Dictionary of immune responses to cytokines at single-cell resolution.

Cytokines mediate cell-cell communication in the immune system and represent important therapeutic targets1-3. A myriad of studies have highlighted their central role in immune function4-13, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap, we created the Immune Dictionary, a compendium of single-cell transcriptomic profiles of more than 17 immune cell types in response to each of 86 cytokines (>1,400 cytokine-cell type combinations) in mouse lymph nodes in vivo. A cytokine-centric view of the dictionary revealed that most cytokines induce highly cell-type-specific responses. For example, the inflammatory cytokine interleukin-1ß induces distinct gene programmes in almost every cell type. A cell-type-centric view of the dictionary identified more than 66 cytokine-driven cellular polarization states across immune cell types, including previously uncharacterized states such as an interleukin-18-induced polyfunctional natural killer cell state. Based on this dictionary, we developed companion software, Immune Response Enrichment Analysis, for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumours following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell-cell communication networks in any immune response.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

Treatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

Macrophages from Rosa26-Integrated Cas9-Expressing C57BL/6J Mice Have a Putative TRIF-Mediated Defect in the TLR-3/4 Signaling.

In this study, we report that the TLR4 ligand, LPS, and TLR3 ligand polyinosinic:polycytidylic acid failed to activate IRF3 or STAT1 in bone marrow-derived macrophages (BMMs) isolated from two independently generated lines of Rosa26-integrated Cas9-expressing C57BL/6J (B6) mice. RNA-sequencing analysis reveals that hundreds to thousands of genes including IFN-stimulated genes were differentially expressed in BMMs from these Cas9 strains compared with B6 upon LPS stimulation. Furthermore, the NF-κB signaling axis and TRIF-mediated necroptosis were also strongly reduced in response to LPS and polyinosinic:polycytidylic acid. In contrast, there were no defects in the responses of BMMs to ligands of the RIG-I, STING, TLR2, TLR9, and IFN receptors. Defects in TLR3 and TLR4 signaling were observed in mice with the B6 but not 129 background, and when Cas9 was integrated at the Rosa26 but not H11 locus. However, integration at the Rosa26 site, CAG promoter-driven Cas9 or eGFP were not individually sufficient to cause the defect. Taken together, the results of this study suggest a putative TRIF-mediated defect in TLR-3/4 signaling in BMMs from commercially available and widely used B6-Cas9-expressing mice.

Differentiation of exhausted CD8+ T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory.

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8+ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory.

Genome-wide enhancer maps link risk variants to disease genes.

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease1. Many of the underlying causal variants may affect enhancers2,3, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types4. Here we apply this ABC model to create enhancer-gene maps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.

Progressive immune dysfunction with advancing disease stage in renal cell carcinoma.

The tumor immune microenvironment plays a critical role in cancer progression and response to immunotherapy in clear cell renal cell carcinoma (ccRCC), yet the composition and phenotypic states of immune cells in this tumor are incompletely characterized. We performed single-cell RNA and T cell receptor sequencing on 164,722 individual cells from tumor and adjacent non-tumor tissue in patients with ccRCC across disease stages: early, locally advanced, and advanced/metastatic. Terminally exhausted CD8+ T cells were enriched in metastatic disease and were restricted in T cell receptor diversity. Within the myeloid compartment, pro-inflammatory macrophages were decreased, and suppressive M2-like macrophages were increased in advanced disease. Terminally exhausted CD8+ T cells and M2-like macrophages co-occurred in advanced disease and expressed ligands and receptors that support T cell dysfunction and M2-like polarization. This immune dysfunction circuit is associated with a worse prognosis in external cohorts and identifies potentially targetable immune inhibitory pathways in ccRCC.

Lysyl oxidase: A colorectal cancer biomarker of lung and hepatic metastasis.

