Molecular Mechanisms Underlying B-cell Differentiation Into Plasma Cells

Background:Abnormal differentiation and proliferation from B cells to plasma cells(PC)and abnormal increasing of PC play crucial roles in autoimmune diseases such as systemic lupus erythematosus(SLE).It is of great significance to deeply investigate the mechanism of abnormal increase of plasma cells for the diagnosis and treatment of autoimmune diseases.To explore the molecular mechanisms in the growth of abnormal plasma,we used LPS-induced plasmablasts as normal plasmablasts and used proliferative(tumor)plasmablast-like SP 2/0 cells as abnormally proliferating plasmablasts.We used mRNA-sequencing assay to explore the new molecules in PC development.We found that plasmablasts(PB)-like SP 2/0 cells expressed a lower level of Loc108168067,Gm40600 and Itch.Loc108168067 and Gm40600 are two new genes with unknown functions.Previous study demonstrated that Itch is an E3 ubiquitin ligase which required for early B-cell development.We used overexpressing in vitro cell,overexpressing in mice,knockouting in mice,etc with these three molecules.Whether these three genes are involved in the abnormal differentiation of B cells into plasma cells provides more possibilities for the diagnosis and treatment of autoimmune diseases.Purpose:To understand the mechanism of differentiation and development of B cells into plasma cells,we explore the biological functions of Loc108168067,Gm40600 and Itch screening out by gene sequencing,which are related to plasma cell production.Methods:1.Gene expression profiles of LPS-induced PB/PC and SP 2/0 cells were determined using RNA-sequencing.Loc108168067,Gm40600 are differentially expressed genes,but their functions are not yet clear.The effect of Gm40600 on SP 2/0 cell proliferation,cell cycle/apoptosis,and tumor progression was assessed by cell counting kit-8(CCK8),flow cytometry(FACS),and the SP 2/0 isograft mouse model,respectively.The effect of Gm40600 on mRNA and protein expression was evaluated by RNA-sequencing and western blotting,respectively.2.To explore the role of Itch deficiency in antigen-driven B cell responses,we used TI Ag LPS and TD Ag SRBC in vitro and in vivo induced B cell activation and antibody production withing Itch KO mice and Itch cKO mice.Results:1.We found that LPS-induced PC expressed a high level of uncharacterized Loc108168067,whereas plasmablasts(PB)-like SP 2/0 cells expressed a lower level of Loc108168067.Loc108168067 overexpression did not affect SP 2/0 cell viability but induced cell apoptosis via G1/S block.Mechanistically,we found that Loc108168067 induced Trim21-CDKN1 c expression to block G1/S by promoting Trim21 promoter activation.On the other hand,we also found that Loc108168067 induced Myc-IRF4-Blimp1 expression to up-regulate cell proliferation/ survival by promoting Myc promoter activation.Thus,comprehensive effect of two aspects resulted into no effect of Loc108168067 on SP 2/0 cell in vitro viability and in vivo xenograft tumor progression.These results suggest that Loc108168067 has many different biological functions.2.We found that SP 2/0 cells expressed lower level of Gm40600 mRNA as compared to LPS-induced PB/PC.Overexpression of Gm40600 significantly suppressed SP 2/0 cell proliferation and isograft tumor progression in an isograft mouse model by promoting apoptosis.In addition,Gm40600 overexpression suppressed transcription of the gene encoding Bcl2.Gm40600 overexpression also reduced the expression of PC-associated transcription factors Blimp1 and Xbp1,which promote transcription of the gene that encodes Bcl2.3.We demonstrated that Itch KO mice showed itchy and scratchy skin,a larger size of spleen and lymph node,high level of antibody-associated genes with high base mutation,whereas spleen and lymph node in Itch cKO mice is only slightly bigger than that in their WT littermates.In addition,base mutation in antibody level and B-cell activation in Itch cKO mice is similar to that in WT littermates.Itch deficiency promoted antigens including LPS and sheep red blood cell(SRBC)induced B-cell activation and antibody production.Finally,we found that Itch deficiency promotes antigen-induced cytokines production by Itch-controlling the proteins with translation initiation factor activity.Conclusion:1.Loc108168067,positively associated with Prdm1 and PC development,induced Myc-IRF4-Blimp1 and Trim21-CDKN1 c expression by promoting Myc and Trim21 promoter activation,respectively,to mutually counter with each other so that Loc108168067 had no effect on SP 2/0 cell in vitro viability and in vivo xenograft tumor progression.2.Gm40600 reduced SP 2/0 cell proliferation and isograft tumor growth and progression by suppressing Blimp1 and Xbp1-mediated Bcl2 transcription to induce apoptosis.3.Our data suggest that Itch deficiency promotes antigen-driven B-cell responses by the proteins with translation initiation factor activity that upregulates cytokines production.This may provide hints to treat patients with autoimmune disease by targeting Itch.