Impact And Mechanism Of WAs Protein Deficiency On The Diversity Of Memory T Cell Receptor And Thymic Output Function

PART?IMPACT OF WAS PROTEIN DEFICIENCY ON THE DIVERSITY OF MEMORY T CELL RECEPTORBackground:The diversity of T cell receptor(TCR)is one of the important guarantees for effective T cell immunity.In the immune system,the T cell receptor repertoire is composed of all T cell clones.Each T cell clone recognizes the corresponding antigens through its TCR specificity,which endows the immune system with the potential to recognize almost all antigens in the surrounding environment.Wiskott Aldrich syndrome(WAS),also known as eczema,thrombocytopenia with immunodeficiency syndrome,is caused by the deficiency of WAS protein(WASp).The main clinical manifestations are eczema,thrombocytopenia,immunodeficiency,autoimmune diseases and lymphoma.WASp,as a regulating factor of actin cytoskeleton polymerization,is involved in the generation and differentiation of hematopoietic cells,which is an important guarantee for normal function of immune system.Our research team and foreign research team have found that the diversity of TCR in patients with WAS is seriously impaired.In both of the studies,the impact of WASp deficiency on TCR diversity of T cell subsets was similar,showing that the impairment of TCR diversity occurred mainly in the memory populations.As we all know,the size of TCR clones is greatly affected by environmental factors such as infection,immunization and autoimmunity,which are the extrinsic factors not determined by the T cells themselves.Despite in the previous study very young WAS patients were recuited to minimize the impact of recurrent infections on their skewed TCR diversity,it is not easy to completely exclude the influence of environmental factors.Therefore,in the present study,we intended to establish chimeric mouse model to ensure that the impairment of TCR diversity in the memory T cell population is mainly due to T cell intrinsic factors.Objective:To verify whether WASp deficiency is the intrinsic factor of limited TCR diversity of memory T cells rather than infection or other extrinsic factors.Methods:We used magnetic beads and flow cytometry to sort the T cell subtypes of the WASKO/WT mice model,WASKO/WT mice(CD45.2+)and wild-type mice(CD45.1+)bone marrow chimeric mice model.The T cell subtypes were detected by using the new UMI quantitative high-throughput sequencing(HTS).The sequence of complementary decision region 3(CDR3)of TCR?chain was comprehensively analyzed by Bioinformatics Method to verify whether WASp deficiency caused the limited TCR diversity of memory T cells.Results:1.The TCRV?diversity of T cell subsets,CD4+TEM?CD4+TCM?CD4+T Na?ve?CD4+Tscm?CD8+TCM?CD8+T Na?ve and CD8+Tscm,from non-chimeric WASKO/WT mice were detected by HST sequencing.The results of bioinformatics analysis are as follows:Compared with WT mice,Shannon index and inverse Simpson index of CD8+T Na?ve and CD8+Tscm were decreased in WASKO mice(P<0.05).The clonality index of CD8+T Na?ve and CD8+Tscm was increased in WASKO mice(P<0.05).There was no significant difference between WASKO and WT mice in Chao1 and ACE index.These five kinds of diversity index was no significant difference between WASKO and WT mice in other T cell subsets.The usage of V/D/J subfamily gene between WASKO and WT mice was no significant difference.There was no significant differences in the sequence length of TCRV?between WASKO and WT mice.2.The UMI quantitative HTS was used to detected the TCRV?diversity of CD4+TEM and CD8+TCM from WASKO/WT mice(CD45.2+)and WT mice(CD45.1+)bone marrow chimeric mice model.In total of 32samples were collected and 5.51 million sequencing readings on average in each sample were obtained(3.05-9.00million).The results of bioinformatics analysis are as follows:(1)Compared with WT,KO-CD4+TEM had increased Simpson index and decreased D50index(P<0.05),but no different in Shannon index and Chao1 index.There was no significant difference between WT-CD8+TCM and KO-CD8+TCM in these four kinds of diversity index.TOP100 was no significant difference between WT and KO chimeric mice in both of CD4+TEM and CD8+TCM.(2)The rank abundance curve of KO-CD4+TEM was shorter and steeper than that of WT.The curve of D~q and q in KO-CD4+TEM was clearly lower.But the position of the two kinds of curve had no obvious change between WT and KO chimeric mice in CD8+TCM.(3)Principal Component Analysis(PCA),which used to make a comprehensive consideration of TCRV?diversity,successfully segregated WT-CD4+TEM from KO-CD4+TEM,failed discriminated between WT-CD8+TCM and KO-CD8+TCM.(4)The usage of TRBV12.2,TRBV30,TRBV31,TRBV4,TRBD1,TRBD2,TRBJ1.1 and TRBJ1.4 had significant difference between WT-CD4+TEM and KO-CD4+TEM(P<0.05).The usage of TRBV12.2,TRBV15 and TRBV20 had significant difference between WT-CD8+TCM and KO-CD8+TCM(P<0.05).(5)PCA,which base on V gene,V-J gene and V-D-J gene,segregated KO-CD4+TEM from WT-CD4+TEM,but with the increase of combination genes,the difference became smaller.The difference of PCA between WT-CD8+TCM and KO-CD8+TCM was not clear as WT-CD4+TEM and KO-CD4+TEM.(6)Compared with WT,the overlap index of KO-CD4+TEM was lower(P<0.001),and that of KO-CD8+TCM was higher(P<0.001).The overlap index of CD4+TEM vs CD8+TCM was higher in KO group(P<0.001).(7)Metrics F2,R and D was analyzed by using VDJTOOLS software.CD4+TEM from WT and KO chimeric mice was more dispersed in D metric.KO-CD8+TCM was more concentrated than WT-CD8+TCM in all of these three metrics.(8)Tyrosine was downregulated,and Phenylalanine,Asparagine and Lysine was upregulated in KO-CD4+TEM(P<0.05).Aspartic acid was downregulated,and Tryptophan was upregulated in KO-CD8+TCM(P<0.05).