Genetic control of the humoral responses to xenografts. III. Identification of the immunoglobulin V(H) genes responsible for encoding rat immunoglobin G xenoantibodies to hamster heart grafts.
Genetic control of the humoral responses to xenografts. III. Identification of the immunoglobulin V(H) genes responsible for encoding rat immunoglobin G xenoantibodies to hamster heart grafts.
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DOI：
10.1097/00007890-199907150-00005
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发表时间：
1999-07
期刊：
影响因子：
6.2
通讯作者：
E. Gochi;G. Wu;S. Wakiyama;M. Kearns-Jonker;J. Swensson;D. Cramer
中科院分区：
文献类型：
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作者：
E. Gochi;G. Wu;S. Wakiyama;M. Kearns-Jonker;J. Swensson;D. Cramer
背景我们以前曾报道大鼠对仓鼠异种移植物的免疫应答的早期阶段的特征是产生由一组有限的IG种系V（H）基因（V（H）HAR家族）编码的IgM异种抗体。在反应的后期阶段，发生IgM到IgG同种型转换，我们的研究检查了在这种同种型转换后用于编码IgG抗体的重排的V（H）HAR基因的结构。方法采用定量聚合酶链反应（PCR）技术，观察异种移植后V（H）HAR+ IgG mRNA水平的变化。从移植后第4、8、21和28天的未处理大鼠和仓鼠心脏异种移植受体的脾细胞中提取总RNA，建立V（H）HAR+ Iggamma链特异性cDNA文库。菌落过滤杂交用于估计使用单个V（H）HAR+ IgG亚类的相对频率。对来自第21天cDNA文库的所选IgG克隆进行测序并分析VH-D-J（H）基因使用和抗体结合位点结构。结果与未处理动物相比，异种移植物受体中V（H）HAR+ IgG的mRNA水平在移植后21天增加6倍。单独使用V（H）HAR+ IgG 1抗体的同种型使用的相对频率从第0天的22.3%增加到第21天PTx的37.4%。对来自第21天cDNA文库的10个IgG克隆进行了重排V（H）-D-J（H）基因的测序。这些IgG克隆中的百分之三十（3/10）使用V（H）HAR基因编码具有有限数量的核酸取代的重链可变区（与其种系祖细胞的同一性>98%），尽管当与种系V（H）基因相比时，其他克隆表现出核苷酸序列的增加的变化（95-97%同一性）。从预测的氨基酸序列的典型结合位点结构的分析表明，大多数的IgG克隆（9/10）显示出相似的模式的保守构型的结合位点。结论：在大鼠对仓鼠异种移植物的体液免疫应答的后期阶段中，IgM至IgG抗体产生的变化与IgM至IgG同种型转换和IgG 1同种型抗体产生的增加有关。大鼠抗仓鼠IgG异种抗体继续表达V（H）基因的V（H）HAR家族，许多处于其原始生殖系构型，以编码仓鼠靶抗原的抗体识别。然而，当与目前可用的种系序列相比时，存在大多数抗体的V（H）基因表达增加的核酸序列变异的证据。这种变异的来源尚不清楚，但可能代表尚未鉴定的种系基因的表达和/或T细胞驱动的体细胞突变的引入。尽管出现了这种变异，但V（H）区内关键抗原结合位点的不寻常保守水平表明，这种变异（与其来源无关）可能对宿主抗体对异种移植物反应的限制性性质产生有限影响。
BACKGROUND We have previously reported that the early phases of the immune response of rats to hamster xenografts are characterized by the production of IgM xenoantibodies encoded by a restricted group of Ig germline V(H) genes (V(H)HAR family). In the later phases of the reaction, an IgM to IgG isotype switch occurs and our study examines the structure of the rearranged V(H)HAR genes used to encode IgG antibodies after this isotype switch. METHODS A quantitative polymerase chain reaction was used to investigate the changes in the levels of V(H)HAR+ IgG mRNA seen after xenotransplantation. cDNA libraries specific for V(H)HAR+ Iggamma chain were established from total RNA extracted from splenocytes of naive rats and xenograft recipients of hamster hearts at days 4, 8, 21, and 28 posttransplantation. Colony filter hybridization was used to estimate the relative frequency of the use of individual V(H)HAR+ IgG subclasses. Selected IgG clones from day 21 cDNA libraries were sequenced and analyzed for VH-D-J(H) gene usage and antibody combining site structure. RESULTS The level of mRNA for V(H)HAR+ IgG increased 6-fold in xenograft recipients at day 21 post-transplantation when compared with naive animals. The relative frequency of isotype usage for V(H)HAR+ IgG1 antibodies alone increased from 22.3% at day 0 to 37.4% at day 21 PTx. Ten IgG clones from the day 21 cDNA libraries have been sequenced for the rearranged V(H)-D-J(H) genes. Thirty percent (3/10) of these IgG clones used V(H)HAR genes for the coding of heavy chain variable region with limited numbers of nucleic acid substitutions (>98% identity with their germline progenitors) although others demonstrated increased variation in nucleotide sequences (95-97% identity) when compared with germline V(H) genes. Analysis of the canonical binding site structure from the predicted amino acid sequences demonstrated that the majority of IgG clones (9/10) displayed a similar pattern of conserved configurations for their combining sites. CONCLUSIONS The change in IgM to IgG antibody production in the later stages of the humoral immune response of rats to hamster xenografts is associated with an IgM to IgG isotype switch and an increased production of antibodies of the IgG1 isotype. Rat anti-hamster IgG xenoantibodies continue to express the V(H)HAR family of V(H) genes, many in their original germline configuration, to encode antibody recognition of the hamster target antigens. There are, however, a majority of antibodies for which the V(H) genes express evidence of increased nucleic acid sequence variation when compared to currently available germline sequences. The source of this variation is not known but may represent the expression of as yet unidentified germline genes and/or the introduction of T cell-driven somatic mutations. Despite the appearance of this variation, the unusual level of conservation in key antigen binding sites within the V(H) region suggests the variation, independent of its origin, may have a limited influence on the restricted nature of the host antibody response to xenografts.