WASP And Mst1 In The Regulation Of BCR Signaling And Characteristics Of 15 Chinese RAG Patients

PART I-II MEMORY B CELLS IN WAS PATIENTS HAVE DEFECTS IN EARLY ACTIVATION VIA POSITIVE REGULATION OF CD19 TRANSCRIPTIONWAS pediatric patients have immunodeficiency. Memory B cells of WAS patients have reduced in vivo proliferation. However, the detailed molecular mechanism underlying WAS memory B cell non-competence remains elusive. Here we collected the peripheral blood mononuclear cells from WAS patients and age matched healthy controls(HCs) and studied the early activation events of na?ve and memory B cells by using TIRFm. In HCs, memory B cells engaged in B cell receptor(BCR) clustering and B cell spreading significantly higher than na?ve B cells. This increased reactivity of memory B cells was lacking in WAS patient samples. We further demonstrated in mechanistic analys is that WAS memory B cells exhibited significantly decreased CD19-Btk mediated signaling and increased FcγRIIB- SHIP-1 mediated signaling. Finally, we showed that WASp positively regulated the transcription of CD19. Overall, our study suggests a new molecular mechanism that WAS memory B cells fail to respond to antigens efficiently.PART III MST1 POSITIVELY REGULATES B CELL RECEPTOR SIGNALING VIA CD19 TRANSCRIPTIONAL LEVELSAs a key regulator of hippo signaling pathway, Mst kinases are emerging as one of the key s ignaling molecules that influence cell proliferation, organ s ize, cell migration, and cell polarity. In B lymphocytes, Mst1 deficiency causes the developmental defect of marginal zone(MZ) B cells, but how Mst1 regulates B cell receptor activation and differentiation remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy(TIRFm), we demonstrated that Mst1 positively regulated B cell receptor(BCR) signaling via modulating CD19 transcriptional levels. Consistent with this, Mst1 deficienct mice exhibited reduced BCR signaling, which was cocurrent with defective BCR clustering and B cell spreading on stimulatory lipid bilayers. The disruption of CD19 mediated Btk signaling by Mst1 deficiency leads to the severe defect in the differentiation of marginal MZ and germinal center(GC) B cells. Mechanistical analys is showed that Mst1 upregulated the m RNA level of CD19 via regulating the transcriptional factor-TEAD2 that directly binds to the consensus motif in the 3’UTR of cd19. Overall our results reveal a new function of Mst1 in B cells and the mechanism by which Mst1 regulates the activation and differentiation of peripheral B cells.PART IV CLINICAL, IMMUNOLOGIC, AND GENETIC CHARACTERISTICS OF RAG MUTATIONS IN 15 CHINESE PATIENTS WITH SCID AND OMENN SYNDROMEObjective:Mutations in Recombination Activating Genes(RAG1 and RAG2) are common genetic causes of severe combined immunodeficiency(SCID) and Omenn syndrome(OS). The clinical, immunologic, and genetic characteristics of RAG mutations in Chinese patients with SCID or OS have not been studied in detail.Methods : From 2008 to 2015, children with SCID or OS from Chinese families were enrolled and heparinized peripheral blood was collected from each patient and an age-matched healthy control. RAG genes were amplified by PCR and sequenced. Single nucleotide polymorphisms of each novel mutation were excluded by screening with the NCBI db SNP database. The potential pathogenicity was evaluated by Mutation Taster and Poly Phen software and the potential structural impact of the novel mutations was further predicted by Swiss-Pdb Viewer. All the patients with OS manifestations were subjected to short tandem repeat analysis to check maternofetal transfusion status. CDR3 spectratyping analys is of TCR Vb was assessed to analysis TCR gene rearrangement divis ity; sj TRECs expression to analyze V(D)J recombination level of T cells; CFSE-labeled lymphocyte proliferative response to phytohemagglutinin to analyze T cell activity and fuction.Results : In this research, 22 RAG mutations were identified in 15 Chinese patients, including 10 novel mutations in RAG1(R108X, M630 T, E510 X, S666 P, E669 K, C730 Y, A857 V, K847 E, L922 Pfs X7, and L1025 Ffs X39) and 4 in RAG2(R73C, I427 Gfs X12, P432 L, and 311 ins L). L1025 Ffs X39 is a potential RAG1 hot-spot mutation in the Chinese population. The distribution of RAG1 mutations rather than mutation type seemed to differ between SCID and OS patients. The thymic output of T lymphocytes, TCR rearrangement, and T cell proliferation were severely impaired in RAG mutant patients.Conclusions : These findings will contribute to the early diagnosis and treatment of SCID and OS to a certain extent.