Abstract
Multi-color flow cytometry is a standard laboratory protocol, which is regularly used to analyze tumor-infiltrating immune cell subsets. Oncolytic herpes simplex virus has shown promise in treating various types of cancers, including deadly glioblastoma. Intracranial/intratumoral treatment with oncolytic herpes simplex virus expressing interleukin 12, i.e., immunovirotherapy results in induction of anti-tumor immune responses and tumor infiltration of a variety of immune cells. Multi-color flow cytometry is employed to characterize immune cells in the tumor microenvironment. Here, we describe a step-by-step 11-color flow cytometry protocol to stain tumor-infiltrating immune cells in glioblastoma following oncolytic herpes virotherapy. We also describe a method to identify HSV-1 glycoprotein-B-specific CD8+ T cells using fluorochrome-conjugated major histocompatibility complex multimers. The multimers carry major histocompatibility peptide complexes, which have the ability to interact and bind to T cell receptors present on the surface of T cells; allowing identification of T cells (e.g., CD8+) reactive to a desired antigen. This multimer staining can be used in conjunction with the multi-parametric flow cytometry staining. Brain tumor quadrants are harvested, minced, enzymatically digested, immune cells are isolated by positive selection, single cells are counted and blocked for Fc receptors, cells are incubated with dye and/or color-conjugated antibodies, and flow cytrometry is performed using a BD LSRII flow cytometer. The protocol described herein is also applicable to stain immune cells in other mouse and human tumors or in any desired tissues.
Highlights
Cancer immunotherapy using immune checkpoint blockade has recently revolutionized the treatment of various cancers [1,2]
Brain tumor inflammation and infiltrating immune cells can be analyzed by multi-color flow cytometry (FC), a commonly used and integral laboratory staining technique in cancer immunology and immunotherapy [15,16]
We describe a method to identify HSV-1 glycoprotein-B-specific CD8+ T cells using fluorochrome-conjugated major histocompatibility complex (MHC) multimers [18]
Summary
Cancer immunotherapy using immune checkpoint blockade has recently revolutionized the treatment of various cancers [1,2]. Multi-parametric flow cytometry staining procedure for analyzing tumor-infiltrating immune cells following oncolytic herpes simplex virus immunotherapy in intracranial glioblastoma. Brain tumor inflammation and infiltrating immune cells can be analyzed by multi-color flow cytometry (FC), a commonly used and integral laboratory staining technique in cancer immunology and immunotherapy [15,16]. We describe a simple step-by-step protocol describing sample preparation and 11-color staining of tumor-infiltrating immune cells in a mouse GBM brain tumor model after treatment with oHSVbased immunotherapy. Surface antibodies (with listed vendor concentrations) APC/Cy7 anti-mouse CD3ε (0.2 mg/ml) PerCP/Cyanine 5.5 anti-mouse CD4 (0.2 mg/ml) Brilliant Violet 510TM anti-mouse CD8a (0.2 mg/ml) Brilliant Violet 605TM anti-mouse/human CD11b (0.2 mg/ml) Alexa Fluor® 700 anti-mouse Ly-6G/Ly-6C (0.5 mg/ml) FITC anti-mouse CD69 (0.5 mg/ml) PE/Cy7 anti-mouse CD152 (0.2 mg/ml) Brilliant Violet 421TM anti-mouse CD274 PE/Dazzle 594 anti-mouse CD279 (0.2 mg/ml) Surface antibody cocktail/sample. From our personal experience, the same listed amount was successfully used multiple times to stain up to 5 × 106 cells in 100 μl volume
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