Immune Checkpoint Discovery With Flow Cytometry

Immune checkpoints regulate the immune system’s ability to distinguish self. Tumor cells use certain immune checkpoint pathways as a method of survival, particularly by inhibiting T cell or NK cell activity. Recent therapeutic developments target checkpoints as a method to increase immune surveillance in the tumor microenvironment.

Explore the immune cell–checkpoint interaction with flow cytometry. Quantitate cells that produce biomarkers of inflammation and immune response from a large catalog of antibodies and kits.

How it works: Inactivating immune cells in the tumor microenvironment

How it works: Inactivating immune cells in the tumor microenvironment

Immune checkpoint flow cytometry workflow

Isolate tumor sample

Isolate tumor sample

Dissociate tumor cells

Dissociate tumor cells

Examine cells for T Cell activity or checkpoint markers

Examine cells for T Cell activity or checkpoint markers

Known targets in immune checkpoint pathways

Antibodies are one tool to probe cellular expression of immune checkpoints. Examine the interaction of cell surface receptors and signal molecules to understand the interaction between cells.

Immune cell Effector cell Function
Cytotoxic T-lymphocyte– associated antigen 4 (CTLA-4) T cells Tumor cells use the CTLA-4 pathway to decrease T cell activation and ability to proliferate into memory T cells
Programed cell death protein (PD-1) T cells PD-1 expression on T cell indicates exhaustion and inability to perform immune responses
OX40 T cells Activating OX40 stimulates T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity
Glucocorticoid-induced TNFR-related (GITR) T cells GITR activation enhances cell reproduction and generate antitumor activity
Indoleamine-2,3 dioxygenase (IDO) Tregs Tumor cells can upregulate IDO activity through the breakdown tryptophan in order to suppress T cell function
CD73 Tregs Tumor cells use CD73 to suppress T cell activity with the production of adenosine
Lymphocyte-activation gene (LAG) T cells and Tregs LAG expression leads to T cell exhaustion and inhibits long-term immune response development
CD137 T cells and NK cells CD137 stimulates NK cells and T cells for anti- tumor response along with immune memory
SLAM family member 7 (SLAMF7) NK cells SLAM7 activation stimulates NK cells and other immune cells in the development of long-term immunity
Killer-cell immunoglobulin- like receptors (KIR) NK cells Tumor cells evade NK cells with KIR expression
Programed cell death protein ligand (PD-L1) Tumor cells PD-L1

Examining inflammation with flow cytometry

Isolate tumor sample

Mince and enzymatically digest organ or tissue overnight

Dilute sample

Dilute sample

Perform immunophenotyping analyses on the Attune NxT Flow Cytometer

Perform immunophenotyping analyses on the Attune NxT Flow Cytometer

Checkpoint inhibitors may induce off-target inflammation in other organs including the gut or heart. Dilute crude cell suspension from organs and tissues to examine millions of cells quickly with the Attune NxT Flow Cytometer. Look for common inflammation markers including prostaglandins, CXCL13, and IFN-γ.

Measuring T cell activity with flow cytometry

The purpose of checkpoint inhibitors is to prevent T cell exhaustion. Assess immune cell function by detecting cytokine production in tumor samples. T cells and NK cells produce interferon gamma (IFN-γ) to exert an immune response under inflammatory conditions or host defense. Quantitate CD8+ or CD4+ T cells that produce IFN-γ in tumor samples with multi-parametric flow cytometry.

Detecting IFN-γ in a subset of CD4+T-cells and NK cells

Detecting IFN-γ in a subset of CD4+T-cells and NK cells. Intracellular staining of stimulated mouse splenocytes for IFN-γ (clone XMG1.2) within a minor subset of CD4+ T cells and a larger subset of NK1.1+. Data obtained using the Attune NxT Flow Cytometer.