Li Wu

Plasmacytoid dendritic cells (pDCs) are a specialized subset of innate immune cells capable of sensing viral nucleic acids and rapidly producing large amounts of type I interferons (IFN‐I). However, excessive IFN‐I production can cause various immunopathogenic conditions. The capacity for IFN‐I production by pDCs is tightly regulated, yet the underlying mechanisms remain incompletely understood. Here, we describe two levels of negative regulatory mechanisms controlling IFN‐I production by pDCs. First, we identified SLC44A2 as a negative regulator of IFN‐I production. Slc44a2 was highly expressed in resting pDCs but significantly downregulated upon activation. Deficiency of Slc44a2 led to excessive IFN‐I production. Mechanistically, SLC44A2 may restrict IFN‐I production by exporting threonine, asparagine, and glutamine, amino acids that we found to be essential for IFN‐I production in pDCs. Second, we uncovered an IFN‐I‐dependent negative feedback mechanism controlling pDC egress. Excessive IFN‐I restrained pDC migration by downregulating CCR2 and CCR5. This feedback was generally observed during viral infections, autoimmune diseases, and in Slc44a2 ‐deficient mice. Taken together, these two regulatory mechanisms are essential for maintaining pDC homeostasis and preventing systemic overactivation of IFN‐I responses.

Transient RNA-labeling tools can reveal transcriptional dynamics with high temporal precision, yet most approaches remain largely confined to cell culture. Here, we report a 5-fluorouridine (5-FUd)-based transient transcriptomics method, 5FU-seq, that surpasses standard strategies by enabling higher metabolic incorporation, greater sensitivity, and robust detection across a broad spectrum of transcripts. Applying 5FU-seq to the murine brain uncovered stimulus-dependent transcriptional networks and revealed a dynamic regulatory program associated with Nrf2 regulation in the hippocampus during anesthesia-induced unconsciousness. We further engineered a transgenic mouse line for cell type-specific transient transcriptomics in situ, enabling simultaneous mapping of gene expression and active enhancer activity with high spatiotemporal resolution. Benchmarking in myeloid cells responding to inflammatory cues against FACS-based RNA-seq and ATAC-seq confirmed the robustness of this dual chemical-genetic toolkit. Together, this work establishes a broadly applicable platform for integrated transcriptomic and epigenetic profiling in complex tissues.

Background: Dendritic cells (DCs) play a predominant role in antitumor immunity. As professional antigen-presenting cells (APCs), DCs can be functionally matured by TLR2 ligand binding to enhance innate immune response and subsequent T cell-dependent adaptive immunity. DC function is often suppressed by the tumor microenvironment, while current TLR2 agonists exhibit suboptimal stability and diminished efficacy in vivo. Therefore, reactivation of suppressed DCs could be a promising strategy for enhancing the efficacy of cancer immunotherapy. Methods: To investigate the antitumor immunity induced by the novel Toll-like receptor 2 (TLR2) agonist SUP3 with better stability, we established murine melanoma, colon cancer and breast cancer tumor models. The hematopoietic growth factor Flt3L-dependent dendritic cells (FLDCs) were generated and utilized to examine their capacities of antigen processing and cross-presentation, and migration to the tumor-draining lymph nodes (TdLNs) in response to SUP3 treatment. To further improve the antitumor response of SUP3 by increasing the abundance and activation of DCs, Flt3L was administrated in vivo in combination with immune checkpoint blockade. Results: SUP3 exhibited stronger inhibition of tumor growth and metastasis than classical TLR2 agonist, Pam3. SUP3 could increase cDC1 antigen cross-presentation and TdLN migration, promoting the proliferation, activation and cytotoxicity of antigen-specific cytotoxic T lymphocytes (CTL). SUP3 promoted the intracellular accumulation of antigens and facilitated the process of antigen cross-presentation, the processe regulated by the small GTPase Rab7. SUP3 induced PD-L1 expression by DCs via an interferon-γ-independent pathway. The combination of SUP3 treatment with immune checkpoint blockade by anti-PD-L1 further improved the antitumor response. Moreover, Flt3L increased DC proliferation and infiltration into the tumor tissues that further enhanced the effects of antitumor immunotherapy when used in combination with SUP3 and anti-PD-L1. Conclusions: This study demonstrated that the modified and more stable TLR2 agonist SUP3 provided an optimal strategy for promoting antitumor immunity via activation of cDC1. SUP3 enhanced antigen cross-presentation by cDC1 and subsequent activation of CTLs. The antitumor effect was further enhanced when SUP3 and Flt3L synergized with PD-L1 blockade. Therefore, reactivation of suppressed DCs in tumor microenvironment would be a promising strategy for designing effective antitumor immunotherapy.

Group 3 innate lymphoid cells (ILC3s) are important for maintaining gut homeostasis. Upon stimulation, ILC3s can rapidly produce cytokines to protect against infections and colitis. However, the regulation of ILC3 quick response is still unclear. Here, we find that eIF6 aggregates with Nsun2 and cytokine mRNA in ILC3s at steady state, which inhibits the methyltransferase activity of Nsun2 and the nuclear export of cytokine mRNA, resulting in the nuclear reservation of cytokine mRNA. Upon stimulation, phosphorylated p38α phosphorylates eIF6, which in turn releases Nsun2 activity, and promotes the nuclear export of cytokine mRNA and rapid cytokine production. Genetic disruption of p38α, Nsun2, or eIF6 in ILC3s influences the mRNA nuclear export and protein expression of the protective cytokines, thus leading to increased susceptibility to colitis. Together, our data identify a crucial role of the p38α–eIF6–Nsun2 axis in regulating rapid ILC3 immune response at the posttranscriptional level, which is critical for gut homeostasis maintenance and protection against gut inflammation.