受体编辑在天然多反应性B细胞发育中的作用及机制研究
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摘要
天然自身抗体(natural autoantibody,NAA)是指在没有任何抗原免疫的情况下,正常机体中针对一种或多种自身抗原的自身抗体;产生NAA的B细胞则被称为天然自身反应性B细胞。NAA及天然自身反应性B细胞广泛存在并发挥重要功能,大量研究提示,自身反应性B细胞存在的耐受机制与自身免疫病的发病机理密切相关。
受体编辑现象是自身反应性B细胞的发育耐受的主导机制。受体编辑通过多种作用方式,一方面保证了人体外周中具有自身反应性的B细胞比例与骨髓相比大大降低;另一方面,发生了受体编辑从而改变特异性的自身反应性B细胞可以逃脱克隆清除或失活的命运而发育成熟。但是,受体编辑现象作为B细胞发育耐受的核心机制,其参与病理性自身免疫即自身免疫病的方式存在巨大争论。因此,研究生理性天然多反应性B细胞发育中受体编辑的作用必将有助于全面揭示自身免疫失耐受的真正原因。
本课题组以往的研究进展,为深入研究受体编辑现象在自身反应性B细胞发育中的作用机制奠定了良好的基础。应用未免疫小鼠B细胞与骨髓瘤细胞融合,我们成功筛选到了分泌抗角蛋白、肌动蛋白等多种自身抗原的自身抗体的3B4等杂交瘤,其中由未免疫小鼠获得的3B4天然多反应性抗体是一种完全意义上的NAA,而表达3B4抗体的B细胞可以代表生理广泛存在的天然自身反应性B细胞。通过克隆3B4可变区基因,我们成功构建了可以表达天然多反应性BCR的3B4重链(TgVH3B4)和轻链(TgVL3B4)转基因小鼠。抗体转基因小鼠的应用,为深入研究自身反应性B细胞发育耐受提供了理想的平台。而来源于未免疫小鼠天然自身反应性IgM的3B4抗体转基因小鼠,为深入研究受体编辑在天然自身反应B细胞发育中的作用机制等问题提供了绝佳模型。
本研究通过交配重链与轻链小鼠,在其子代中筛选到了完整表达天然多反应性3B4抗体的转基因重轻链小鼠(TgVH/L3B4)。TgVH/L3B4小鼠中表达转基因完整BCR的B细胞复制了3B4抗体分泌细胞在野生小鼠中的发育过程;而表达内源轻链的TgVH3B4小鼠由于表达转基因重链与不同内源轻链所组成的BCR,更有利于观察不同特异性自身反应性B细胞发育耐受的普遍规律。利用这个平台,通过比较研究TgVH3B4小鼠和TgVH/L3B4小鼠,我们就受体编辑中的等位相容现象对自身反应性B细胞发育分化及其功能的影响进行了分析。
一、受体编辑在天然自身反应性B细胞发育中的检测
利用流式分析转基因重链与内源性重链表达,并且检测不同来源B细胞轻链的表达情况,发现H链鼠外周B细胞发生明显的重链等位相容现象,并且伴随活跃的轻链受体编辑,而HL链鼠B细胞发育中并没有明显的受体编辑和等位相容现象,始终完整表达3B4抗体基因。
二、转基因小鼠中天然自身反应性B细胞的发育和分化
通过对骨髓中不同发育阶段的B细胞的表面标记进行抗体染色和流式分析,发现HL链鼠B细胞在骨髓中得到阳性选择;而H链小鼠B细胞在中枢发育中受到严格的阴性选择,相应阶段细胞发育受阻并诱导了受体编辑的发生。同样利用B细胞外周不同发育阶段和成熟亚群的不同表型,通过流式和免疫荧光染色,明确了自身反应性B细胞在H链鼠的脾脏和腹腔分别向边缘带(MZ)和B-1方向分化;在HL链小鼠表达3B4的B细胞则主要发育为T2’的特殊表型。
三、等位相容在自身反应性B细胞发育中的作用分析
同样利用流式和激光共聚焦等方法,明确了发生显著重链等位相容的H链小鼠中,等位相容可以诱导B细胞向MZ和B-1a细胞发育分化。四、自身反应性B细胞功能状态分析
体内分析:利用ELISA检测到H链和HL链小鼠血清中同时存在高表达的天然多反应性自身抗体;流式分析发现H链鼠中发生等位相容的B细胞表面活化分子表达水平升高;凋亡检测明确了B细胞向不同亚群分化由阳性选择决定。体外分析:通过体外培养不同组织或流式分选的不同亚群B细胞,明确了天然多反应性自身抗体可以同时来自脾脏和腹腔,并且在H链小鼠中,发生重链等位相容的B细胞可以被抗原刺激而引起胞内Ca2+浓度改变,提示BCR信号通路的活化;并且其可以在体外分泌天然多反应性自身抗体。
总结这些结果,我们可以得出如下结论:受体编辑现象很可能并未参与表达胚系基因编码的3B4抗体的B细胞发育过程;但是重链等位相容可以作为特殊的耐受机制影响B细胞在外周的分化方向并且保持其天然多反应性。这些发现不仅有助于理解受体编辑在生理性自身反应性B细胞发育耐受中的作用,并且对于尽早揭示其在自身免疫病发病中的作用提供了积极的线索。
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.
