Critical Role Of ATG7 In Germinal Center B Cells

Germinal centres(GCs)are transient structures that form within peripheral lymphoid organs in response to T cell-dependent antigen.Within GCs,B cells expressing high-affinity antibodies develop and differentiate into antibody-secreting plasma cells and memory B cells that mediate and sustain protection against invading pathogens.The importance of the GC reaction is best shown by the immunodeficiency syndromes that are observed in patients who are unable to form GCs.Activation Induced cytidine Deaminase(AID)initiates Immunoglobulin(Ig)heavy chain(IgH)class switch recombination(CSR)and Ig variable region somatic hypermutation(SHM)in B lymphocytes by deaminating cytidines on DNA substrates,leads to mismatch dU:dG,followed by error-prone mismatch-or base-excision DNA repair.Autophagy is an evolutionarily conserved process that involves the engulfment and delivery of cytosolic contents to the lysosome for degradation.In order to study the function of ATG7 in the germinal center,we used the Cre-Loxp system to conditionally knockout ATG7 of the mice germinal center B cells,got the mouse model.We immunized mouse model through the T-dependent specific antigen(NP-CGG),and analyzed the development and function of the splenic GCs by flow cytometry after 14 days.We found that germinal center developed normally,but the function of the germinal center(CSR)decreased significantly in the Atg7-/-GC B cells.And we found AID protein levels significantly decreased by western blot,but the AID mRNA level no obviously changed by Q-PCR,which meant that Atg7 can affect AID protein stability,and this process was through the post-translation level rather than at the transcriptional level.In order to more clearly understand the molecular mechanism by witch ATG7 influenced AID protein stability,we constructed a stable expressed Flag-AID-293 T cells,and then knocked down ATG7.Through a series of molecular and biochemical experiments,we proved the ATG7 can impair E3 ubiquitin ligase CHIP combined with AID,affected the ubiquitination of AID,and made the AID protein stability.Because AID stability plays an important role in the function of CSR,so ATG7 is essential to maintain the function of the germinal center.