Ph.D Thesis


Ph.D StudentHilla Azulay-Debby
SubjectCpG DNA Activates Developing B-lymphocytes in the Bone
Marrow to Break Tolerance and to Secrete
Autoantibodies
DepartmentDepartment of Biotechnology
Supervisors Full Professors Melamed Doron
Full Professors Machluf Marcelle
Full Thesis textFull thesis text - English Version


Abstract

The classical immunological dogma excludes bone marrow (BM) B cells from participating in an immune response, since most of these cells (55-75%) are autoreactive. However, it has been shown that following immunization or infection immature BM B cells migrate to the periphery. The appearance of these immature B cells in the periphery led us to hypothesize that non-tolerant developing B cells may also initiate/participate in an autoimmune response. However, the competence of these cells to respond to an external immunologic stimulus is unknown. To test our hypothesis we used CpG DNA as an immunologic stimulus and determined the ability of developing BM B cells to undergo polyclonal activation, to secrete autoantibodies and to participate in an autoimmune response.

Here we show that developing B cells express TLR9 and undergo a polyclonal response to CpG DNA stimulation. In vitro and ex vivo experiments revealed that stimulation with CpG DNA protects immature B cells from negative selection imposed by apoptosis and by receptor editing and results in the production of autoantibodies. We also found that in vivo administration of CpG DNA activates immature B cells in the bone marrow and suppresses the expression of recombination-activating genes (RAG) in a mouse model of central tolerance and receptor editing. In addition, in the same model of central tolerance, we show that immature B cells participate in an inflammatory response as reflected by autoantibodies secretion in vivo. Finally, using a mouse model for experimental autoimmune encephalomyelitis (EAE), we found that the depletion of immature BM B cells, prior the induction of the disease, reduces the levels of autoantibodies that are produced.

Since immunologic stimuli are regulated in B lymphocytes by co-receptors, we studied the involvement of CD19, which is a positive response regulator, in regulating TLR9 signaling. Here we used the model of CD19 deficient mouse and found that in the absence of CD19, immature and mature B cells exhibit reduced responsiveness to CpG DNA as reflected by reduced proliferation and lower levels of antibody secretion. Further analysis revealed that CD19 is phosphorylated in B lymphocytes following stimulation with CpG DNA, thereby suggesting that TLR9 signaling is regulated by CD19.

Collectively, our results suggest that immunologic signals can stimulate nontolerant immature B cells in the bone marrow to proliferate and to secrete autoantibodies. This activation has the potential to interfere with the establishment of central tolerance as well as to initiate or promote autoimmune processes.