Immune checkpoint proteins and immune cell functionality

Discovering cytokines and checkpoint inhibitors

Targeting immune checkpoint protein interactions is one form of cancer immunotherapy that offers a novel way to attack tumor cells. The discovery of those druggable protein targets relies on an in-depth understanding of cell signaling pathways. Markers present during immune cell activation are often engaged by cancer cells to alter activation pathways and to ultimately attenuate the immune response. To help unveil those interactions, Thermo Fisher Scientific offers researchers the necessary resources for checkpoint protein identification, discovery, and characterization.

What are immune checkpoint inhibitor proteins?

Immune checkpoint proteins function to maintain cellular homeostasis by regulating immune cell activation pathways. Checkpoint proteins can be stimulatory or inhibitory, but those targeted for treatment of cancer have typically been inhibitory in nature. The programmed death protein 1 (PD-1), also known as CD279, is a receptor that is expressed at the cell surface of activated T cells, B cells, and macrophages that acts as a negative regulator of apoptosis. Inhibitory functions of PD-1 are critical for maintaining expansion of activated T cells as well as pro-inflammatory cytokines that would target the cancer cell. Expression of PD-1 on cells actively involved in the immune response is exploited by many cancers to attenuate those anti-tumor effects, as many cancer cells express the ligand of PD-1 (PD-L1). Cancerous cells bind PD-1, blocking the activation and expansion of T cell populations. Therefore, the disruption of this interaction using biotherapeutics has become a target for anti-cancer treatments.

Clinical data shows PD-L1–expressing cancers correlate with a reduction in survival, making it a valuable target for cancer immunotherapy. Studies using combination therapy with other checkpoint inhibitors, such as CTLA-4, have shown to be even more effective than either monotherapy. Mutations can genetically alter tumors over time, therefore therapies effective in some stages of cancer can become ineffective in others. As combination therapies evolve to become more personalized, effective attenuation of mutating cancers will result in prevention of immune evasion and reduced resistance to treatment. For this reason, biomarker discovery and analysis continue to be a critical component of checkpoint inhibitor immuno-oncology research.

Tools for characterizing the role of identified checkpoint proteins

Characterizing the role of identified checkpoint proteins requires tools that elucidate protein processing and maturation, where they are localized, and the kinetics of their protein–protein interactions. These studies are critical for mapping trafficking and their ultimate destination where they may mediate interactions with other cells, including immune cells and cancerous cells. See Figure 1 and Table 1 for an overview of some of the identified immune checkpoint pathway proteins. Analysis of activated cells and associated checkpoint molecules can be identified using the protein and cell analysis tools from Thermo Fisher Scientific.

Charts illustrating the interaction of multiple pairs of T cell and antigen-presenting cell checkpoint proteins

Figure 1. Multiple co-stimulatory and co-inhibitory receptor–ligand interactions between antigen-presenting cells (APCs) and T cells. T cell receptors (TCRs) detect antigens on the surface of APCs in the form of antigen-complexed major histocompatibility complexes (MHCs), and this antigen-specific recognition is necessary but not sufficient for an effective T cell response. For T cell activation or suppression, T cells must recognize their cognate antigens through TCRs and then respond to co-stimulatory (for activation) or co-inhibitory (for suppression) receptor–ligand interactions, examples of which are shown in this schematic. One important family of membrane-bound molecules that binds both co-stimulatory and co-inhibitory receptors is the B7-CD28 family shown in purple boxes; all of the B7 family members and their known ligands belong to the immunoglobulin superfamily. Another major category of signals arises from tumor necrosis factor (TNF) family members (shown in green boxes), which regulate the activation of T cells in response to cytokines.

Table 1. Mediators of immune checkpoint pathways.

Co-stimulatory target Co-stimulatory target Co-stimulatory target Function
CD28 A B7 receptor expressed on naïve T cells. Required for T cell activation. Results in the production of interleukins, like IL-2 and IL-6. PD1 PD-1 expression on T cell indicates exhaustion and inability to perform immune responses
ICOS (CD278) Expressed on activated T cells. Mediates cell to cell signaling and proliferation in the immune response. BTLA (CD272) Binding to HVEM negatively regulates T cell immune response. Recruits SHP-1 and SHP-2 and inhibits signaling cascades.
OX40 (CD134) Activating OX40 stimulates T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity. VISTA (B7-H5) Expressed highly on regulatory T cells and myeloid-derived suppressor cells. Blockade results in anti-cancer activity in some models.
4-1BB (CD137) CD137 stimulates NK cells and T cells for anti-tumor response along with immune memory. LIGHT (CD258) As opposed to BTLA, binding to HVEM induces a positive T cell immune response.
CD27 Tumor cells use CD73 to suppress T cell activity with the production of adenosine. CTLA-4 (CD152) Tumor cells use the CTLA-4 pathway to decrease T cell activation and ability to proliferate into memory T cells
CD40L (CD154) Expressed on activated T cells and T follicular helper cells. Promotes B cell maturation and has a role in humoral and cellular immunity. LAG3 LAG expression leads to T cell exhaustion and inhibits long-term immune response development
GITR (CD357) GITR activation enhances cell reproduction and generates antitumor activity. TIM3 Expression on IFN-gamma secreting T cells indicates T cell exhaustion.
CD30 A positive regulator of apoptosis expressed on activated T cells. Protects against autoimmunity.    
DR3 Mediates differentiation and apoptosis. Reduces inflammation by stimulating Tregs when engaged.    
DNAM (CD226) Mediates cellular adhesion to target cells, and has a role in T cell activation.    
CD96 Expressed on T cells and NK cells late in activation and promotes cell adhesion to target cells.    

Application Spotlight: Detection of soluble isoforms of immuno-oncology checkpoint proteins

This application note highlights the simultaneous detection of multiple biomarkers from liquid samples (plasma or serum), which allows correlation of their levels with progression of disease in longitudinal studies, and begins to associate biomarker levels with checkpoint blockade therapy response.

The Invitrogen ProcartaPlex Immuno-Oncology Checkpoint Panels target a set of selected molecules, including stimulatory factors that promote immunity and inhibitory factors to reduce immune activity and prevent autoimmunity. These panels allow the simultaneous detection of multiple soluble immune checkpoint proteins and help give a comprehensive picture of cancer immunity in a blood sample.

Learn how to detect soluble checkpoint proteins with ProcartaPlex Immuno-Oncology Checkpoint Panels

Simplified illustration of the process of checkpoint protein capture, labeling, and analysis using ProcartaPlex Immuno-Oncology Checkpoint Panels - step 1

Use small amount of plasma or serum

Simplified illustration of the process of checkpoint protein capture, labeling, and analysis using ProcartaPlex Immuno-Oncology Checkpoint Panels - step 2

Capture and detect proteins using ProcartaPlex panels

Simplified illustration of the process of checkpoint protein capture, labeling, and analysis using ProcartaPlex Immuno-Oncology Checkpoint Panels - step 3

Analyze results on the Luminex instrument; choose from FlexMap-3D (shown above), MAGPIX, or the Luminex 200 system

Protein and cell analysis products for immuno-oncology research