Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8 T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8 T cells and Tregs and represses the CD8 T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers.

MicroRNAs regulate gene expression posttranscriptionally and function within the cells in which they are transcribed. However, recent evidence suggests that microRNAs can be transferred between cells and mediate target gene repression. We find that endogenous miR-155 and miR-146a, two critical microRNAs that regulate inflammation, are released from dendritic cells within exosomes and are subsequently taken up by recipient dendritic cells. Following uptake, exogenous microRNAs mediate target gene repression and can reprogramme the cellular response to endotoxin, where exosome-delivered miR-155 enhances while miR-146a reduces inflammatory gene expression. We also find that miR-155 and miR-146a are present in exosomes and pass between immune cells in vivo, as well as demonstrate that exosomal miR-146a inhibits while miR-155 promotes endotoxin-induced inflammation in mice. Together, our findings provide strong evidence that endogenous microRNAs undergo a functional transfer between immune cells and constitute a mechanism of regulating the inflammatory response.

Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer.

To detect and repair damaged DNA, DNA damage response proteins need to overcome the barrier of condensed chromatin to gain access to DNA lesions1. ATP-dependent chromatin remodeling is one of the fundamental mechanisms used by cells to relax chromatin in DNA repair2–3. However, the mechanism mediating their recruitment to DNA lesions remains largely unknown. BRIT1 (also known as MCPH1) is an early DNA damage response protein that is mutated in human primary microcephaly4–8. We report here a previously unknown function of BRIT1 as a regulator of ATP-dependent chromatin remodeling complex SWI/SNF in DNA repair. Upon DNA damage, BRIT1 increases its interaction with SWI/SNF through the ATM/ATR-dependent phosphorylation on the BAF170 subunit. This increase of binding affinity provides a means by which SWI/SNF can be specifically recruited to and maintained at DNA lesions. Loss of BRIT1 causes impaired chromatin relaxation owing to reduced association of SWI/SNF with chromatin. This explains the decreased recruitment of repair proteins to DNA lesions and reduced efficiency of repair in BRIT1-deficient cells, resulting in impaired survival from DNA damage. Our findings, therefore, identify BRIT1 as a key molecule that links chromatin remodeling with DNA damage response in the control of DNA repair, and its dysfunction contributes to human disease.

SUMMARY An increased understanding of antitumor immunity is necessary to improve cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs), miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits IFNγ responses by T cells and reduced solid tumor growth in vivo. Using a novel double knockout (DKO) mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155−/− mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4+ and CD8+ T cell-mediated antitumor immunity, and demonstrates the dominant nature of miR-155 during its promotion of immune responses.

Summary Expression of PTEN tumor suppressor is frequently lost in breast cancer in the absence of mutation or promoter methylation through as yet undetermined mechanisms. In this study, we demonstrated that the Rak tyrosine kinase physically interacts with PTEN and phosphorylates PTEN on Tyr 336. The knockdown of Rak enhanced the binding of PTEN to its E3 ligase, NEDD4-1, and promoted PTEN polyubiquitination, leading to PTEN protein degradation. Notably, ectopic expression of Rak effectively suppressed breast cancer cell proliferation, invasion, and colony formation in vitro and tumor growth in vivo. Furthermore, Rak knockdown was sufficient to transform normal mammary epithelial cells. Therefore, Rak acts as a bona fide tumor suppressor gene through the mechanism of regulating PTEN protein stability and function.

Summary Chronic inflammation is a contributing factor to most life shortening human diseases. However, the molecular and cellular mechanisms that sustain chronic inflammatory responses remain poorly understood making it difficult to treat this deleterious condition. Using a mouse model of age-dependent inflammation that results from a deficiency in miR-146a, we demonstrate that miR-155 contributed to the progressive inflammatory disease that emerged as Mir146a−/− mice grew older. Upon analyzing lymphocytes from inflamed versus healthy middle-aged mice we found elevated numbers of T follicular helper (Tfh) cells, germinal center (GC) B cells and autoantibodies, all occurring in a miR-155-dependent manner. Further, Cd4-cre Mir155fl/fl mice were generated and demonstrated that miR-155 functions in T cells, in addition to its established role in B cells, to promote humoral immunity in a variety of contexts. Taken together, our study discovers that miR-146a and miR-155 counter-regulate Tfh cell development that drives aberrant GC reactions during chronic inflammation.

Th17 cells are central to the pathogenesis of autoimmune disease, and recently specific noncoding microRNAs (miRNAs) have been shown to regulate their development. However, it remains unclear if miRNAs are also involved in modulating Th17 cell effector functions. Consequently, we examined the role of miR-155 in differentiated Th17 cells during their induction of Experimental Autoimmune Encephalomyelitis (EAE). Using adoptive transfer experiments, we found that highly purified, MOG antigen-specific Th17 cells lacking miR-155 were defective in their capacity to cause EAE. Gene expression profiling of purified miR-155−/− IL-17F+ Th17 cells identified a subset of effector genes that are dependent upon miR-155 for their proper expression through a mechanism involving repression of the transcription factor Ets1. Among the genes reduced in the absence of miR-155 was IL-23R, resulting in miR-155−/− Th17 cells being hypo-responsive to IL-23. Taken together, our study demonstrates a critical role for miR-155 in Th17 cells as they unleash autoimmune inflammation, and finds that this occurs through a signaling network involving miR-155, Ets1 and the clinically relevant IL-23-IL-23R pathway.