受体编辑现象是自身反应性B细胞的发育耐受的主导机制。受体编辑通过多种作用方式,一方面保证了人体外周中具有自身反应性的B细胞比例与骨髓相比大大降低;另一方面,发生了受体编辑从而改变特异性的自身反应性B细胞可以逃脱克隆清除或失活的命运而发育成熟。但是,受体编辑现象作为B细胞发育耐受的核心机制,其参与病理性自身免疫即自身免疫病的方式存在巨大争论。因此,研究生理性天然多反应性B细胞发育中受体编辑的作用必将有助于全面揭示自身免疫失耐受的真正原因。
本课题组以往的研究进展,为深入研究受体编辑现象在自身反应性B细胞发育中的作用机制奠定了良好的基础。应用未免疫小鼠B细胞与骨髓瘤细胞融合,我们成功筛选到了分泌抗角蛋白、肌动蛋白等多种自身抗原的自身抗体的3B4等杂交瘤,其中由未免疫小鼠获得的3B4天然多反应性抗体是一种完全意义上的NAA,而表达3B4抗体的B细胞可以代表生理广泛存在的天然自身反应性B细胞。通过克隆3B4可变区基因,我们成功构建了可以表达天然多反应性BCR的3B4重链(TgVH3B4)和轻链(TgVL3B4)转基因小鼠。抗体转基因小鼠的应用,为深入研究自身反应性B细胞发育耐受提供了理想的平台。而来源于未免疫小鼠天然自身反应性IgM的3B4抗体转基因小鼠,为深入研究受体编辑在天然自身反应B细胞发育中的作用机制等问题提供了绝佳模型。
本研究通过交配重链与轻链小鼠,在其子代中筛选到了完整表达天然多反应性3B4抗体的转基因重轻链小鼠(TgVH/L3B4)。TgVH/L3B4小鼠中表达转基因完整BCR的B细胞复制了3B4抗体分泌细胞在野生小鼠中的发育过程;而表达内源轻链的TgVH3B4小鼠由于表达转基因重链与不同内源轻链所组成的BCR,更有利于观察不同特异性自身反应性B细胞发育耐受的普遍规律。利用这个平台,通过比较研究TgVH3B4小鼠和TgVH/L3B4小鼠,我们就受体编辑中的等位相容现象对自身反应性B细胞发育分化及其功能的影响进行了分析。
一、受体编辑在天然自身反应性B细胞发育中的检测
利用流式分析转基因重链与内源性重链表达,并且检测不同来源B细胞轻链的表达情况,发现H链鼠外周B细胞发生明显的重链等位相容现象,并且伴随活跃的轻链受体编辑,而HL链鼠B细胞发育中并没有明显的受体编辑和等位相容现象,始终完整表达3B4抗体基因。
二、转基因小鼠中天然自身反应性B细胞的发育和分化
通过对骨髓中不同发育阶段的B细胞的表面标记进行抗体染色和流式分析,发现HL链鼠B细胞在骨髓中得到阳性选择;而H链小鼠B细胞在中枢发育中受到严格的阴性选择,相应阶段细胞发育受阻并诱导了受体编辑的发生。同样利用B细胞外周不同发育阶段和成熟亚群的不同表型,通过流式和免疫荧光染色,明确了自身反应性B细胞在H链鼠的脾脏和腹腔分别向边缘带(MZ)和B-1方向分化;在HL链小鼠表达3B4的B细胞则主要发育为T2’的特殊表型。
三、等位相容在自身反应性B细胞发育中的作用分析
同样利用流式和激光共聚焦等方法,明确了发生显著重链等位相容的H链小鼠中,等位相容可以诱导B细胞向MZ和B-1a细胞发育分化。四、自身反应性B细胞功能状态分析
体内分析:利用ELISA检测到H链和HL链小鼠血清中同时存在高表达的天然多反应性自身抗体;流式分析发现H链鼠中发生等位相容的B细胞表面活化分子表达水平升高;凋亡检测明确了B细胞向不同亚群分化由阳性选择决定。体外分析:通过体外培养不同组织或流式分选的不同亚群B细胞,明确了天然多反应性自身抗体可以同时来自脾脏和腹腔,并且在H链小鼠中,发生重链等位相容的B细胞可以被抗原刺激而引起胞内Ca2+浓度改变,提示BCR信号通路的活化;并且其可以在体外分泌天然多反应性自身抗体。
总结这些结果,我们可以得出如下结论:受体编辑现象很可能并未参与表达胚系基因编码的3B4抗体的B细胞发育过程;但是重链等位相容可以作为特殊的耐受机制影响B细胞在外周的分化方向并且保持其天然多反应性。这些发现不仅有助于理解受体编辑在生理性自身反应性B细胞发育耐受中的作用,并且对于尽早揭示其在自身免疫病发病中的作用提供了积极的线索。
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.
