Jackson S M, Harp N, Patel D, Henderson M, Roy N M, Courtney M A, Johnson A, Capra J D
Molecular Immunogenetics Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Scand J Immunol. 2007 Aug-Sep;66(2-3):249-60. doi: 10.1111/j.1365-3083.2007.01985.x.
We previously showed that IgH sequence alone minimally influenced germinal centre (GC) B-cell survival fate. As end-stage effector B cells are typically more mutated than founder GC B cells, we worked to develop an assay that would enrich for populations of GC B cells with progressively increasing numbers of somatic mutations, which could potentially be used as an indicator of positive selection. We targeted CD45 as it has been shown to influence activation-induced cytidine deaminase (AID) expression. In this study, anti-CD77 and anti-CD45RO (RO) were used to subdivide CD19(+)IgD(-)CD38(+)CD77(+) centroblasts (CB) and CD19(+)IgD(-)CD38(+)CD77(-) centrocytes (CC) into three contiguous RO fractions (RO(-), RO(+/-) and RO(+)) and assessed whether mutation frequency and characteristics associated with selection varied with respect to increasing RO expression. Here, we show that the average number of mutations per IgV(H)4 transcript increased concordantly with RO for CC, but not for CB. CC also exhibited an RO-associated increase in replacement mutations. Comparative analysis of clonally related sequences revealed that increased mutations were not due to the exclusive persistence of surface RO on highly mutated cells. RO-expressing CC and CB pools showed increased signs of activation (CD69(+)) and were enriched for surface Ig(+) cells. BCR-crosslinking induced a significant increase in surface RO on total tonsillar and GC B cells, which collectively suggests that the RO-associated increase in mutations is attributable, at least in part, to the cycling of cells that may have recently undergone BCR-mediated selection, or are potentially in developmental transition between CC and CB stages.
我们之前表明，仅免疫球蛋白重链（IgH）序列对生发中心（GC）B细胞存活命运的影响极小。由于终末效应B细胞通常比起始GC B细胞发生更多突变，我们致力于开发一种检测方法，以富集体细胞突变数量逐渐增加的GC B细胞群体，这些细胞可能用作阳性选择的指标。我们将目标锁定在CD45，因为已有研究表明它会影响激活诱导的胞苷脱氨酶（AID）表达。在本研究中，抗CD77和抗CD45RO（RO）被用于将CD19(+)IgD(-)CD38(+)CD77(+)中心母细胞（CB）和CD19(+)IgD(-)CD38(+)CD77(-)中心细胞（CC）细分为三个连续的RO亚群（RO(-)、RO(+/-)和RO(+)），并评估与选择相关的突变频率和特征是否随RO表达增加而变化。在此，我们表明，每个IgV(H)4转录本的平均突变数在CC中与RO呈一致增加，但在CB中并非如此。CC还表现出与RO相关的置换突变增加。对克隆相关序列的比较分析表明，突变增加并非由于高突变细胞上表面RO的排他性持续存在。表达RO的CC和CB群体显示出激活迹象（CD69(+)）增加，并且表面Ig(+)细胞富集。BCR交联诱导总扁桃体和GC B细胞表面RO显著增加，这共同表明与RO相关的突变增加至少部分归因于可能最近经历了BCR介导选择或可能处于CC和CB阶段之间发育转变的细胞循环。
