Microarray expression data from WT and IRF4 KO Sirp-a+ DCs

Cross-presentation of cell-associated antigens is carried out by classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs), but whether a similar or distinct program exists for this process is unknown. In examining this issue, we discovered that only Ly-6ChiTremL4– monocytes, but not Ly-6ChiTremL4+ monocytes, can differentiate into Zbtb46+ Mo-DCs in response to GM-CSF and IL-4. However, Ly-6ChiTremL4+ monocytes were committed to Nur77-dependent development of Ly-6CloTremL4+ monocytes. Further, differentiation of monocytes with GM-CSF required addition of IL-4 to generate Zbtb46+ Mo-DCs that cross-presented as efficiently as CD24+ cDCs, which was accompanied by increased Batf3 and Irf4 expression. Unlike cDCs, Mo-DCs required only IRF4, and not Batf3, for cross-presentation. Further, Irf4–/– monocytes failed to develop into Zbtb46+ Mo-DCs, and instead developed into macrophages. Thus, cDCs and Mo-DCs use distinct transcriptional programs for cross-presentation that may drive different antigen-processing pathways. These differences may influence development of therapeutic DC vaccines based on Mo-DCs. Analysis of Sirp-a+ DCs from WT and IRF4KO DCs Splenocytes were harvested from WT and IRF4-/- C57Bl/6 mice and Sirp-a+ DCs were sorted to >95% purity on a FacsAria Fusion

交叉呈递（cross-presentation）细胞相关抗原的功能由经典树突状细胞（classical DCs, cDCs）与单核细胞衍生树突状细胞（monocyte-derived DCs, Mo-DCs）介导，但目前尚不明确该过程是否存在相似或差异化的调控程序。针对该问题开展研究后，我们发现仅Ly-6C高表达TremL4阴性（Ly-6ChiTremL4⁻）单核细胞，而非Ly-6C高表达TremL4阳性（Ly-6ChiTremL4+）单核细胞，可在粒细胞-巨噬细胞集落刺激因子（GM-CSF）与白细胞介素4（IL-4）诱导下分化为Zbtb46阳性单核细胞衍生树突状细胞。然而，Ly-6ChiTremL4+单核细胞会定向参与依赖于核受体77（Nur77）的Ly-6C低表达TremL4阳性（Ly-6CloTremL4+）单核细胞的发育过程。进一步研究表明，利用GM-CSF诱导单核细胞分化时，需联合添加IL-4才能生成交叉呈递效率与CD24阳性cDCs相当的Zbtb46+ Mo-DCs，该过程伴随碱性亮氨酸拉链ATF样转录因子3（Batf3）与干扰素调节因子4（Irf4）的表达水平显著上调。与cDCs不同，Mo-DCs的交叉呈递仅依赖于Irf4，而非Batf3。此外，Irf4敲除（Irf4–/–）的单核细胞无法分化为Zbtb46+ Mo-DCs，而是向巨噬细胞方向发育。综上，cDCs与Mo-DCs采用截然不同的转录程序介导交叉呈递，这可能对应不同的抗原加工通路。上述差异或可影响基于Mo-DCs的治疗性树突状细胞疫苗的开发。针对野生型（WT）与IRF4敲除（IRF4KO）小鼠的信号调节蛋白α阳性（Sirp-α+）树突状细胞的分析：从野生型及Irf4–/– C57BL/6小鼠中收获脾细胞，通过FACSAria Fusion流式细胞分选仪将Sirp-α阳性DCs分选至纯度>95%。