引文
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28. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers, and R. Carsetti. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J Exp Med 190:75-89.
29. Nemazee, D. 2006. Receptor editing in lymphocyte development and central tolerance. Nat Rev Immunol 6:728-740.
30. Gay, D., T. Saunders, S. Camper, and M. Weigert. 1993. Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med 177:999-1008.
31. Tiegs, S. L., D. M. Russell, and D. Nemazee. 1993. Receptor editing in self-reactive bone marrow B cells. J Exp Med 177:1009-1020.
32. Edry, E., and D. Melamed. 2004. Receptor editing in positive and negative selection of B lymphopoiesis. J Immunol 173:4265-4271.
33. Liu, S., M. G. Velez, J. Humann, S. Rowland, F. J. Conrad, R. Halverson, R. M. Torres, and R. Pelanda. 2005. Receptor editing can lead to allelic inclusion and development of B cells that retain antibodies reacting with high avidity autoantigens. J Immunol 175:5067-5076.
34. Li, Y., H. Li, and M. Weigert. 2002. Autoreactive B cells in the marginal zone that express dual receptors. J Exp Med 195:181-188.
35. Sekiguchi, D. R., R. A. Eisenberg, and M. Weigert. 2003. Secondary heavy chain rearrangement: a mechanism for generating anti-double-stranded DNA B cells. J Exp Med 197:27-39.
36. Fields, M. L., and J. Erikson. 2003. The regulation of lupus-associated autoantibodies: immunoglobulin transgenic models. Curr Opin Immunol 15:709-717.
37. Itoh, K., E. Meffre, E. Albesiano, A. Farber, D. Dines, P. Stein, S. E. Asnis, R. A. Furie, R. I. Jain, and N. Chiorazzi. 2000. Immunoglobulin heavy chain variable region gene replacement As a mechanism for receptor revision in rheumatoid arthritis synovial tissue B lymphocytes. J Exp Med 192:1151-1164.
38. Sening, W., R. Lisner, and G. Niedobitek. 2004. Rare detection of phenotypically immature lymphocytes in Hashimoto thyroiditis and rheumatoid arthritis. J Autoimmun 22:147-152.
39. Yachimovich-Cohen, N., R. Fischel, N. Bachar, Y. Yarkoni, and D. Eilat. 2003. Autoimmune NZB/NZW F1 mice utilize B cell receptor editing for generating high-affinity anti-dsDNA autoantibodies from low-affinity precursors. Eur J Immunol 33:2469-2478.
40. Li, H., Y. Jiang, E. L. Prak, M. Radic, and M. Weigert. 2001. Editors and editing of anti-DNA receptors. Immunity 15:947-957.
41. Gerdes, T., and M. Wabl. 2004. Autoreactivity and allelic inclusion in a B cell nuclear transfer mouse. Nat Immunol 5:1282-1287.
42. Casellas, R., Q. Zhang, N. Y. Zheng, M. D. Mathias, K. Smith, and P. C. Wilson. 2007. Igkappa allelic inclusion is a consequence of receptor editing. J Exp Med 204:153-160.
43. Heltemes-Harris, L., X. Liu, and T. Manser. 2005. An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion.Int Immunol 17:1447-1461.
