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Aims: Atherosclerosis is characterized by the abundant infiltration of myeloid cells starting at early stages of disease. Myeloid cells are key players in vascular immunity during atherogenesis. However, the subsets of vascular myeloid cells have eluded resolution due to shared marker expression and atypical heterogeneity in vascular tissues. We applied the high-dimensionality of mass cytometry to the study of myeloid cell subsets in atherosclerosis., Methods and Results: Apolipoprotein E-deficient (ApoE-/-) mice were fed a chow or a high fat (western) diet for 12 weeks. Single-cell aortic preparations were probed with a panel of 35 metal-conjugated antibodies using cytometry by time of flight (CyTOF). Clustering of marker expression on live CD45+ cells from the aortas of ApoE-/- mice identified 13 broad populations of leucocytes. Monocyte, macrophage, type 1 and type 2 conventional dendritic cell (cDC1 and cDC2), plasmacytoid dendritic cell (pDC), neutrophil, eosinophil, B cell, CD4+ and CD8+ T cell, γδ T cell, natural killer (NK) cell, and innate lymphoid cell (ILC) populations accounted for approximately 95% of the live CD45+ aortic cells. Automated clustering algorithms applied to the Lin-CD11blo-hi cells revealed 20 clusters of myeloid cells. Comparison between chow and high fat fed animals revealed increases in monocytes (both Ly6C+ and Ly6C-), pDC, and a CD11c+ macrophage subset with high fat feeding. Concomitantly, the proportions of CD206+ CD169+ subsets of macrophages were significantly reduced as were cDC2., Conclusions: A CyTOF-based comprehensive mapping of the immune cell subsets within atherosclerotic aortas from ApoE-/- mice offers tools for myeloid cell discrimination within the vascular compartment and it reveals that high fat feeding skews the myeloid cell repertoire toward inflammatory monocyte-macrophage populations rather than resident macrophage phenotypes and cDC2 during atherogenesis.

Objective To examine the migratory properties of cytokine-activated T (Tck) cells. Methods Tck cells were generated by culture of peripheral blood T cells in the presence of interleukin-6 (IL-6), tumor necrosis factor α, and IL-2. Changes in cell surface phenotype were analyzed by flow cytometry. Chemotactic responsiveness was measured using in vitro chemotaxis assays and transendothelial migration through human umbilical vein endothelial cell monolayers. Levels of vascular cell adhesion molecule 1 (VCAM-1) were measured by sandwich enzyme-linked immunosorbent assay. Results Cytokine stimulation up-regulated the expression of chemokine receptors and integrins on Tck cells, including CXCR4, very late activation antigen 4 (VLA-4), and lymphocyte function-associated antigen 1. Increased expression of CXCR4 and VLA-4 integrin resulted in concentration-dependent chemotaxis to their ligands, stromal cell-derived factor 1 (SDF-1) and VCAM-1, which could be selectively blocked using a specific CXCR4 inhibitor and antibodies against VLA-4. Increased expression of VLA-4 also resulted in increased transendothelial migration of Tck cells, which could be abrogated using blocking antibodies against VLA-4. Tck cells also showed an increased chemotactic response to rheumatoid arthritis (RA) fibroblast-like synoviocytes cultured in vitro, which could be blocked using inhibitors against VLA-4 and CXCR4. Conclusion The activated phenotype of Tck cells results in increased migratory responsiveness to SDF-1 and soluble VCAM-1, which are among the chemokines and proteins found elevated in the RA synovial joint environment. Cytokine-dependent activation may contribute to RA pathogenicity by promoting T cell recruitment to and retention in the joint, perpetuating the inflammatory cascade in RA. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic cancer has the worst survival of any human cancer. Checkpoint blockade has not yielded much benefit in pancreatic cancer. We explored immune cell phenotypes with this disease to identify new targets for checkpoint blockade therapy. We created a checkpoint-focused panel to analyse immune cells from eight pancreatic cancer patients. This showed us the majority of T-cells are senescent. Further T-cell investigation demonstrated the majority of cytotoxic T-cells have intermediate to low PD1 expression suggesting why PD1 may not work as a pancreatic cancer therapy strategy. Our data has also highlighted a regulatory T-cell population which is highly activated and can mediate immunosuppression. The checkpoints that are highly expressed on these cells are TIGIT, ICOS and CD39, suggesting inhibition of these may be a viable therapeutic strategy. Furthermore, we showed that Tregs were retained amongst the fibroblast stroma of the tumour. Our work suggests there are key checkpoints on Tregs that may help guide therapeutic strategies in pancreatic cancer. Pancreatic cancer has one of the worst prognoses of any human malignancy and leukocyte infiltration is a major prognostic marker of the disease. As current immunotherapies confer negligible survival benefits, there is a need to better characterise leukocytes in pancreatic cancer to identify better therapeutic strategies. In this study, we analysed 32 human pancreatic cancer patients from two independent cohorts. A multi-parameter mass-cytometry analysis was performed on 32,000 T-cells from eight patients. Single-cell RNA sequencing dataset analysis was performed on a cohort of 24 patients. Multiplex immunohistochemistry imaging and spatial analysis were performed to map immune infiltration into the tumour microenvironment. Regulatory T-cell populations demonstrated highly immunosuppressive states with high TIGIT, ICOS and CD39 expression. CD8+ T-cells were found to be either in senescence or an exhausted state. The exhausted CD8 T-cells had low PD-1 expression but high TIGIT and CD39 expression. These findings were corroborated in an independent pancreatic cancer single-cell RNA dataset. These data suggest that T-cells are major players in the suppressive microenvironment of pancreatic cancer. Our work identifies multiple novel therapeutic targets that should form the basis for rational design of a new generation of clinical trials in pancreatic ductal adenocarcinoma. [ABSTRACT FROM AUTHOR]