Cooperstone B G, Rahman M M, Rudolph E H, Foster M H
Department of Paediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Immunol Cell Biol. 2001 Jun;79(3):222-30. doi: 10.1046/j.1440-1711.2001.01001.x.
Lymphocyte antigen receptors are promising targets for immune intervention strategies in disorders marked by repertoire skewing or expansion of lymphocyte subsets. Appropriate application of immune receptor modulation is predicated on understanding the role of a particular receptor in pathogenesis and disease regulation. The VHB/W16 gene, restricted to mice carrying the j haplotype for the J558 family, is overexpressed by murine lupus anti-DNA Ig. This gene is also expressed recurrently among nephritogenic anti-DNA Ig recovered from several autoimmune strains, suggesting that cells expressing this pathogenic receptor are positively selected during disease progression. To explore the extent and mechanisms by which Ig H chains expressing this gene contribute to autoimmunity, an Ig H chain gene was engineered for in vitro and in vivo recombination studies. Site-directed mutagenesis generated unique restriction sites to link PCR-amplified V region (VDJ) cDNA to previously isolated genomic fragments containing Ig regulatory and signal sequences. The new 3 kb VDJ gene was then ligated to a 9 kb fragment encoding the IgM constant region. Transfection of H chain loss variant myeloma with the complete 12 kb construct, termed 238H-Cmicro, resulted in secretion of intact Ig pairing 238H-Cmicro, with a lambda L chain; however, transfectant Ig lacked autoreactivity and pathogenicity. Introduction of the 238H-Cmicro H chain as a transgene onto the non-autoimmune C57BL/6 background resulted in abundant B cell surface expression of 238H-Cmicro, however, four transgenic Ig recovered by fusion of LPS-stimulated splenocytes and formed by combination of 238H-Cmicro, with endogenous kappa chains do not bind DNA or laminin. These results indicate that the antigen binding sites encoded by this disease-associated gene and/or H chain must associate with permissive L chains to specify autoimmunity. The 238H-Cmicro, transgenic model should prove useful in dissecting the in vivo fate of 238H-Cmicro, L combinations that produce pathogenic autoreactive receptors and in evaluating receptor-targeted interventions.
淋巴细胞抗原受体是免疫干预策略的理想靶点，这些策略用于治疗以淋巴细胞亚群库偏移或扩增为特征的疾病。免疫受体调节的恰当应用取决于对特定受体在发病机制和疾病调控中作用的理解。VHB/W16基因仅限于携带J558家族j单倍型的小鼠，它在小鼠狼疮抗DNA Ig中过度表达。该基因在从几种自身免疫品系中回收的致肾炎抗DNA Ig中也反复表达，这表明表达这种致病受体的细胞在疾病进展过程中被阳性选择。为了探究表达该基因的Ig重链在多大程度上以及通过何种机制导致自身免疫，构建了一个Ig重链基因用于体外和体内重组研究。定点诱变产生独特的限制性酶切位点，以将PCR扩增的V区（VDJ）cDNA连接到先前分离的包含Ig调节和信号序列的基因组片段上。然后将新的3 kb VDJ基因连接到一个9 kb编码IgM恒定区的片段上。用完整的12 kb构建体（称为238H-Cμ）转染重链缺失变异骨髓瘤细胞，导致分泌完整的与λ轻链配对的Ig 238H-Cμ；然而，转染细胞的Ig缺乏自身反应性和致病性。将238H-Cμ重链作为转基因导入非自身免疫的C57BL/6背景中，导致238H-Cμ在B细胞表面大量表达，然而，通过LPS刺激的脾细胞融合回收的四种转基因Ig，由238H-Cμ与内源性κ链组合形成，不结合DNA或层粘连蛋白。这些结果表明，由该疾病相关基因和/或重链编码的抗原结合位点必须与允许的轻链结合，才能确定自身免疫性。238H-Cμ转基因模型在剖析产生致病自身反应性受体的238H-Cμ轻链组合的体内命运以及评估受体靶向干预方面应是有用的。
