Yen-Rei A. Yu

Abstract Purpose: T-cell dysfunction is a hallmark of glioblastoma (GBM). Although anergy and tolerance have been well characterized, T-cell exhaustion remains relatively unexplored. Exhaustion, characterized in part by the upregulation of multiple immune checkpoints, is a known contributor to failures amid immune checkpoint blockade, a strategy that has lacked success thus far in GBM. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating human and murine GBM. Experimental Design: Tumor-infiltrating and peripheral blood lymphocytes (TILs and PBLs) were isolated from patients with GBM. Levels of exhaustion-associated inhibitory receptors and poststimulation levels of the cytokines IFNγ, TNFα, and IL2 were assessed by flow cytometry. T-cell receptor Vβ chain expansion was also assessed in TILs and PBLs. Similar analysis was extended to TILs isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers. Results: Our data reveal that GBM elicits a particularly severe T-cell exhaustion signature among infiltrating T cells characterized by: (1) prominent upregulation of multiple immune checkpoints; (2) stereotyped T-cell transcriptional programs matching classical virus-induced exhaustion; and (3) notable T-cell hyporesponsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations. Conclusions: Distinct cancers possess similarly distinct mechanisms for exhausting T cells. The poor TIL function and severe exhaustion observed in GBM highlight the need to better understand this tumor-imposed mode of T-cell dysfunction in order to formulate effective immunotherapeutic strategies targeting GBM. Clin Cancer Res; 24(17); 4175–86. ©2018 AACR. See related commentary by Jackson and Lim, p. 4059

Abstract Obesity and elevated circulating cholesterol are risk factors for breast cancer recurrence, while the use of statins, cholesterol biosynthesis inhibitors widely used for treating hypercholesterolemia, is associated with improved disease-free survival. Here, we show that cholesterol mediates the metastatic effects of a high-fat diet via its oxysterol metabolite, 27-hydroxycholesterol. Ablation or inhibition of CYP27A1, the enzyme responsible for the rate-limiting step in 27-hydroxycholesterol biosynthesis, significantly reduces metastasis in relevant animal models of cancer. The robust effects of 27-hydroxycholesterol on metastasis requires myeloid immune cell function, and it was found that this oxysterol increases the number of polymorphonuclear-neutrophils and γδ-T cells at distal metastatic sites. The pro-metastatic actions of 27-hydroxycholesterol requires both polymorphonuclear-neutrophils and γδ-T cells, and 27-hydroxycholesterol treatment results in a decreased number of cytotoxic CD8 + T lymphocytes. Therefore, through its actions on γδ-T cells and polymorphonuclear-neutrophils, 27-hydroxycholesterol functions as a biochemical mediator of the metastatic effects of hypercholesterolemia.

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.

Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry-based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206(+)) subsets and other CD206(-) leukocytes. The CD206(-) cells include: (1) three monocyte (CD14(+)) subsets, (2) CD11c(+) dendritic cells (CD14(-), CD11c(+), HLA-DR(+)), (3) plasmacytoid dendritic cells (CD14(-), CD11c(-), HLA-DR(+), CD123(+)), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169(+) and CD169(-) macrophages. In lung tissue, CD169(-) macrophages were a prominent cell type. Using confocal microscopy, CD169(+) macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169(-) macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.

OBJECTIVE: Dendritic cells (DCs) have recently been found in atherosclerosis-predisposed regions of arteries and have been proposed to be causal in atherosclerosis. The chemokine receptor CX3CR1 is associated with arterial injury and atherosclerosis. We sought to determine whether a link exists between arterial DC accumulation, CX3CR1, and atherosclerosis. METHODS AND RESULTS: Mouse aortas were isolated and subjected to en face immunofluorescence analysis. We found that DCs were located predominantly in the intimal regions of arterial branch points and curvatures. Consistent with the increased accumulation of intimal DCs in aged and ApoE-/- aortas compared with young WT aortas (P=0.004 and 0.05, respectively), the incidence of atherosclerosis was 88.9% for aged WT and 100% for ApoE-/- mice compared with 0% for young WT mice. CX3CR1 was expressed on intimal DCs and DC numbers were decreased in CX3CR1-deficient aortas of young, aged, and ApoE-/- mice (P=0.0008, 0.013, and 0.0099). The reduced DC accumulation in CX3CR1-deficiency was also correlated with decreased atherosclerosis in these animals. CONCLUSIONS: The accumulation of intimal DC increases in aged and ApoE-/- aortas and correlates with the generation of atherosclerosis. CX3CR1-deficiency impairs the accumulation of DC in the aortic wall and markedly reduces the atherosclerotic burden.