(9)There was no significant difference in hydrophobicity of amino acids(aa)residues at positions 6 and 7 of the 13 aa-long CDR3?between WT and KO chimeric mice in CD4+TEM or CD8+TCM.(10)There was no significant difference in distribution of the length of CDR3?region between WT and KO chimeric mice in CD4+TEM or CD8+TCM.Conclusion:The limitation of TCRV?diversity of CD4+TEM and CD8+TCM caused by WASp deficiency is intrinsic.WASp deficiency has little impact on TCRV?diversity of CD8+TCM.WASp deficiency can result in the difference usage of V/D/J subfamily genes,the gene combination of TRB and amino acid composition in CD4+TEM and CD8+TCM.But WASp defects not cause significant differences in the hydrophobicity and sequence length of TCRV?.PART ? IMPACT AND MECHANISM OF WAS PROTEIN DEFICIENCY ON THYMIC OUTPUTBackground: T cells originate from bone marrow and mature in thymus.The V / D / J rearrangement of TRB is also completed in thymus.TCR rearrangement excision circles(TRECs)present in thymic output cells stably,which is used as a molecular marker for quantitatively assessing thymic output.Wiskott Aldrich syndrome(WAS),also known as eczema,thrombocytopenia with immunodeficiency syndrome,can be classified clinically into classic WAS,intermittent X-linked thrombocytopenia(IXLT),X-linked thrombocytopenia(XLT)and X-linked neutropenia(XLN).WASp,as a regulating factor of actin cytoskeleton polymerization,possibly takes parts in the generation and differentiation of hematopoietic cells and T cell maturation in the thymus.Defective T cell function is a major cause for immune deficiency in WAS patients.Many studies have confirmed that children with WAS have T cell lymphopenia,which gradually aggravates with age.Several studies have suggested that the early slightly impaired development of T cells,the impaired function of T cell activation and proliferation,and the increase of T cell apoptosis may attribute to lymphopenia in WAS.Jilkina et al.previously examined the TRECs copy numbers in one WAS and one XLT patient and found normal TRECs in the WAS and XLT patient when compared to a healthy control.Due to the limited sample size of this study,it is not fully convincing.So whether WASp deficiency leads to the decrease of thymic output function,needs to be further clarified,and the relationship between thymic output deficiency and F-actin needs to be further studied.Objective: In this study,young children with WAS,XLT and WASKO mice were used as models to determine the effect of WASp deficiency on thymus output of T cells,and to explore the relationship between thymus output and F-actin polymerization.Methods: Sanger generation gene sequencing was used to detect the mutation site of WAS gene.Four children with classic WAS,four children with XLT and age-matched healthy controls(HC1and HC2)were included in the present study.The expression of WASp protein in Peripheral blood mononuclear cells(PBMCs)was measured by flow cytometry.Flow cytometry was used to detect the distribution of T cell subtypes in peripheral blood of children with WAS or healthy donors,including CD4+ T cells and CD8+ T cells,na?ve T cells,memory T cells and terminal T cells.The copy numbers of TRECs in peripheral blood of patients or healthy controls and WASKO / WT mice were detected by q RT-PCR.Magnetic beads was used to sort T cells from patients or healthy controls,and then confocal microscopy was used to detect the MFI,TFI and the subcellular localization of F-actin in T cells after stimulated with PMA and ionomycin at 0 min,5min,30 min.The MFI of F-actin in T cell was also detected by flow cytometry at same time.Results: The percentage and number of T cell subsets in total lymphocyte of classic WAS patients and XLT patients were altered,including the number of CD4+ T cells and naive CD8+ T cells in classic WAS patients decreased significantly(P<0.05).Compared to healthy controls,the MFI of WASp was lower in the PBMC of classic WAS patients(P<0.01)and XLT patients(P<0.05).The MFI of WASp in PBMC was no statistical difference between classic WAS and XLT patients.The copy number of TRECs in peripheral blood was decreased in classic WAS patients(P<0.05)and XLT patients(P<0.05).But the copy number of TRECs was no statistical difference between classic WAS and XLT patients.Compared with WT mice,the copy number of TRECs in the peripheral blood of WASKO mice was decreased(P<0.01).We found the MFI or TFI of F-actin determined by NIS-Elements AR 3.2 software on the membrane and in the cytoplasma was reduced in XLT patients(P<0.01)and more severely reduced in WAS patients(P<0.01)when compared to HCs.We took the 3D images of the T cells from HC1,HC2,XLT and WAS patients and quantified the F-actin level of each slice with NIS-Elements AR 3.2 software.We found the F-actin level of each slice in HC1 or HC2 group was significant higher than that in XLT patients and WAS patients(P<0.001).The F-actin level of each slice in XLT patients was higher than WAS patients(P<0.01).We found the levels of F-actin in XLT were decreased and further decreased in WAS compared to that of HC1 or HC2 by using flow cytometry(P<0.01).Conclusion: All these results collectively imply that WASp defects affect the thymic output of T cells.The current results obtained from peripheral blood T cells of WAS patients imply that the degree of actin alteration is associated with WASp expression and indicate the disrupted F-actin organization may account for the impaired function of thymic output.But it is not clear the impairment of thymic output in WAS patients mainly due to which abnormal stage of T cell development in thymus.