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

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 GC B cells, PCs, YFP+ and RFPhigh MBCs generated at day7 post NP-OVA immunization by B1-8hi PA-GFP+ donor B cells from a BARBE mouse and naïve B cells from unimmunized mouse with the same genotype.

长期体液免疫的维持部分依赖于记忆B细胞（memory B cells, MBCs）。MBCs可被召回以产生抗体分泌浆细胞（antibody-producing plasma cells, PCs），或形成继发性生发中心（secondary germinal centers, GCs）。前者是二次应答中抗原特异性抗体显著升高的基础，而后者则允许持续的体细胞超突变（somatic hypermutation）与亲和力成熟（affinity maturation）。人们认为，MBC重新参与生发中心反应，对于针对流感、HIV这类高度变异病毒产生广泛中和抗体（broadly neutralizing antibodies）至关重要。然而，目前尚不清楚MBC的召回是如何在转录或表观遗传层面被编程，以产生继发性PC或GC的。

本研究发现，BACH2——一种参与生发中心形成与维持的转录因子（transcription factor）1,2——在B细胞活化后的各个阶段（从初始naive状态到生发中心、记忆细胞，终末浆细胞阶段）表达水平逐渐降低。与之相反，BLIMP-1——一种可促进PC形成3,4且与BACH2相互拮抗5的转录因子——在B细胞经历上述阶段时表达水平逐渐升高。在初次应答过程中，生发中心与MBC内BLIMP-1与BACH2的相对强度逐渐向BLIMP-1倾斜。

相较于易向PC分化的MBC亚群，那些优先分化为继发性GC的同型或亚型MBC，其BACH2表达水平相对更高，而BLIMP-1表达水平则更低。通过在MBC中植入四环素调控回路，我们证实：若将BLIMP-1与BACH2的平衡偏向BACH2，原本易向PC分化的MBC的GC形成能力将显著增强；反之，若将平衡偏向BLIMP-1，则易参与GC反应的MBC会转向PC形成。这表明，单个MBC内BLIMP-1相对于BACH2的相对强度，决定了其重新参与GC反应或向PC分化的潜能。

在初始naive B细胞、生发中心、MBC与PC中，这种BLIMP-1相对于BACH2的相对强度背后，我们观察到：PC特异性染色质位点的可及性（chromatin accessibility）逐渐升高，尤其是那些包含ISRE元件（ISRE elements）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在B细胞中普遍上调。通过对带时间标记的生发中心B细胞进行分析，我们证实：随着时间推移，PC特异性、受IRF-4调控的基因位点的染色质可及性逐渐升高。

我们的研究结果支持一种渐进式IRF-4印记模型：B细胞的累积刺激史以IRF-4依赖的方式被表观遗传记录，进而决定单个MBC内BLIMP-1与BACH2的相对强度，并最终决定其在再次受到刺激时分化为GC或PC的个体概率。

整体实验设计：对经NP-OVA免疫7天后，由BARBE小鼠来源的B1-8hi PA-GFP+供体B细胞所产生的生发中心B细胞、PC、YFP+及RFPhigh MBC，以及同基因型未免疫小鼠的初始naive B细胞进行ATAC-seq分析。