BACKGROUND: Colorectal cancer (CRC) is a common and lethal disease in which distant metastasis remains the primary cause of death. Paradoxical roles of LOX have been reported in CRC, and the intracellular function of LOX has also recently been determined. Correlations of LOX expression and its intracellular localization with clinicopathological features in CRC patients remain largely unknown. The aim of the present study was to explore the potential roles of LOX in CRC. METHODS: LOX messenger RNA expression was assayed by quantitative PCR in eight paired normal mucosa and tumor tissues. Immunohistochemistry was conducted using tissue arrays to investigate LOX expression in 201 CRC patients. Regulation of LOX by YAP and TEAD4 was explored by YAP or TEAD4 short hairpin RNA interference in a LoVo cell line. RESULTS: LOX messenger RNA expression was elevated in some CRC specimens, and LOX nuclear localization was detected in CRC tumor tissues. LOX nuclear localization was found to correlate with lung/hepatic metastasis, elevated serum carcinoembryonic antigen concentration, and mucinous tumor type (P < 0.05). Nuclear LOX expression was found to be associated with poor overall and disease-free survival (P < 0.05), and postoperative lung/hepatic metastasis (P < 0.05). Knockdown of YAP or TEAD4 induced downregulation of LOX expression. CONCLUSIONS: LOX nuclear localization was significantly associated with poor survival in patients with CRC. Nuclear LOX expression was correlated with synchronous or postoperative lung/hepatic metastasis. LOX may prove to be a potential target gene of YAP and TEAD4.

Monitoring T cell-dendritic cell interactions in vivo by intercellular enzymatic labelling.

Interactions between different cell types are essential for multiple biological processes, including immunity, embryonic development and neuronal signalling. Although the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy, this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells for downstream analysis. Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor-ligand interactions between cells in living mice, by generating a signal that can subsequently be detected ex vivo by flow cytometry. We call this approach for the labelling of 'kiss-and-run' interactions between immune cells 'Labelling Immune Partnerships by SorTagging Intercellular Contacts' (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells and CD4+ T cells during T-cell priming in vivo occur in two distinct modalities: an early, cognate stage, during which CD40-CD40L interactions occur specifically between T cells and antigen-loaded dendritic cells; and a later, non-cognate stage during which these interactions no longer require prior engagement of the T-cell receptor. Therefore, LIPSTIC enables the direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor-ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology requiring quantification of intercellular communication.

Prognostic value of IRF-2 expression in colorectal cancer.

Interferon regulatory factor 2 (IRF-2) is known to play a pivotal role in the development and progression of several malignancies. As a crucial member of interferon regulatory factor family, the association between the expression of IRF-2 and clinical prognostic significance has not been fully explored in colorectal cancer (CRC). The purpose of our study was to investigate the expression profile of IRF-2 in CRC and to examine its association with clinical features. The expression levels of IRF-2 in 18 paired CRC and non-cancerous colorectal tissues were measured by quantitative real-time PCR (qRT-PCR) and those in 4 paired samples by Western blotting. The results showed a significant increase in IRF-2 mRNA expression and protein expression in CRC tissues compared to those in paired normal tissues. Besides, high expression of IRF-2 was significantly associated with distant metastasis (P = 0.041) and preoperative serum CEA level (P = 0.045). Kaplan-Meier survival analysis showed that patients with high expression of IRF-2 had a significantly worse overall survival than those with low expression of IRF-2 (P = 0.006). Further multivariate analysis indicated that IRF-2 and TNM stage were independent prognostic factors for overall survival in patients with CRC. Our study primarily suggests IRF-2 as a potential prognostic biomarker in CRC.

Anal malignant proliferative trichilemmoma: report of a rare case with review of literature.

Trichilemmoma is a rare type of benign cutaneous neoplasm, which derives from outer sheath of hair follicle. It barely develops malignant progression and has rarely been reported in anal cancer. In this article, we report a case of a 73-year-old woman who presented to the outer-patient department with complaints of a ruptured and longstanding anal phyma. All the appearances were atypical. Blood routine examination showed that neutrophilic granulocyte percentage was elevated and suggest it was a simple inflammation response. No evidence of malignancy was detected upon the laboratory examinations. Then we performed an abscess incision drainage for the patient. A few days later, the biopsy pathological report suggested the specimen is a malignant proliferative trichilemmoma. We decided to perform a wide local excision instead of an extended radical operation in order to preserve anus. After the surgery, we chose not to give chemoradio-treatment for fear of side effects and complications. Careful follow-up indicates that peri-anal malignant proliferative trichilemmoma may have a good prognosis and our treatment is a good choice for the patients with this tumor. Because of the low occurrence rate of anal cancer, especially malignant trichilemmoma, any clinical manifestation and experience are valuable. On one hand, our case may help to take the consideration of the diagnosis of malignant trichilemmoma in case of longtime-suffered peri-anal mass, on the other hand it propose a different treatment method from other anal cancers for clinical doctors.