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1.
STAR Protoc ; 3(2): 101444, 2022 06 17.
Article in English | MEDLINE | ID: mdl-35677615

ABSTRACT

Here, we present a protocol for flow cytometry analysis of endothelial cells (ECs) and CD8+ T cells in murine tumor models, at baseline and after cancer immunotherapy with anti-PD-1/anti-CTLA-4 antibodies. We provide gating strategies for identification of specific cell subsets including ECs from tumor-associated high endothelial venules (TA-HEVs), stem-like, and terminally exhausted CD8+ T cells. This protocol represents a valuable tool for the analysis of rare subsets of tumor ECs and CD8+ T cells with critical roles in antitumor immunity. For complete details on the use and execution of this protocol, please refer to Asrir et al. (2022).


Subject(s)
Neoplasms , Programmed Cell Death 1 Receptor , Animals , CD8-Positive T-Lymphocytes , Endothelial Cells , Flow Cytometry , Immunotherapy/methods , Mice , Neoplasms/therapy
2.
Cancer Cell ; 40(3): 318-334.e9, 2022 03 14.
Article in English | MEDLINE | ID: mdl-35120598

ABSTRACT

Recruitment of lymphocytes into tumors is critical for anti-tumor immunity and efficacious immunotherapy. We show in murine models that tumor-associated high endothelial venules (TA-HEVs) are major sites of lymphocyte entry into tumors at baseline and upon treatment with anti-PD-1/anti-CTLA-4 immune checkpoint blockade (ICB). TA-HEV endothelial cells (TA-HECs) derive from post-capillary venules, co-express MECA-79+ HEV sialomucins and E/P-selectins, and are associated with homing and infiltration into tumors of various T cell subsets. Intravital microscopy further shows that TA-HEVs are the main sites of lymphocyte arrest and extravasation into ICB-treated tumors. Increasing TA-HEC frequency and maturation increases the proportion of tumor-infiltrating stem-like CD8+ T cells, and ameliorates ICB efficacy. Analysis of tumor biopsies from 93 patients with metastatic melanoma reveals that TA-HEVs are predictive of better response and survival upon treatment with anti-PD-1/anti-CTLA-4 combination. These studies provide critical insights into the mechanisms governing lymphocyte trafficking in cancer immunity and immunotherapy.


Subject(s)
Melanoma , Programmed Cell Death 1 Receptor , Animals , CD8-Positive T-Lymphocytes , CTLA-4 Antigen , Endothelial Cells , Humans , Immunologic Factors , Immunotherapy , Lymphocytes, Tumor-Infiltrating , Melanoma/pathology , Mice , T-Lymphocyte Subsets , Venules/pathology
3.
Cell Rep ; 26(11): 3116-3131.e5, 2019 03 12.
Article in English | MEDLINE | ID: mdl-30865898

ABSTRACT

High-endothelial venules (HEVs) are specialized blood vessels allowing recirculation of naive lymphocytes through lymphoid organs. Here, using full-length, single-cell RNA sequencing, RNA fluorescence in situ hybridization (FISH), flow cytometry, and immunohistofluorescence, we reveal the heterogeneity of HEVs in adult mouse peripheral lymph nodes (PLNs) under conditions of homeostasis, antigenic stimulation, and after inhibition of lymphotoxin-ß receptor (LTßR) signaling. We demonstrate that HEV endothelial cells are in an activated state during homeostasis, and we identify the genes characteristic of the differentiated HEV phenotype. We show that LTßR signaling regulates many HEV genes and pathways in resting PLNs and that immune stimulation induces a global and temporary inflammatory phenotype in HEVs without compromising their ability to recruit naive lymphocytes. Most importantly, we uncover differences in the regulation of genes controlling lymphocyte trafficking, Glycam1, Fut7, Gcnt1, Chst4, B3gnt3, and Ccl21a, that have implications for HEV function and regulation in health and disease.


Subject(s)
Cell Movement/genetics , Endothelium, Vascular/metabolism , Homeostasis , Lymphocytes/physiology , Transcriptome , Venules/metabolism , Animals , Chemokine CCL21/genetics , Chemokine CCL21/metabolism , Endothelium, Vascular/cytology , Female , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Genetic Heterogeneity , Lymph Nodes/cytology , Lymphocytes/metabolism , Lymphotoxin beta Receptor/genetics , Lymphotoxin beta Receptor/metabolism , Mice , Mice, Inbred C57BL , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Single-Cell Analysis , Sulfotransferases/genetics , Sulfotransferases/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Venules/cytology , Carbohydrate Sulfotransferases
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