Epigenetic recording of stimulation history reveals BLIMP1-BACH2 balance in determining memory B cell fate upon recall challenge (ATAC-Seq II)

Long-term humoral immunity is partly maintained by memory B cells (MBCs). MBCs can be recalled to produce antibody-producing plasma cells (PCs) or form secondary germinal centers (GCs). The former process underlies markedly increased antigen-specific antibodies seen in the secondary response, while the latter process allows continuous somatic hypermutation and affinity maturation. MBC re-participation in the GC reaction is thought to be important for generating broadly neutralizing antibodies against highly mutating viruses such as influenza and HIV. However, how MBC recall is transcriptionally or epigenetically programmed to produce secondary PCs or GCs is not yet understood. Here we show that BACH2, a transcription factor required for GC formation and maintenance1,2, decreases across post-activation stages of B cells, from the naïve state to GC, memory, and the terminal PC state. In contrast, BLIMP-1, a transcription factor that promotes PC formation3,4 and is antagonistic to BACH2 (ref. 5), increases as B cells traverse the same course. The relative strength between BLIMP-1 and BACH2 progressively increases in favor of BLIMP-1 in GCs and MBCs through the primary response. MBCs of the isotype or subset identity that preferentially give rise to secondary GCs exhibit comparatively higher BACH2 but lower BLIMP-1 expression than those predisposed for PC formation. Using a tetracycline-controlled circuit implanted in MBCs, we show that skewing the BLIMP-1-BACH2 balance in favor of BACH2 markedly increases GC formation by otherwise PC-predisposed MBCs, whereas skewing the balance in favor of BLIMP-1 drives PC formation by GC-prone MBCs, indicating that the potential for GC re-participation or PC development rests in the relative strength of BLIMP-1 over BACH2 within individual MBCs. Underneath this relative BLIMP-1-over-BACH2 strength in naïve B cells, GCs, MBCs and PCs, we observe progressively increased accessibilities to chromatin loci that are specifically opened in PCs, particularly those that contain ISRE elements and are controlled by IRF-4. IRF-4 is universally upregulated in B cells when stimulated through the BCR, the CD40 receptor, or by innate stimuli. By analyzing time-stamped GC B cells, we demonstrate a progressive increase in chromatin accessibilities at PC-specific, IRF-4-controlled gene loci over time. Our results support a model of progressive IRF-4 imprinting in which the cumulative stimulation history of B cells is epigenetically recorded in an IRF-4-dependent manner, determines the relative strength between BLIMP-1 and BACH2 in individual MBCs, and thereby dictates their individual probabilities to develop into GCs or PCs upon re-stimulation. Overall design: ATAC-seq analysis of pulse-labeled tdTomato+ light zone GC B cells by tamoxifen adiministration from day6 to day8 and collected at day10 and day21 post NP-KLH immunization using mice of a S1pr2-creERT2;Rosa26-Ai14 genotype.

体液免疫的长期维持，部分依赖于记忆B细胞（memory B cells, MBCs）。记忆B细胞可被再次激活，分化为产生抗体的浆细胞（plasma cells, PCs），或是形成次级生发中心（secondary germinal centers, GCs）。前者是二次免疫应答中抗原特异性抗体水平显著升高的核心机制，后者则支持持续的体细胞超突变与亲和力成熟过程。记忆B细胞重新参与生发中心反应，被认为是针对流感、HIV等高度变异病毒产生广谱中和抗体的关键环节。然而，记忆B细胞的再次激活如何在转录或表观遗传层面被调控，以分化为次级浆细胞或生发中心，目前仍不明确。本研究发现，在B细胞激活后的各个阶段——从初始态（naive state）到生发中心、记忆B细胞，再至终末分化的浆细胞阶段——生发中心形成与维持所必需的转录因子BACH2的表达水平持续下调。与之相反，促进浆细胞分化且与BACH2存在拮抗作用的转录因子BLIMP-1，在B细胞经历上述分化进程时表达水平持续上调。在初次免疫应答过程中，生发中心与记忆B细胞内BLIMP-1与BACH2的表达相对强度逐渐向BLIMP-1倾斜。相较于倾向于分化为浆细胞的记忆B细胞亚群，那些更易生成次级生发中心的同型或亚型记忆B细胞，其BACH2表达水平更高，而BLIMP-1表达水平更低。本研究通过植入记忆B细胞的四环素调控系统发现，将BLIMP-1与BACH2的表达平衡向BACH2倾斜，可显著提升原本倾向于分化为浆细胞的记忆B细胞形成生发中心的能力；反之，将平衡向BLIMP-1倾斜，则会使倾向于生成生发中心的记忆B细胞转向浆细胞分化。这表明记忆B细胞的生发中心重新参与能力或浆细胞分化潜能，取决于单个记忆B细胞内BLIMP-1相对于BACH2的表达强度。我们观察到，在初始B细胞、生发中心B细胞、记忆B细胞与浆细胞中，BLIMP-1相对于BACH2的表达强度变化，伴随着浆细胞特异性开放的染色质位点的可及性持续升高，尤其是那些含有ISRE元件并受IRF-4调控的位点。当B细胞通过BCR、CD40受体或先天刺激被激活时，IRF-4的表达普遍上调。通过对标记有时间戳的生发中心B细胞进行分析，我们发现随着时间推移，浆细胞特异性、受IRF-4调控的基因位点的染色质可及性持续升高。本研究结果支持渐进式IRF-4印记模型：B细胞的累积刺激历史以IRF-4依赖的方式被表观遗传记录下来，进而决定单个记忆B细胞内BLIMP-1与BACH2的相对表达强度，最终决定其在再次受到刺激时分化为生发中心B细胞或是浆细胞的概率。实验整体设计：本研究采用S1pr2-creERT2;Rosa26-Ai14基因型小鼠，于NP-KLH免疫后第6天至第8天通过他莫昔芬给药对荧光素tdTomato阳性的生发中心明区B细胞进行脉冲标记，并分别于免疫后第10天与第21天收集样本，进行ATAC-seq分析。