Autoactivation of lineage-determining transcription factors mediates bistable expression, generating distinct cell phenotypes essential for complex body plans. Classical type 1 dendritic cell (cDC1) and type 2 dendritic cell (cDC2) subsets provide nonredundant functions for defense against distinct immune challenges. Interferon regulatory factor 8 (IRF8), the cDC1 lineage-determining transcription factor, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32-kb enhancer, responsible for IRF8 autoactivation, is naturally suboptimized with low-affinity IRF8 binding sites. Introducing multiple high-affinity IRF8 sites into the Irf8 +32-kb enhancer causes a gain-of-function effect, leading to erroneous IRF8 autoactivation in specified cDC2 progenitors, redirecting them toward cDC1 and a novel hybrid DC subset with mixed-lineage phenotypes. Further, this also causes a loss-of-function effect, reducing Irf8 expression in cDC1s. These developmental alterations critically impair both cDC1-dependent and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of cDCs required for normal immune function.

谱系决定转录因子的自动激活介导双稳态表达，产生对复杂身体计划至关重要的不同细胞表型。经典 1 型树突状细胞 （cDC1） 和 2 型树突状细胞 （cDC2） 亚群为防御不同的免疫挑战提供了非冗余功能。干扰素调节因子 8 （IRF8） 是 cDC1 谱系决定转录因子，在 cDC1 祖细胞中发生自激活以建立 cDC1 身份，但其表达在 cDC2 分化过程中通过未知机制下调。这项研究表明，负责 IRF8 自动激活的 Irf8 +32-kb 增强子自然地与低亲和力 IRF8 结合位点进行了次优化。将多个高亲和力 IRF8 位点引入 Irf8 +32-kb 增强子会导致功能获得效应，导致特定 cDC2 祖细胞中错误的 IRF8 自动激活，将它们重定向到 cDC1 和具有混合谱系表型的新型杂交 DC 亚群。此外，这也会导致功能丧失效应，降低 cDC1 中 Irf8 的表达。这些发育改变严重损害了 cDC1 依赖性和 cDC2 依赖性免疫组。总的来说，我们的研究结果强调了增强子次优化在正常免疫功能所需的 cDC 发育分离中的重要性。