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Biology subjects

Tough, D. F.

Publications and source records attributed to Tough, D. F..

5 recordsLinked to original sources

Discovery of an orally available potent ER aminopeptidase 1 (ERAP1) inhibitor that enhances anti-tumor responses and limits inflammatory autoimmunity in vivo.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can regulate immune responses primarily by proteolytically processing peptides before loading and presentation on the cell surface by major histocompatibility class I molecules (MHC-I). ERAP1 activity can either reduce the immunogenicity of cancer cells by over-trimming cancer-associated antigenic peptides or contribute to autoimmunity by generating self-antigenic peptides. As a result, ERAP1 inhibition has emerged as a tractable approach for cancer immunotherapy and specific classes of autoimmunity. Here, we describe the discovery, after hit-to-lead optimization, of a potent and selective ERAP1 inhibitor based on the pyrrolidine 3-carboxylic acid scaffold that targets the regulatory allosteric site. The compound has favourable in vivo pharmacokinetics, including oral bioavailability, and can regulate the immunopeptidome of cancer cells and enhance cancer cell antigenicity in vivo in a dose-dependent manner, controlling tumor growth. In addition, when administered in the murine collagen-induced arthritis model, it does not induce any exacerbation of autoimmune responses but rather results in a dose-dependent therapeutic benefit. Our results demonstrate that ERAP1 inhibition can constitute a tractable approach to modulating immune responses for therapeutic applications, providing mechanistic insight and a valuable lead and in vivo tool for further drug development efforts and for interrogating ERAP1 biology.

immunology↗

Robust temporal map of human in vitro myelopoiesis using single-cell genomics

Myeloid cells have a central role in homeostasis and tissue defence. Characterising the current in vitro protocols of myelopoiesis is imperative for their use in research and immunotherapy as well as for understanding the early stages of myeloid differentiation in humans. Here, we profiled the transcriptome of more than 400k cells and generated a robust molecular map of the differentiation of human induced pluripotent stem cells (iPSC) into macrophages. By integrating our in vitro datasets with in vivo single-cell developmental atlases, we found that in vitro macrophage differentiation recapitulates features of in vivo yolk sac hematopoiesis, which happens prior to the appearance of definitive hematopoietic stem cells (HSC). During in vitro myelopoiesis, a wide range of myeloid cells are generated, including erythrocytes, mast cells and monocytes, suggesting that, during early human development, the HSC-independent immune wave gives rise to multiple myeloid cell lineages. We leveraged this model to characterize the transition of hemogenic endothelium into myeloid cells, uncovering poorly described myeloid progenitors and regulatory programs. Taking advantage of the variety of myeloid cells produced, we developed a new protocol to produce type 2 conventional dendritic cells (cDC2) in vitro. We found that the underlying regulatory networks coding for myeloid identity are conserved in vivo and in vitro. Using genetic engineering techniques, we validated the effects of key transcription factors important for cDC2 and macrophage identity and ontogeny. This roadmap of early myeloid differentiation will serve as an important resource for investigating the initial stages of hematopoiesis, which are largely unexplored in humans, and will open up new therapeutic opportunities.

developmental biology↗

Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140

Speckled 140 KDa (SP140) is a nuclear body protein, mainly expressed in immune cells, which contains multiple domains suggestive of an epigenetic reader function; namely a bromodomain, a PHD domain and a SAND domain. Single nucleotide polymorphisms and epigenetic modifications in the SP140 locus have been linked to autoimmune and inflammatory diseases including Crohns disease (CD). However, little is known about the cellular function of SP140; this is due in part to the fact that, unlike for other many other epigenetic proteins, no small molecule inhibitors have been available to investigate the biological role of SP140. We report the discovery of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in innate immune cells. We show that SP140 is highly expressed in CD68+ CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte differentiation into inflammatory macrophages and lipopolysaccharide (LPS)-induced inflammatory activation, whilst inducing the generation of CD206+ regulatory macrophages that mark anti-TNF remission induction in CD patients. ChIP-seq analyses revealed that SP140 preferentially occupies transcriptional start sites (TSS) in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduced SP140 binding and thereby expression of SP140-dependent downstream inflammatory genes. Notably, in CD14+ macrophages isolated from CD intestinal-mucosa, GSK761 inhibited the spontaneous expression of cytokines, including TNF. Together, this study identifies SP140 as a druggable epigenetic reader and therapeutic target for CD.

immunology↗

Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines

Naive CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. We used quantitative proteomics, bulk RNA-seq and single-cell RNA-seq of over 40,000 human naive and memory CD4+ T cells to generate a detailed map of cytokine-regulated gene expression programs. We demonstrated that cytokine response differs substantially between naive and memory T cells and showed that memory cells are unable to differentiate into the Th2 phenotype. Moreover, memory T cells acquire a Th17-like phenotype in response to iTreg polarization. At the single-cell level, we demonstrated that T cells form a continuum which progresses from naive to effector memory T cells. This continuum is accompanied by a gradual increase in the expression levels of chemokines and cytokines and thus represents an effectorness gradient. Finally, we found that T cell cytokine responses are determined by where the cells lie in the effectorness gradient and identified genes whose expression is controlled by cytokines in an effectorness-dependent manner. Our results shed light on the heterogeneity of T cells and their responses to cytokines, provide insight into immune disease inflammation and could inform drug development.

immunology↗

Chromatin activity at GWAS loci identifies T cell states driving complex immune diseases

Complex immune disease variants are enriched in active chromatin regions of T cells and macrophages. However, whether these variants function in specific cell states or stages of cell activation is unknown. We stimulated T cells and macrophages in the presence of thirteen different cytokine cocktails linked to immune diseases and profiled active enhancers and promoters together with regions of open chromatin. We observed that T cell activation induced major chromatin remodelling, while additional exposure to cytokines fine-tuned the magnitude of these changes. Therefore, we developed a new statistical method that accounts for subtle changes in chromatin landscape to identify SNP enrichment across cell states. Our results point towards the role of immune disease variants in early rather than late activation of memory CD4+ T cells, and with limited differences across polarizing cytokines. Furthermore, we demonstrate that inflammatory bowel disease variants are enriched in chromatin regions active in Th1 cells, while asthma variants overlap regions active in Th2 cells. We also show that Alzheimers disease variants are enriched in different macrophage cell states. Our results represent the first in-depth analysis of immune disease variants across a comprehensive panel of activation states of T cells and macrophages.

genetics↗