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Advancements in rational drug design over the past decades have consistently produced new cancer therapies, but such treatments are inevitably countered through an adaptive process that fosters therapy resistance. Malignant cells achieve drug resistance through intrinsic and acquired mechanisms, rooted in genetic and non-genetic determinants. In particular, recent work has highlighted the role of intrinsic cellular heterogeneity in the emergence of transient drug-tolerant persister cells that survive drug treatment, as well as non-genetically driven cell plasticity toward stable resistance. However, these models do not account for the role of dose and treatment duration as extrinsic forces in eliciting cancer cell adaptation. Here, we show that these two components together drive the resistance of ovarian cancer cells to targeted therapy along a trajectory of cellular adaptation, that we denote the ‘resistance continuum’. We report that gradual dose exposure and prolonged treatment promote a continuous increase in fitness, and show that this process is mediated by evolving transcriptional, epigenetic and genetic changes that promote multiple cell state transitions. The resistance continuum is underpinned by the assembly of gene expression programs and epigenetically reinforced stress response regulation. Using both in vivo and in vitro models, we found that this process involves widespread reprogramming of cell survival pathways, including interferon response, lineage reprogramming, metabolic rewiring and oxidative stress regulation. Together, the resistance continuum reveals the dynamic nature of cellular adaptation, and carries implications for cancer therapies, as initial exposure to lower doses primes cells over time for increased resistance to higher doses. Beyond cancer, such continuous adaptation exposes a basic aspect of cellular plasticity, which may also be deployed in other biological systems such as development, immune response and host-pathogen interactions.

Innate immune recognition of bacterial pathogens is a key determinant of the ensuing systemic response, and host or pathogen heterogeneity in this early interaction can impact the course of infection. To gain insight into host response heterogeneity, we investigate macrophage inflammatory dynamics using primary human macrophages infected with Group B Streptococcus. Transcriptomic analysis reveals discrete cellular states within responding macrophages, one of which consists of four sub-states, reflecting inflammatory activation. Infection with six additional bacterial species-Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Yersinia pseudotuberculosis, Shigella flexneri, and Salmonella enterica-recapitulates these states, though at different frequencies. We show that modulating the duration of infection and the presence of a toxin impacts inflammatory trajectory dynamics. We provide evidence for this trajectory in infected macrophages in an in vivo model of Staphylococcus aureus infection. Our cell-state analysis defines a framework for understanding inflammatory activation dynamics in response to bacterial infection.

Heterogeneity in host-pathogen interaction includes bacterial and host cell diversity, which together can lead to different infection outcomes. We report on macrophage inflammatory dynamics using primary human macrophages infected with Group B Streptococcus. We found a highly inflammatory state which included four conserved transcriptional programs – response to stimulus, activation of immune response, antigen presentation, and T cell activation – induced sequentially and punctuated by a ‘mid-infection transition’ between bacterial sensing and signaling. We validated these modules and their dynamic relationship in an in vivo mouse model of infection. Analysis of stimulation with 6 additional species – Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Yersinia pseudotuberculosis, Shigella flexneri and Salmonella enterica – reveals conservation of this program with distinct rates of module expression between species. Our work defines the hallmarks of host-pathogen interactions by identifying recurring properties of infection, providing insights into clinical diagnostics and therapeutic timing.

A large-scale, high-resolution cell atlas of gene expression and regulation in human embryos enables innovative investigation of development through multi‑organ and multi‑modal analysis. A multi-modal, pan-organ cell atlas for human development.

Objective— T cell immunoglobulin and mucin domain (Tim) proteins are expressed by numerous immune cells, recognize phosphatidylserine on apoptotic cells, and function as costimulators or coinhibitors. Tim-1 is expressed by activated T cells but is also found on dendritic cells and B cells. Tim-4, present on macrophages and dendritic cells, plays a critical role in apoptotic cell clearance, regulates the number of phosphatidylserine-expressing activated T cells, and is genetically associated with low low-density lipoprotein and triglyceride levels. Because these functions of Tim-1 and Tim-4 could affect atherosclerosis, their modulation has potential therapeutic value in cardiovascular disease. Approach and Results— ldlr −/− mice were fed a high-fat diet for 4 weeks while being treated with control (rat immunoglobulin G1) or anti-Tim-1 (3D10) or -Tim-4 (21H12) monoclonal antibodies that block phosphatidylserine recognition and phagocytosis. Both anti-Tim-1 and anti-Tim-4 treatments enhanced atherosclerosis by 45% compared with controls by impairment of efferocytosis and increasing aortic CD4 + T cells. Consistently, anti-Tim-4-treated mice showed increased percentages of activated T cells and late apoptotic cells in the circulation. Moreover, in vitro blockade of Tim-4 inhibited efferocytosis of oxidized low-density lipoprotein–induced apoptotic macrophages. Although anti-Tim-4 treatment increased T helper cell (Th)1 and Th2 responses, anti-Tim-1 induced Th2 responses but dramatically reduced the percentage of regulatory T cells. Finally, combined blockade of Tim-1 and Tim-4 increased atherosclerotic lesion size by 59%. Conclusions— Blockade of Tim-4 aggravates atherosclerosis likely by prevention of phagocytosis of phosphatidylserine-expressing apoptotic cells and activated T cells by Tim-4-expressing cells, whereas Tim-1-associated effects on atherosclerosis are related to changes in Th1/Th2 balance and reduced circulating regulatory T cells.