44. Sonoda, E., Y. Pewzner-Jung, S. Schwers, S. Taki, S. Jung, D. Eilat, and K. Rajewsky. 1997. B cell development under the condition of allelic inclusion. Immunity 6:225-233.
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46. Li, H., Y. Jiang, H. Cao, M. Radic, E. L. Prak, and M. Weigert. 2003. Regulation of anti-phosphatidylserine antibodies. Immunity 18:185-192.
47. Fu, M., P. S. Fan, W. Li, C. X. Li, Y. Xing, J. G. An, G. Wang, X. L. Fan, T. W. Gao, Y. F. Liu, and S. Ikeda. 2007. Identification of poly-reactive natural IgM antibody that recognizes late apoptotic cells and promotes phagocytosis of the cells. Apoptosis 12:355-362.
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49. Li, Y. S., K. Hayakawa, and R. R. Hardy. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J Exp Med 178:951-960.
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51. Chen, C., Z. Nagy, M. Z. Radic, R. R. Hardy, D. Huszar, S. A. Camper, and M. Weigert. 1995. The site and stage of anti-DNA B-cell deletion. Nature 373:252-255.
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31. Tiegs, S. L., D. M. Russell, and D. Nemazee. 1993. Receptor editing in self-reactive bone marrow B cells. J Exp Med 177:1009-1020.
32. Edry, E., and D. Melamed. 2004. Receptor editing in positive and negative selection of B lymphopoiesis. J Immunol 173:4265-4271.
33. Liu, S., M. G. Velez, J. Humann, S. Rowland, F. J. Conrad, R. Halverson, R. M. Torres, and R. Pelanda. 2005. Receptor editing can lead to allelic inclusion and development of B cells that retain antibodies reacting with high avidity autoantigens. J Immunol 175:5067-5076.
34. Li, Y., H. Li, and M. Weigert. 2002. Autoreactive B cells in the marginal zone that express dual receptors. J Exp Med 195:181-188.
35. Sekiguchi, D. R., R. A. Eisenberg, and M. Weigert. 2003. Secondary heavy chain rearrangement: a mechanism for generating anti-double-stranded DNA B cells. J Exp Med 197:27-39.
36. Fields, M. L., and J. Erikson. 2003. The regulation of lupus-associated autoantibodies: immunoglobulin transgenic models. Curr Opin Immunol 15:709-717.
37. Itoh, K., E. Meffre, E. Albesiano, A. Farber, D. Dines, P. Stein, S. E. Asnis, R. A. Furie, R. I. Jain, and N. Chiorazzi. 2000. Immunoglobulin heavy chain variable region gene replacement As a mechanism for receptor revision in rheumatoid arthritis synovial tissue B lymphocytes. J Exp Med 192:1151-1164.
38. Sening, W., R. Lisner, and G. Niedobitek. 2004. Rare detection of phenotypically immature lymphocytes in Hashimoto thyroiditis and rheumatoid arthritis. J Autoimmun 22:147-152.
39. Yachimovich-Cohen, N., R. Fischel, N. Bachar, Y. Yarkoni, and D. Eilat. 2003. Autoimmune NZB/NZW F1 mice utilize B cell receptor editing for generating high-affinity anti-dsDNA autoantibodies from low-affinity precursors. Eur J Immunol 33:2469-2478.
40. Li, H., Y. Jiang, E. L. Prak, M. Radic, and M. Weigert. 2001. Editors and editing of anti-DNA receptors. Immunity 15:947-957.
41. Gerdes, T., and M. Wabl. 2004. Autoreactivity and allelic inclusion in a B cell nuclear transfer mouse. Nat Immunol 5:1282-1287.
42. Casellas, R., Q. Zhang, N. Y. Zheng, M. D. Mathias, K. Smith, and P. C. Wilson. 2007. Igkappa allelic inclusion is a consequence of receptor editing. J Exp Med 204:153-160.
43. Heltemes-Harris, L., X. Liu, and T. Manser. 2005. An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion.Int Immunol 17:1447-1461.
44. Sonoda, E., Y. Pewzner-Jung, S. Schwers, S. Taki, S. Jung, D. Eilat, and K. Rajewsky. 1997. B cell development under the condition of allelic inclusion. Immunity 6:225-233.
45. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, and et al. 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676-682.
46. Li, H., Y. Jiang, H. Cao, M. Radic, E. L. Prak, and M. Weigert. 2003. Regulation of anti-phosphatidylserine antibodies. Immunity 18:185-192.
