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细胞相关抗原的交叉呈递（cross-presentation）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独特的调控程序。针对这一科学问题开展研究后，我们发现仅Ly-6C^hiTremL4^-单核细胞，而非Ly-6C^hiTremL4^+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）的诱导下分化为Zbtb46+单核细胞衍生树突状细胞。然而，Ly-6C^hiTremL4^+单核细胞的发育则依赖于Nur77，定向分化为Ly-6C^loTremL4^+单核细胞。进一步研究显示，利用粒细胞-巨噬细胞集落刺激因子诱导单核细胞分化时，需添加白细胞介素4才能生成Zbtb46+单核细胞衍生树突状细胞，其交叉呈递效率可与CD24+经典树突状细胞相当，且该过程伴随Batf3与Irf4表达水平的显著上调。与经典树突状细胞不同，单核细胞衍生树突状细胞的交叉呈递仅需IRF4参与，而非Batf3。此外，Irf4基因敲除（Irf4^-/-）的单核细胞无法分化为Zbtb46+单核细胞衍生树突状细胞，反而会向巨噬细胞谱系发育。综上，经典树突状细胞与单核细胞衍生树突状细胞采用截然不同的转录程序调控交叉呈递过程，这一差异可能对应不同的抗原加工通路。上述发现或可影响基于单核细胞衍生树突状细胞的治疗性树突状细胞疫苗的开发。针对野生型（WT）与IRF4基因敲除小鼠的Sirp-a+树突状细胞的分析实验：从野生型与IRF4^-/- C57Bl/6小鼠体内分离脾脏淋巴细胞，随后通过FACSAria Fusion流式细胞分选仪将Sirp-a+树突状细胞分选至纯度>95%。