Optimization of the Irf8 +32 kb enhancer disrupts dendritic cell lineage segregation [scRNA-seq]

Autoactivation of lineage-determining transcription factors (TFs) mediates bistable expression to generate distinct cell phenotypes essential for complex body plans. Classical dendritic cells type 1 (cDC1) and type 2 (cDC2) provide non-redundant functions required for defense against distinct immune challenges. Interferon Regulatory Factor 8 (IRF8), the cDC1 lineage-determining TF, 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, has been tuned to possess low-affinity IRF8 binding sites. Incorporation of multiple high-affinity IRF8 binding sites into the Irf8 +32 kb enhancer induces erroneous IRF8 autoactivation in specified cDC2 progenitors, causing their redirection towards cDC1 and a novel hybrid DC subset with mixed lineage phenotypes. These developmental alterations critically impair both cDC1- and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of classical dendritic cells required for normal immune function. BM and spleens were harvested from two WT and two Irf8 +32H/H mice. The BM samples were depleted of lin+ cells expressing CD3e, CD8b, CD11b, CD19, B220, ly-6G, TER-119, and NK1.1. Each sample was stained with fluorochrome-conjugated antibodies and labeled with a unique Antibody Capture TotalSeq B antibody (Biolegend) before sorting for lin- Flt3+ Kitint-lo cells. Two samples from each genotype were pooled and resuspended in PBS with 0.04% BSA at a final concentration of approximately 1,000 cells/mL. The spleen samples were enriched for CD11c-expressing cells, similarly stained with fluorochrome-conjugated antibodies and labeled with a unique Antibody Capture TotalSeq B antibody (Biolegend), before further sort-purification for CD11c+ cells. All four samples were pooled and resuspended in PBS with 0.04% BSA at approximately 1,000 cells/mL. These were loaded on a 10× Genomics Chromium Single Cell Controller at GTAC@MGI at Washington University School of Medicine. Library preparation was performed using the 10× Genomics Next GEM Single Cell 3’ Reagents Kit v3. Libraries were sequenced using an Illumina Novaseq 6000 sequencer, producing 150 bp paired-end reads. Sequencing targeted depths of 1B reads for the gene expression (GEX) library and 100M reads for the hashtag oligo (HTO) library, respectively. The Cell Ranger pipeline v8.0.0 was used for aligning reads to the mm10 reference genome, single cell counting, barcode processing, and sample demultiplexing. scRNA-seq downstream analysis was conducted using Seurat v5.0.3 in R. After removing unwanted cells, UMI counts in each cell were normalized using log transformation with a scale factor of 10,000. For each dataset, 2,000 highly variable genes were identified and used for PCA. For the BM dataset, cell cycle phase scores were calculated, and the difference between G2M and S phase scores was regressed out. Dimensional reduction and clustering were performed using the FindNeighbors, RunUMAP, and FindClusters functions, utilizing the top 30 PCs. The clustering resolution was set at 0.6 for the BM dataset and 0.5 for the spleen dataset.

谱系决定性转录因子（lineage-determining transcription factors, TFs）的自激活可介导双稳态表达，进而产生复杂躯体模式必需的独特细胞表型。经典1型树突状细胞（classical dendritic cells type 1, cDC1）与经典2型树突状细胞（classical dendritic cells type 2, cDC2）可发挥针对不同免疫挑战的非冗余防御功能。干扰素调节因子8（Interferon Regulatory Factor 8, IRF8）作为cDC1的谱系决定性转录因子，可在cDC1前体中发生自激活以确立cDC1的谱系特性，但其在cDC2分化过程中的表达会被下调，具体调控机制尚未明确。本研究发现，负责IRF8自激活的Irf8 +32 kb增强子，经演化微调后仅具备低亲和力的IRF8结合位点。将多个高亲和力IRF8结合位点插入Irf8 +32 kb增强子，会在已定向分化的cDC2前体中诱导异常的IRF8自激活，导致这些前体重定向至cDC1谱系，并产生一类兼具混合谱系表型的新型混合DC亚群。此类发育异常会严重损害依赖cDC1与cDC2的两条免疫效应通路。综上，本研究结果证实了增强子亚优化在经典树突状细胞发育分化中的关键意义，该过程是维持机体正常免疫功能所必需的。

本研究从2只野生型（wild type, WT）小鼠与2只Irf8 +32H/H小鼠中采集骨髓（bone marrow, BM）与脾脏组织。骨髓样本中去除表达CD3e、CD8b、CD11b、CD19、B220、Ly-6G、TER-119及NK1.1的谱系阳性（lineage-positive, lin+）细胞。每份样本均用荧光素偶联抗体染色，并使用独特的抗体捕获TotalSeq B抗体（Biolegend）进行标记，随后分选得到lin- Flt3+ Kitint-lo细胞。将每种基因型的两份样本混合，重悬于含0.04%牛血清白蛋白（bovine serum albumin, BSA）的磷酸盐缓冲液（phosphate-buffered saline, PBS）中，终浓度约为1000个细胞/mL。脾脏样本先富集CD11c阳性细胞，同样用荧光素偶联抗体染色并标记独特的TotalSeq B抗体（Biolegend），随后进一步分选纯化得到CD11c+细胞。将四份样本混合并重悬于含0.04% BSA的PBS中，终浓度约为1000个细胞/mL。随后将样本置于华盛顿大学医学院GTAC@MGI的10× Genomics Chromium单细胞控制器上进行上机捕获。使用10× Genomics Next GEM Single Cell 3’ Reagents Kit v3完成文库制备。使用Illumina Novaseq 6000测序仪进行测序，获得150 bp双端读长序列。测序的靶向深度分别为：基因表达（gene expression, GEX）文库10亿条读长，标签寡核苷酸（hashtag oligo, HTO）文库1亿条读长。使用Cell Ranger分析流程v8.0.0将读段比对至mm10参考基因组，完成单细胞计数、条形码处理及样本解多重。单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）的下游分析使用R语言中的Seurat v5.0.3完成。去除无关细胞后，使用缩放因子为10000的对数转换对每个细胞的唯一分子标识符（unique molecular identifier, UMI）计数进行归一化。对每个数据集，筛选2000个高可变基因并用于主成分分析（Principal Component Analysis, PCA）。对于骨髓数据集，计算细胞周期阶段得分，并对G2M期与S期得分的差值进行回归校正。使用FindNeighbors、RunUMAP及FindClusters函数，基于前30个主成分进行降维与聚类。骨髓数据集的聚类分辨率设为0.6，脾脏数据集设为0.5。