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The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially localized multicellular ‘immunity hubs’ defined by expression of the T cell-attracting chemokines CXCL10/CXCL11and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens and found an association with beneficial response to PD-1 blockade. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcome. This hub is distinct from mature tertiary lymphoid structures and is enriched for stem-like TCF7+PD-1+CD8+T cells, activated CCR7+LAMP3+dendritic cells and CCL19+fibroblasts as well as chemokines that organize these cells. Within the stem-immunity hub, we find preferential interactions between CXCL10+macrophages and TCF7−CD8+T cells as well as between mature regulatory dendritic cells and TCF7+CD4+and regulatory T cells. These results provide a picture of the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.

Atherosclerosis is driven by multifaceted contributions of the immune system within the circulation and at vascular focal sites. However, specific characteristics of dysregulated immune cells within atherosclerotic lesions that lead to clinical events such as ischemic stroke or myocardial infarction are poorly understood. Here, using single-cell proteomic and transcriptomic analyses, we uncovered distinct features of both T cells and macrophages in carotid artery plaques of patients with clinically symptomatic disease (recent stroke or transient ischemic attack) compared to asymptomatic disease (no recent stroke). Plaques from symptomatic patients were characterized by a distinct subset of CD4+T cells and by T cells that were activated and differentiated. Moreover, some T cell subsets in these plaques presented markers of T cell exhaustion. Additionally, macrophages from these plaques contained alternatively activated phenotypes, including subsets associated with plaque vulnerability. In plaques from asymptomatic patients, T cells and macrophages were activated and displayed evidence of interleukin-1β signaling. The identification of specific features of innate and adaptive immune cells in plaques that are associated with cerebrovascular events may enable the design of more precisely tailored cardiovascular immunotherapies.

Arterioles and sinusoids of the bone marrow (BM) are accompanied by stromal cells that express nerve/glial antigen 2 (NG2) and leptin receptor (LepR), and constitute specialized niches that regulate quiescence and proliferation of haematopoietic stem cells (HSCs). However, how niche cells differentially regulate HSC functions remains unknown. Here, we show that the effects of cytokines regulating HSC functions are dependent on the producing cell sources. Deletion of chemokine C-X-C motif ligand 12 (Cxcl12) or stem cell factor (Scf) from all perivascular cells marked by nestin-GFP dramatically depleted BM HSCs. Selective Cxcl12 deletion from arteriolar NG2+cells, but not from sinusoidal LepR+cells, caused HSC reductions and altered HSC localization in BM. By contrast, deletion of Scf in LepR+cells, but not NG2+cells, led to reductions in BM HSC numbers. These results uncover distinct contributions of cytokines derived from perivascular cells in separate vascular niches to HSC maintenance.

Introduction:Inflammation is a key driver of atherosclerosis. The identification of new treatments and strategies targeting the specific inflammatory signaling that persists in optimally treated atherosclerotic patients remain an unmet clinical need.Hypothesis:The integration of time-of-flight mass cytometry (CyTOF), gene expression analysis, and drug repositioning computational approaches applied to pre-clinical validation in models of the disease using non-invasive imaging will help to identify drugs tailored to residual inflammatory signaling in patients with atherosclerosis.Methods:We developed a new systems biology pipeline to identify new therapeutic/indication pair that combines CyTOF and RNA sequencing (RNA-seq) data analysis of clinical samples from patients with carotid and coronary artery disease (CVD) (Fig.1A). This approach allowed the identification of new uses for existing compounds. The predicted anti-atherosclerotic effect was validated in ApoE-/- mice and in a rabbit model of atherosclerosis using 18F-Fluorodeoxyglucose (FDG) PET-MRI.Results:We found a specific inflammatory signature in CD14+monocytes stimulated with CVD plasma that was used to identify 50 small molecules predicted to reverse this signature. CyTOF screening of the top eight candidate compounds showed that Saracatinib—a phase 2a-ready Src/Abl inhibitor—blunted the inflammatory response found in CD14+monocytes (Fig.1B). The anti-atherosclerotic efficacy of Saracatinib was confirmed in ApoE-/- mice fed a high fat diet (Fig.1C). In the atherosclerotic rabbit model, Saracatinib reduced plaque inflammation measured as reduction of 18F-FDG uptake by PET-MRI (Fig. 1D).Conclusions:These data support the use of our integrated systems biology pipeline as a powerful approach to identify new immune signature specific to patients with atherosclerosis that can be leveraged to identify new indications for existing drugs to treat cardiovascular disease.