Research On The Function And Mechanism Of Receptor Editing In The Development Of Natural Poly-reactive B Cells

Natural autoantibodies can be defined as antibodies produced by healthy individual against one or more autoantigens without immunization. B cells which can produce NAA are regarded as natural autoreactive B cells.NAA and atural autoreactive B cells are widely exist and have an important function in normal individuals. Extensive evidence suggested that the tolerance mechanisms are closely related to the genesis of autoimmune diseases.Receptor editing plays a central role in the tolerance mechanism of autoreactive B cells development. Autoreactive B cells decreased significantly in B cells development through negative selection including receptor editing. Moreover, receptor editing has been considered as the dominant mechanism of central B cell tolerance. Autoreactive B cells that are destined to clonal deletion or anergy can be rescued by successfully secondly rearrangement of B cell receptor genes. Similar to the intensive debate in autoimmunity, the function of receptor editing in the development of natural autoreactive B cells is also unclear. So, understanding the mechanism that lead to the breakdown of self-tolerance in these poly-reactive B cells is therefore crucial to understanding the genesis of autoimmunity.The previous work by our group has made a solid foundation to this study.Through fusion of spleen cells from unimmuned mice with myeloma cells,we successfully obtained several lines of hybridoma cells whigh can secreat NAA. Poly-reactive natural antibody 3B4 secreated by Hybridoma 3B4 can identify keratin, actin, myosin and many foreign antigens. Recently, we have successfully establishedμchain transgenic mice (TgVH3B4) with the VH gene from 3B4 hybridoma, and high titers of transgene encoded natural poly-reactive antibodies can be detected in the serum of TgVH3B4 mice. In this work, we have constructedκchain transgenic mice (TgVL3B4) with the VL gene from the same hybridoma. In addition, double transgenic mice (TgVH/L3B4) which can express integrated natural IgM were screened among the offspring of the TgVH3B4 and TgVL3B4 mice. Here, we sought to determine whether receptor editing play an important role in the development of natural poly-reactive B cells by studying the development and tolerance of transgene expressing B cells in TgVH3B4 and TgVH/L3B4 mice.1. The detection of receptor editing in the development of natural autoreactive B cells.We detected the expression of transgenic or endogenous heavy and light chains. While the 3B4 heavy chain and light chain exerts good allelic exclusion in TgVH/L3B4 mice, we found that significant heavy chain allelic inclusion and light chain receptor editing happens in a significant fraction of B cells from each population of TgVH3B4 mice,2. The development of antoreactive B cells in Tg mice.Through immunofluoresence and FACS analysis, we found 3B4 B cells got positive selection in the BM of TgVH/L3B4 mice while apparent negative selection happens in the BM B cell development of TgVH3B4 mice. In periphery, B cells expressing transgene develop into MZ and B-1 subsets in TgVH3B4 mice, but B cells expressing integrated 3B4 develop into a special phenotype like T2.3. Analysis of the infection of allelic inclusion on B cell development.Through FACS and con-focal analysis, we found heavy chain allelic inclusion can induce B cells to differentiation into MZ and B-1a subsets.4. Detection the function status of autoreactive B cells in Tg mice.ELISA was performed over varied Ags and we found high level of Tg expressed IgMa in the serum of both lines which had a binding pattern similar to that of the original 3B4 mAb. Allelic included B cells seemed express slightly higher level of some activation markers. Apoptosis analysis confirmed the hypothesis that B cells were positive selected in periphery. Moreover, B cells from both spleen and peritoneal cavity in either TgVH3B4 or TgVH/L3B4 mice can produce much higher level of autoantibodies in vitro, under the stimulation of LPS or CD40. Further analysis demonstrated that autoantibody production in spleen of TgVH3B4 mice was mainly contributed by the allelic included cells.Taken together, our findings suggest that receptor editing plays a minor role in the positive selection of B cells expressing natural poly-reactive antibodies, and more importantly these B cells can also be positive selected through heavy chain allelic inclusion to retain this poly-reactivity in periphery. These innovations not only help the understanding of the function in the tolerance mechanism of natural poly-reactive B cells development, but also provide clues to elucidate the genesis of autoimmune disease.