47. Fu, M., P. S. Fan, W. Li, C. X. Li, Y. Xing, J. G. An, G. Wang, X. L. Fan, T. W. Gao, Y. F. Liu, and S. Ikeda. 2007. Identification of poly-reactive natural IgM antibody that recognizes late apoptotic cells and promotes phagocytosis of the cells. Apoptosis 12:355-362.
48. Li, W., M. Fu, J. G. An, Y. Xing, P. Zhang, X. Zhang, Y. C. Wang, C. X. Li, R. Tian, W. J. Su, H. H. Guan, G. Wang, T. W. Gao, H. Han, and Y. F. Liu. 2007. Host defence against C. albicans infections in IgH transgenic mice with V(H) derived from a natural anti-keratin antibody. Cell Microbiol 9:306-315.
49. Li, Y. S., K. Hayakawa, and R. R. Hardy. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J Exp Med 178:951-960.
50. Yu, W., H. Nagaoka, M. Jankovic, Z. Misulovin, H. Suh, A. Rolink, F. Melchers, E. Meffre, and M. C. Nussenzweig. 1999. Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization. Nature 400:682-687.
51. Chen, C., Z. Nagy, M. Z. Radic, R. R. Hardy, D. Huszar, S. A. Camper, and M. Weigert. 1995. The site and stage of anti-DNA B-cell deletion. Nature 373:252-255.
52. Radic, M. Z., J. Erikson, S. Litwin, and M. Weigert. 1993. B lymphocytes may escape tolerance by revising their antigen receptors. J Exp Med 177:1165-1173.
53. Era, T., M. Ogawa, S. Nishikawa, M. Okamoto, T. Honjo, K. Akagi, J. Miyazaki, and K. Yamamura. 1991. Differentiation of growth signal requirement of B lymphocyte precursor is directed by expression of immunoglobulin. Embo J 10:337-342.
54. Arakawa, H., and S. Takeda. 1996. Early expression of Ig mu chain from a transgene significantly reduces the duration of the pro-B stage but doesnot affect the small pre-B stage. Int Immunol 8:1319-1328.
55. Wortis, H. H., M. Teutsch, M. Higer, J. Zheng, and D. C. Parker. 1995. B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes. Proc Natl Acad Sci U S A 92:3348-3352.
56. Roehm, N. W., H. J. Leibson, A. Zlotnik, J. Kappler, P. Marrack, and J. C. Cambier. 1984. Interleukin-induced increase in Ia expression by normal mouse B cells. J Exp Med 160:679-694.
57. Besredka, M. 1901. Les antihemolysines naturelles. Ann. Inst. Pasteur (Paris) 15:785-807.
58. Lamoureux, J. L., L. C. Watson, M. Cherrier, P. Skog, D. Nemazee, and A. J. Feeney. 2007. Reduced receptor editing in lupus-prone MRL/lpr mice. J Exp Med 204:2853-2864.
59. Yurasov, S., H. Wardemann, J. Hammersen, M. Tsuiji, E. Meffre, V. Pascual, and M. C. Nussenzweig. 2005. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J Exp Med 201:703-711.
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62. Chu, Y. P., L. Spatz, and B. Diamond. 2004. A second heavy chain permits survival of high affinity autoreactive B cells. Autoimmunity 37:27-32.
63. Kenny, J. J., L. J. Rezanka, A. Lustig, R. T. Fischer, J. Yoder, S. Marshall, and D. L. Longo. 2000. Autoreactive B cells escape clonal deletion by expressing multiple antigen receptors. J Immunol 164:4111-4119.
64. Nemazee, D., and M. Weigert. 2000. Revising B cell receptors. J Exp Med 191:1813-1817.
65. Gaudin, E., Y. Hao, M. M. Rosado, R. Chaby, R. Girard, and A. A. Freitas. 2004. Positive selection of B cells expressing low densities of self-reactive BCRs. J Exp Med 199:843-853.
66. Casellas, R., T. A. Shih, M. Kleinewietfeld, J. Rakonjac, D. Nemazee, K. Rajewsky, and M. C. Nussenzweig. 2001. Contribution of receptor editing to the antibody repertoire. Science 291:1541-1544.
67. Carsetti, R., G. Kohler, and M. C. Lamers. 1995. Transitional B cells are the target of negative selection in the B cell compartment. J Exp Med 181:2129-2140.
68. Alugupalli, K. R., J. M. Leong, R. T. Woodland, M. Muramatsu, T. Honjo, and R. M. Gerstein. 2004. B1b lymphocytes confer T cell-independent long-lasting immunity. Immunity 21:379-390.
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