-of-function(GOF)mutation in PIK3 CD,which encodes p110 delta,a catalytic subunit of PI3 K delta.PI3 K delta is a heterodimer containing p110 delta catalytic subunit and p85 a regulatory subunit,which catalyzes the production of phosphatidylinositol 3,4,5-triphosphate,and is predominantly expressed in leukocytes and plays an important role in their proliferation,survival and activation.The clinical manifestations include recurrent sinopulmonary infections,bronchiectasis,benign lymphocytic hyperplasia,herpes virus infection and autoimmunity diseases.The significant immunologic features of APDS patients are increased level of Ig M but decreased levels of Ig G,CD4 T cells lymphopenia,and increased senescened and exhausted CD8 T lymphocytes.Currently,studies have elucidated that abnormalities in Tfh and GCB which led to low Ig G in patients,but the mechanism of high levels of Ig M in patients was not very clear.MZB and B1 cells are the main sources of Ig M production,Increased MZB and B1 cells and significantly decreased follicular B cells in the B cell subpopulations have been found in patients and mouse models,which are associated with autoimmunity and susceptibility of pulmonary infections in APDS,but the mechanism of excessive activation of p110 delta leading to increased MZB and B1 cells remains unclear.Early developed B cells in the bone marrow migrate into the spleen for further development,differentiation and maturation,gradually from T1 and T2 stage B cells then into mature follicular B cells and MZB cells.The latest studies showed that B cells in the spleen undergone with the stage of ADAM10 positive T1 B as MZB precursor cells and professional development into MZB cells,and the precursors of CD19+B220low CD5+Ig M+CD93+ transitional B cells professionally developed into B1 cells.The purpose of this study was to investigate whether the excessive activation of PI3 K delta affects the differentiation of professional MZB progenitor cells and B1 progenitor cells at the stage of tansitional B cells,thereby leading to the abnormal differentiation and development of B cells.Methods: The proportions of spleen T1 B,T2B,ADAM10+ T1 B and CD19+B220low CD5+Ig M+CD93+ B cells as well as other B cell subsets were detected in APDS mice and WT mice by flow cytometry.The bone marrow chimeric mouse model was used to verify whether the abnormal differentiation of each subgroup of B cells was inherent.Results: Compared with WT mice,APDS mice had intrinsicly increased proportions of spleen CD19+B220+CD21+CD23-CD93-or CD19+B220+CD21+CD1d+CD93-MZB cells and CD19+B220low CD5+ B1 cells and CD19+B220+CD21-CD23-CD93-DNB cells while inherently decreased mature follicular B cells in mature B cells subsets.In immature transitional B cells subsets,APDS mice had immanently multiplied proportions of spleen CD19+B220+Ig M+CD23-CD93+ T1 B cells while lessened CD19+B220+Ig M+CD23+CD93+ T2 B cells.APDS mice had intrinsicly strengthened ADAM10 positive T1 B cells as professional MZB precursor cells and CD19+B220low CD5+Ig M+CD93+ transitional B cells as professional spleen B1 precursor cells resulting in abnormal differentiation as early as in the stage of immature transitional B cells.Conclusion: The excessive activation of p110 delta leads to inherently increased expression of ADAM10 expression in T1 B cells resulting in enhanced differantiation of professional MZB precursor cells as early as in the stage of immature transitional B cells,which is one of the important mechanisms leads to strengthen MZB cells development and attenuated mature follicular B cells development.The excessive activation of p110 delta leads to intrinsicly amplified CD19+B220low CD5+Ig M+CD93+professional B1 precursor cells in the stage of transitional B cells,leading to the abnormal increased development of spleen B1 cells.PART ? CLINICAL STUDY OF A CHILD WITH SPONTANEOUS MUTATION OF RAC2 GENEResearch background: RAC2 protein(ras-related C3 botulinum toxin substrate 2)is a member of the Rho superfamily of GTP enzymes and predominantly expressed in hematopoietic system,which integrates receptors mediated signals and regulate vital cellular functions via differential binding to effectors and regulators in the active versus inactive conformation,such as compositions of NADPH in neutrophils for ROS production,as a molecular switch by adjusting the GTP-bound state(activation state)and GDP-bound state(inactivation state)to control the downstream cascades and play immportnant biological roles in immune cell skeleton rearrangement,cellular pseudopod formation,the phagocytosis,adhesion,chemotaxis,T lymphocytes and B lymphocytes development,migration,activation and engaging in a variety of signaling pathways such as PAK1 signal transduction and so on.RAC2 gene mutations are extremely rare.It has been reported that RAC2 gene mutations include autosomal dominant loss of function(LOF)mutations that cause severe neutrophil dysfunctions and present clinical symptoms similar to CGD and severe reduction of T lymphocytes,and also include autosomal recessive loss of function mutations that lead to the clinical manifestations of CVID.The expression of RAC2 protein caused by these two genetic loss of function mutations is obviously decreased or absent.With the development and the widespread application of gene sequencing technology,there are increasing cases reports about RAC2 autosomal dominant function mutations lead to increasing RAC2 protein activity(gain of function,GOF)and such children suffered with different clinical manifestations while commonly strengthen granulocytes ROS activity and significantly decreased T lymphocytes and B lymphocytes.The mechanisms for significantly decreased T lymphocytes and B lymphocytes are not clear.One patient with spontaneous mutation of RAC2 gene was diagnosised in our center,the mutation site had not been reported.The clinical manifestations were repeated pulmonary infection and bronchiectasis.We conducted some preliminary studies on the clinical and partial immune cell functions.Methods: Peripheral blood mononuclear lymphocytes and neutrophils were isolated and sequenced.Lymphocyte subgroups were detected by flow cytometry.T lymphocyte proliferation was detected by CD3/CD28 and PMA/ionomycin stimulations in vitro.The production of neutrophils ROS was detected by luminol amplification chemiluminescence.The expression of neutrophils actin was detected by flow cytometry.The polarization of neutrophils and T lymphocytes was observed by confocal microscope.RAC2 protein expression was detected by western blot.The apoptosis of neutrophils and lymphocytes was detected by flow cytometry,as well as the expression of RAC2 in lymphocyte subgroups.Results: The spontaneous missense RAC2 gene mutation was found in the patient,both PBMC and neutrophils undergone with this mutation.The immunophenotype was combined immunodeficiency,T lymphocytes proliferation was reduced,the ROS activity of granulocytes showed strengthen function and the F-actin expression was enhanced.Defects of polarization of actin and RAC2 in neutrophils and T lymphocytes during in vitro stimulation wrer observed.The expression of RAC2 protein in neutrophils was increased.The apoptosis of neutrophils and lymphocytes was enhanced.RAC2 were strongly expressed in B lymphocyte subsets in the patient.Conclusion: In this study,we found a rare spontaneous mutation of RAC2 gene,presenting as an activated mutation.The immunophenotype was combined immunodeficiency,and the functions of lymphocytes and neutrophils were affected.