Background Interleukin‐23 ( IL ‐23) has been implicated in inflammatory and autoimmune diseases by skewing CD 4 + T helper cells towards a pathogenic Th17 phenotype. In this study we investigated the presence of IL ‐23 receptor ( IL ‐23R)‐expressing cells in the atherosclerotic aorta and evaluated the effect of IL ‐23R deficiency on atherosclerosis development in mice. Methods and Results We used heterozygous Ldlr −/− Il23r e GFP / WT knock‐in mice to identify IL ‐23R‐expressing cells by flow cytometry and homozygous Ldlr −/− Il23r e GFP / eGFP ( Ldlr −/− Il23r −/− ) mice to investigate the effect of lack of IL ‐23R in atherosclerosis. We demonstrate the presence of relatively rare IL ‐23R‐expressing cells in lymphoid tissue and aorta (≈0.1–1% IL 23R + cells of all CD 45 + leukocytes). After 10 weeks on a high‐fat diet, production of IL ‐17, but not interferon‐γ, by CD 4 + T cells and other lymphocytes was reduced in Ldlr −/− Il23r −/− compared with Ldlr −/− controls. However, Ldlr −/− and Ldlr −/− Il23r −/− mice had equivalent amounts of aortic sinus and descending aorta lesions. Adoptive transfer of IL ‐23R‐deficient CD 4 + T cells to lymphopenic Ldlr −/− Rag1 −/− resulted in dramatically reduced IL ‐17‐producing T cells but did not reduce atherosclerosis, compared with transfer of IL ‐23R‐sufficient CD 4 + T cells. Conclusions These data demonstrate that loss of IL ‐23R does not affect development of experimental atherosclerosis in LDL r‐deficient mice, despite a role for IL ‐23 in differentiation of IL ‐17‐producing T cells.

Dendritic cells (DCs) have been implicated as important regulators of innate and adaptive inflammation in many diseases, including atherosclerosis. However, the molecular mechanisms by which DCs mitigate or promote inflammatory pathogenesis are only partially understood. Previous studies have shown an important anti-inflammatory role for the transcription factor Krüppel-like factor 2 (KLF2) in regulating activation of various cell types that participate in atherosclerotic lesion development, including endothelial cells, macrophages, and T cells. We used a pan-DC, CD11c-specific cre-lox gene knockout mouse model to assess the role of KLF2 in DC activation, function, and control of inflammation in the context of hypercholesterolemia and atherosclerosis. We found that KLF2 deficiency enhanced surface expression of costimulatory molecules CD40 and CD86 in DCs and promoted increased T cell proliferation and apoptosis. Transplant of bone marrow from mice with KLF2-deficient DCs into Ldlr−/− mice aggravated atherosclerosis compared with control mice, most likely due to heightened vascular inflammation evidenced by increased DC presence within lesions, enhanced T cell activation and cytokine production, and increased cell death in atherosclerotic lesions. Taken together, these data indicate that KLF2 governs the degree of DC activation and hence the intensity of proatherogenic T cell responses.

Objective— Innate lymphoid cells (ILCs) are a newly discovered subset of immune cells that promote tissue homeostasis and protect against pathogens. ILCs produce cytokines also produced by T lymphocytes that have been shown to affect atherosclerosis, but the influence of ILCs on atherosclerosis has not been explored. Approach and Results— We demonstrate that CD25 + ILCs that produce type 2 cytokines (ILC2s) are present in the aorta of atherosclerotic immunodeficient ldlr −/− rag1 −/− mice. To investigate the role of ILCs in atherosclerosis, ldlr −/− rag1 −/− mice were concurrently fed an atherogenic diet and treated with either ILC-depleting anti-CD90.2 antibodies or IL-2/anti-IL-2 complexes that expand CD25 + ILCs. Lesion development was not affected by anti-CD90.2 treatment, but was reduced in IL-2/anti–IL-2-treated mice. These IL-2-treated mice had reduced very low–density lipoprotein cholesterol and increased triglycerides compared with controls and reduced apolipoprotein B100 gene expression in the liver. IL-2/anti-IL-2 treatment caused expansion of ILC2s in aorta and other tissues, elevated levels of IL-5, systemic eosinophila, and hepatic eosinophilic inflammation. Blockade of IL-5 reversed the IL-2 complex–induced eosinophilia but did not change lesion size. Conclusions— This study demonstrates that expansion of CD25-expressing ILCs by IL-2/anti-IL-2 complexes leads to a reduction in very low–density lipoprotein cholesterol and atherosclerosis. Global depletion of ILCs by anti-CD90.2 did not significantly affect lesion size indicating that different ILC subsets may have divergent effects on atherosclerosis.