Immune checkpoint analysis | Flow cytometry assays

Immune checkpoints are important regulators that maintain immune homeostasis and prevent autoimmunity. They consist of both stimulatory and inhibitory pathways that are important for maintaining self-tolerance as well as regulating the type, magnitude, and duration of immune responses. Under normal circumstances, immune checkpoint molecules maintain homeostasis and fight against disease conditions; however, some of them also play a significant role in promoting tumor progression.

Therefore, targeting immune checkpoints to treat cancer is extensively studied in the field of immuno-oncology. Although the immune checkpoint targets that have been studied the most are inhibitory pathways, identification of novel stimulatory pathways also encourage scientists to develop drugs targeting those pathways.

To study immune checkpoints and compounds that have the potential to target these molecules, flow cytometric analysis yields high-content and multi-parametric information. Even though it is still considered a state-of-the-art technique, traditional, hybridoma-derived antibody clones used for flow cytometric evaluation show a tendency of unspecific binding to immune cells via naturally expressed Fcγ receptors (FcγR). This can lead to an inaccurate and biased estimation of immune checkpoint protein expression.

Blocking reagents, including commercially available immunoglobulins, are often used to avoid FcγR-mediated background binding. This adds additional complexity to the analysis, as there is no defined consensus on the best titer and type of blocking reagent to be used for complex tissues like tumors. In addition, blocking reagents increase staining time and hinder scale up or automation of multiple sample analyses.

Our recombinantly generated REAfinity™ Antibodies provide several benefits over hybridoma-derived antibody clones, for example, a specifically mutated human IgG1 Fc region that abolishes their binding to FcγRs. This enables more reliable and reproducible flow cytometric analyses of immune cells.

Find flow cytometry data on immune checkpoints using REAfinity Antibodies below.

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Flow cytometry basics

Antibodies

Immune checkpoint targets

Immune checkpoints and their ligands that are currently considered for drug development
(click on a specificity to find product information on available REAfinity Antibody clones)

Immune checkpoint	Mode	Key function	Source	Respective ligands
CD152 (CTLA-4)	Inhibitory	Downregulation of immune responses	T cells	CD80 (B7-1), CD86 (B7-2)
CD279 (PD1)	Inhibitory	Downregulation of immune responses and promoting self-tolerance by suppressing T cell inflammatory activity	Activated T cells	CD274 (PD-L1), CD273 (PD-L2)
CD223 (LAG-3)	Inhibitory	Downregulation of T cell function to prevent tissue damage and autoimmunity	T cells	MHC-II
TIM-3	Inhibitory	Promoting immunosuppression by inducing expansion of myeloid-derived suppressor cells (MDSCs)	T cells	GAL9
TIGIT	Inhibitory	Indirectly increasing the release of immunregulatory cytokines (e.g., IL-10), and thereby preventing maturation of dendritic cells (DCs)	T, NK cells	CD155 (PVR), CD112 (Nectin-2)
CD276 (B7-H3)	Inhibitory	Inhibition of T cell activation, proliferation, and cytokine production	APCs, NK, B, and T cells
CD73	Inhibitory	Co-signal for T cell activation	Most tissues
CD272 (BTLA)	Inhibitory	Blocking B and T cell activation, proliferation, and cytokine production	Lymphocytes	CD270 (HVEM)
TGF-β	Inhibitory	Suppression of cytotoxic T cells, which can promote cancer cell proliferation, invasion, and metastases during tumorigenesis (functional switch known as the TGF-β paradox)	Leukocytes, macrophages	TGF-βR
KIR	Inhibitory	Promoting self-tolerance by dampening lymphocyte activation, cytotoxic activity, and cytokine release	NK cells	MHC-I
CD47	Inhibitory	Inhibition of macrophages and other myeloid cells	All human cells	CD172a (SIRPα)
CD134 (OX40)	Co-stimulatory	Activation, potentiation, proliferation, and survival of T cells and modulation of NK cell function	T cells	CD252 (OX40L)
CD357 (GITR)	Co-stimulatory	Treg activation, leukocyte adhesion and migration	T, NK cells	GITRL
CD278 (ICOS)	Co-stimulatory	Co-stimulation of proliferation and cytokine production	CD4⁺ T cells	CD275 (B7-H2)
CD137 (4-1BB)	Co-stimulatory	Stimulation of immune cell proliferation and activation, particularly of T and NK cells	T, NK cells	CD137L (4-1BBL)
CD27	Co-stimulatory	Activation and differentiation of T cells into effector and memory cells, and boosting B cells	T cells	CD70
CD40	Co-stimulatory	Inducing dendritic cell maturation and thereby triggering T cell activation and differentiation	T cells	CD154 (CD40L)
CD28	Co-stimulatory	Stimulation of T cell expansion	T cells	CD80 (B7-1), CD86 (B7-2)
IDO	Other	Promoting the differentiation and activation of Treg cells and decreasing the activity of CD8⁺ T cells leading to an immunosuppressed environment	Tumor cells
TLR	Other	Recognition of pathogens and control of immune response	DCs, Macrophages
CD25 (IL2R)	Other	Promoting the differentiation of T cells	T cells
IL-10	Other	Inhibiting secretion of pro-inflammatory cytokines as well as expression of MHC and co-stimulatory molecules, leading to inhibition of T cell function	Monocytes

Flow cytometry data using REAfinity™ Recombinant Antibodies

Background staining with hybridoma-derived antibodies leads to biased phenotypical characterization of critical tumor-infiltrating T cell subpopulations.

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Precise analysis of T cell populations without the need for FcR blocking

It has been shown that chronically-activated T cells, such as tumor-reactive T cells, can express FcγRs⁴, which is relevant for T cell function within the tumor microenvironment1. Our results show that FcγR expression on T cells lead to unspecific binding of hybridoma-derived antibodies, resulting in false characterization of T cell subtypes.

Addition of a FcR blocking reagent reduced the amount of background binding; however, FcγR blocking is often suboptimal, as it has been shown to affect T cell function in vivo1. This issue can be resolved by using REAfinity™ Antibodies, which enable an accurate analysis of T cell subtypes without the need for FcR blocking.