Highlights d scRNA-seq analyses highlight conserved myeloid subsets in human and murine CRC d Two distinct TAM subsets show inflammatory and angiogenic signatures, respectively d Two distinct TAM subsets show differential sensitivity to CSF1R blockade d Anti-CD40 activates specific cDC1s and expands Th1-like and CD8 + memory T cells

SUMMARY Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and post-transcriptional mechanisms contributing to the regulatory network. We examined pro-inflammatory gene regulation in activated macrophages by performing RNA-Seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the sub-cellular locations of coding and non-coding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of co-expressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.

SUMMARY We describe a broad mechanistic framework for the transcriptional induction of mammalian primary response genes by Toll-like receptors and other stimuli. One major class of primary response genes is characterized by CpG-island promoters, which facilitate promiscuous induction from constitutively active chromatin without a requirement for SWI/SNF nucleosome remodeling complexes. The low nucleosome occupancy at promoters in this class can be attributed to the assembly of CpG islands into unstable nucleosomes, which may lead to SWI/SNF independence. Another major class consists of non-CpG-island promoters that assemble into stable nucleosomes, resulting in SWI/SNF dependence and a requirement for transcription factors that promote selective nucleosome remodeling. Some stimuli, including serum and tumor necrosis factor-α, exhibit a strong bias toward activation of SWI/SNF-independent CpG-island genes. In contrast, interferon-β is strongly biased toward SWI/SNF-dependent non-CpG-island genes. By activating a diverse set of transcription factors, Toll-like-receptors induce both classes and others for an optimal response to microbial pathogens.

Summary Regulatory T cells (Tregs) play a pivotal role in the inhibition of anti-tumor immune responses. Understanding the mechanisms governing Treg homeostasis may therefore be important for development of effective tumor immunotherapy. We have recently demonstrated a key role for the canonical nuclear factor κB (NF-κB) subunits, p65 and c-Rel, in Treg identity and function. In this report, we show that NF-κB c-Rel ablation specifically impairs the generation and maintenance of the activated Treg (aTreg) subset, which is known to be enriched at sites of tumors. Using mouse models, we demonstrate that melanoma growth is drastically reduced in mice lacking c-Rel, but not p65, in Tregs. Moreover, chemical inhibition of c-Rel function delayed melanoma growth by impairing aTreg-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. Our studies therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.

Summary Both conventional T (Tconv) cells and regulatory T (Treg) cells are activated through ligation of the T cell receptor (TCR) complex, leading to the induction of the transcription factor NF-κB. In Tconv cells, NF-κB regulates expression of genes essential for T cell activation, proliferation and function. However the role of NF-κB in Treg function remains unclear. We conditionally deleted canonical NF-κB members p65 and c-Rel in developing and mature Treg cells and found they have unique but partially redundant roles. c-Rel was critical for thymic Treg development while p65 was essential for mature Treg identity and maintenance of immune tolerance. Transcriptome and NF-κB p65 binding analyses demonstrated a lineage specific, NF-κB-dependent transcriptional program, enabled by enhanced chromatin accessibility. These dual roles of canonical NF-κB in Tconv and Treg cells highlight the functional plasticity of the NF-κB signaling pathway and underscores the need for more selective strategies to therapeutically target NF-κB.