EZH2 mutations reprogram the immune response to yield a pre-malignant lymphoma niche [RNA-Seq]. EZH2 mutations reprogram the immune response to yield a pre-malignant lymphoma niche [RNA-Seq]

The mechanism through which lymphoma-associated EZH2 gain-of-function mutations disrupt the immune system to initiate malignant transformation is poorly understood. Conditional expression of mutant Ezh2 in germinal center (GC) B-cells provides a competitive advantage to activated B-cells in expanding the GC light zone (LZ) that is not caused by failure to differentiate and exit the GC. Instead, the LZ enlargement is explained by reduced apoptosis of Ezh2 mutant centrocytes, aberrant LZ proliferation, and failure of these centrocytes to re-enter the dark zone (DZ). Ezh2 mutant GCs fail to engage T-cells and to undergo clonal diversification, exhibiting a reduced IgV mutation frequency. Hence the dominant competitive advantage effect of Ezh2 mutation during early stages of transformation is to uncouple GC B-cells from T-cell help checkpoint, which allows GC B-cells entering the LZ to persist and expand regardless of their immunoglobulin status, reflecting the biology of low-grade follicular lymphomas. Overall design: Twenty-four samples total: centroblast Ezh2(Y641F)fl/wt;Cγ1-cre (four biological replicates), centroblast Cγ1-cre (four biological replicates), centrocytes Ezh2(Y641F)fl/wt;Cγ1-cre (four biological replicates), centrocytes Cγ1-cre (four biological replicates), naïve B-cells Ezh2(Y641F)fl/wt;Cγ1-cre (four biological replicates), naïve B-cells Cγ1-cre (four biological replicates) of 3 months-old mice immunized with SRBC and euthanized 8 days post-immunization

目前，淋巴瘤相关EZH2功能获得性突变（gain-of-function mutations）破坏免疫系统以启动恶性转化的具体机制尚未完全阐明。在生发中心（germinal center, GC）B细胞中条件性表达突变型Ezh2，可使活化B细胞在拓展生发中心亮区（light zone, LZ）的过程中获得竞争优势，该优势并非源于分化障碍及脱离生发中心。相反，亮区扩大的原因在于：Ezh2突变型中心细胞（centrocyte）的凋亡水平降低、亮区出现异常增殖，以及这些中心细胞无法重新进入暗区（dark zone, DZ）。携带Ezh2突变的生发中心无法激活T细胞，也不能发生克隆多样化，表现为IgV突变频率降低。因此，在恶性转化早期阶段，Ezh2突变的主要竞争优势效应是使生发中心B细胞脱离T细胞辅助检查点（checkpoint），这使得进入亮区的生发中心B细胞无需依赖自身免疫球蛋白状态即可存活并扩增，该特征与低度恶性滤泡性淋巴瘤的生物学行为相符。总体实验设计：本研究共纳入24份样本，所有样本均取自经绵羊红细胞（sheep red blood cell, SRBC）免疫、于免疫后8天实施安乐死的3月龄小鼠，具体分组如下：成中心细胞（centroblast）组：Ezh2(Y641F)fl/wt;Cγ1-cre（4份生物学重复）、Cγ1-cre（4份生物学重复）；中心细胞（centrocyte）组：Ezh2(Y641F)fl/wt;Cγ1-cre（4份生物学重复）、Cγ1-cre（4份生物学重复）；初始B细胞（naïve B-cells）组：Ezh2(Y641F)fl/wt;Cγ1-cre（4份生物学重复）、Cγ1-cre（4份生物学重复）