Tracing the development of single memory-lineage B cells in a highly defined immune response.
Tracing the development of single memory-lineage B cells in a highly defined immune response.
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DOI：
10.1084/jem.183.5.2053
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发表时间：
1996-05-01
期刊：
影响因子：
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通讯作者：
Wysocki LJ
中科院分区：
文献类型：
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作者：
Liu AH;Jena PK;Wysocki LJ
为了研究B淋巴细胞记忆的发育，我们鉴定并分离出表达一种高度明确的抗体可变区的脾脏B细胞，该可变区构成了对苯胂酸偶氮苯（Ars）的记忆性抗体应答中可重复且占主导地位的成分。在初次免疫应答期间，通过使用一种名为E4的特异性抗独特型抗体进行表面染色和流式细胞术实现了分离，E4可识别由一组V基因片段编码的这种规范的V区。分离出的E4⁺细胞显示出生发中心中心细胞的所有表型特征，包括表面Ig水平低、缺乏表面IgD、花生凝集素受体水平高以及突变的抗体V基因表达。初次免疫后7 - 8天在脾脏中首次检测到E4⁺ B细胞，在第10 - 13天达到数量峰值，并在第16天减少。令人惊讶的是，在峰值时，E4⁺细胞在所有脾细胞中仅占40,000个，其中一半不能结合Ars。根据这个数字，我们估计脾脏中Ars特异性记忆谱系细胞的总数在任何时候都不超过50,000个（0.1%），并且推测进入记忆库的细胞数量要少得多。通过巢式PCR扩增单个E4⁺细胞重排的V基因的染色体拷贝，并且在不克隆的情况下使用标准化条件直接对扩增产物进行测序，这些条件几乎不会显示出Taq聚合酶错误。对从单个小鼠分离出的E4⁺细胞的V基因序列分析证实了它们的规范性质，并揭示它们来自少数前体细胞。在平均每只小鼠中，E4⁺细胞群来自不到五个规范前体细胞。在几乎所有细胞分离物的V基因内都发现了体细胞突变。在第13天，相当一部分E4⁺细胞具有已知可增加对Ars的抗体亲和力的突变，这表明它们是至少一轮突变后抗原驱动选择的产物。然而，克隆相关细胞缺乏共同突变表明，记忆应答中典型的突变亚克隆的选择性扩增尚未发生。一半的E4⁺细胞不能结合Ars这一观察结果支持了这一点。总体而言，我们的结果表明，即使在初次免疫应答的相对早期阶段，当体细胞突变和克隆选择仍在进行时，记忆区室也是一个高度选择性的实体。如果生发中心是记忆B细胞的来源，我们的数据表明，B细胞记忆可能仅来自所有生发中心的一小部分。
To study the development of B lymphocyte memory, we identified and isolated splenic B cells expressing a highly defined antibody variable region that constitutes a reproducible and predominant component of the memory antibody response to p-azophenylarsonate (Ars). Isolation was achieved during the primary immune response by surface staining and flow cytometry using a specific anti-idiotypic antibody called E4, which recognizes this canonical V region, encoded by one set of V gene segments. The isolated E4+ cells displayed all of the phenotypic characteristics of germinal center centrocytes, including a low level of surface Ig, a lack of surface IgD, a high level of receptor for peanut agglutinin, and expression of mutated antibody V genes. E4+ B cells were first detected in the spleen 7-8 d after primary immunization, reached peak numbers from days 10-13, and waned by day 16. Surprisingly, at their peak, E4+ cells comprised only 40,000 of all splenocytes, and half of these failed to bind Ars. Using this number, we estimate the total number of Ars-specific memory-lineage cells in the spleen to be no more than 50,000 (0.1%) at any one time, and presumably far fewer that are committed to the memory pool. Chromosomal copies of rearranged V genes from single E4+ cells were amplified by nested PCR, and the amplified products were sequenced directly without cloning, using standardized conditions that disclose virtually no Taq polymerase errors. V gene sequence analyses of E4+ cells isolated from single mice confirmed their canonical nature and revealed that they were derived from few precursors. In the average mouse, the E4+ pool was derived from fewer than five canonical precursors. Somatic mutations were found within the V genes of almost all cell isolates. At day 13, a significant fraction of E4+ cells had mutations known to increase antibody affinity for Ars, suggesting they were products of at least one cycle of post-mutational antigen-driven selection. However, the lack of shared mutations by clonally related cells indicated that the selective expansion of mutant subclones typical of memory responses had not yet taken place. This was supported by the observation that half of the E4+ cells failed to bind Ars. Collectively, our results indicate that the memory compartment is a highly selected entity, even at relatively early stages of the primary immune response when somatic mutation and clonal selection are still in progress. If germinal centers are the source of memory B cells, our data suggest that B cell memory may be derived from only a small fraction of all germinal centers.