- Flow cytometric analysis for high-content, multi-parametric information in immune checkpoint studies
- Reliable estimation of checkpoint protein expression without blocking reagents
- Simple and fast sample analyses, while improving results
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™ Recombinant 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.
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 |
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 FcγRs, which is relevant for T cell function within the tumor microenvironment. 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 vivo. This issue can be resolved by using REAfinity™ Antibodies, which enable an accurate analysis of T cell subtypes without the need for FcR blocking.
