Mini-review: Immune Checkpoints

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Role of Immune Checkpoints in Immunity and Cancer: Mini-Review

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Immune checkpoints identified: purpose, mechanism and detection

In order to keep the immune response in check, thereby avoiding chronic inflammation and autoimmunity, several immune signaling pathways are employed that stimulate or inhibit the immune response to maintain homeostasis. These signaling pathways are designed to allow optimal immune response to foreign antigens but prevent self-harm from an over reacting immune response. These signals that regulate an immune response are known as immune checkpoints. For detailed information on immune checkpoints, read our mini-review entitled “The Role of Immune Checkpoints in Immunity and Cancer”. Table 1 lists examples of positive and negative immune checkpoint receptors and their ligands.

Table 1: Immune checkpoint receptors and ligands

CTLA-4, cytotoxic T lymphocyte antigen 4; GITR, glucocorticoid-induced tumor necrosis factor receptor; GITRL, GITR ligand; KIR, killer IgG-like receptor; LAG-3, lymphocyte-activation gene 3; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; PDL-1, programmed cell death ligand 1; TIM-3, T cell immunoglobulin mucin 3; TIGIT, T cell immunoreceptor with Ig and ITIM domains.

Cancerous cells have been shown to exploit a number of these self-regulating immune checkpoint responses, manipulating them to negatively regulate the immune response. Examples of these are:

• Downregulation of T cell response by stimulating  regulatory T cells (T regs), myeloid derived T cells and inhibitory molecules such as Fas ligand and programmed death ligand – 1 (PDL-1)
• Decrease in production of antibodies
• Increase in production of immunosuppressive cytokines e.g. interleukin (IL)-10 and tumor growth factor (TGF) beta (β)

Thereby avoiding an immune response and permitting tumor growth.

Just as cancer cells manipulate immune checkpoints for their own ends; these checkpoints can also be manipulated to enhance the immune response against the tumor more efficiently. A major drawback for the therapeutic use of immune check blockage in immunotherapy is that an immune system that is no longer kept in check can go on to cause tissue damage by mechanisms such as chronic inflammation. Cancer immunotherapy, the manipulation of the immune response to fight this disease, has focussed on utilizing these checkpoints to not only prevent the negative regulation of the immune response but also  enhance their positive regulatory effects. Many different immune checkpoints have been targeted with this approach, these include:

1. Cytotoxic T lymphocyte antigen 4 (CTLA-4)

Negative regulation of immune response.

CTLA-4 (CD152) is a receptor that is expressed mainly on T cells, including CD4⁺, CD8⁺ and T regs. Once bound to B7-1 (CD80) and B7-2 (CD86) proteins, it negatively regulates T cell activation. Therefore it is an ideal receptor for tumors to target.

During an immune response, CD80 and CD86 present on antigen presenting cells (APCs) bind to CD28 on T cells and provide the co-stimulatory signal for T cell activation by the binding of MHC class II to the T cell receptor (TCR).  However when CTLA-4 is expressed by T cells (CTLA-4 expression is upregulated upon T cell activation), it will preferentially bind to the B7 protein of CD80 (B7-1) and CD86 (B7-2) as it has higher affinity than CD28. Moreover, this binding of CTLA-4 to CD80 and CD86 will also prevent CD28 interacting with other B7 proteins. This results in the prevention of T cell activation, thus damping down T cell activation and negatively regulating the immune response.

Blocking of CTLA-4 would remove the possibility of T cell inhibition by this route and help ensure the continued immune response to the tumor. Immunotherapy using the antibody ipilimumab has been used to aid T cell activation. By binding directly to CTLA-4, ipilimumab permits unhindered binding of CD80 and CD86 to CD28 and the subsequent co-stimulation of the MHC class II/TCR T cell activation process.

Table 2: Key markers for the study of CTLA-4

Applications include: ELISA, flow cytometry, functional assay, immunofluorescence/immunocytochemistry, immunohistochemistry – frozen, immunoprecipitation and western blotting.

2. Programmed cell death protein 1 (PD-1)

Negative regulation of immune response.

The function of PD-1, also known as CD279, as an immune checkpoint is to down regulate the immune response, thereby reducing autoimmunity and promoting self-tolerance. PD-1 is expressed on most mature T cells located in peripheral tissue.

During an immune response PD-1 binds to two ligands:

1. PD ligand 1 (PDL-1) – also known as B7-H1 and CD274, found on most tissues.
2. PD ligand 2 (PDL-2) – also known as B7-DC and CD273, found only on cells from the hematopoietic lineage.

Binding of PD-1 to its ligands results in a signaling pathway mediated by tyrosine phosphatase SHP-2 suppression of the TCR receptor signals. PD-1 initiated signaling pathways have a number of different effects including:

• Blocking TCR mediated signals by preventing B7 protein binding to CD28, which is the co-stimulator required for effective TCR signaling
• Inducing T cell anergy and exhaustion
• Promoting apoptosis of tumor specific T cells
• Reducing apoptosis in T regs (suppressor T cells)
• Increasing IL-10 secretion by T cells (IL-10 is an anti-inflammatory cytokine)
• Decreasing IL-2 and IFN-γ secretion by T cells (pro-inflammatory cytokines)

Ultimately these lead to a reduced immune response.

Owing to the immunosuppressive function of PD-1, it is an ideal mechanism for cancerous cells to exploit in order to avoid an immune response. Tumor cells are known to highly express PD-1 in response to IFN-γ secretion which occurs during an immune response.  

Immunotherapy has also focused on PD-1 as a target. Several antibodies have been generated against this immune checkpoint such as nivolumab and pidilizumab. These antibodies prevent binding to the PD ligands and as a consequence ensure continuation of the anti-tumor immune response.

Table 3: Key markers for the study of PD-1

Applications include: ELISA, flow cytometry, functional assay, immunohistochemistry – frozen, immunohistochemistry – paraffin, immunoprecipitation and western blotting.

3. Lymphocyte-activation gene 3 (LAG-3)

Negative regulation of immune response.

LAG-3, also known as CD223, is a cell surface receptor expressed on activated T cells (CD4⁺ and CD8⁺) and NK cells. During an immune response, LAG-3 is upregulated by pro-inflammatory cytokines such as interferon gamma (IFN-γ). LAG-3 binds MHC class II thereby preventing MHC class II binding with the TCR and thus inhibiting T cell stimulation.  The binding of LAG-3 to MHC class II on dendritic cells (DCs) may result in signaling pathways that upregulate TNF-α and IL-12.

LAG-3, either alone or in combination with other immune checkpoints, is an ideal target for immune checkpoint blockade immunotherapy.

4. Killer IgG-like receptors (KIR)

Negative regulation of immune response.

KIRs, also known as CD158, are mainly expressed on NK cells, but have been detected on tumor specific cytotoxic T cells. KIRs induce NK tolerance to self, they bind to MHC class I molecules on the cell surface, which results in the negative regulation of the NK cell function, reducing NK cell mediated lysis. Antibodies generated against KIR have been shown to induce NK cell mediated lysis and therefore they make an ideal target for immunotherapy.

5. T cell immunoglobulin mucin 3 (TIM-3)

Negative regulation of immune response.

TIM-3 is a marker found on CD4⁺ and CD8⁺ T cells and many other tissues. Upon binding to galectin, it is thought to initiate a number of signaling pathways involved in the negative regulation of the immune response such as:

• Reducing T cell function
• Increasing CD4⁺ T cell death
• Promoting the development of myeloid derived suppressor cells

Anti-TIM-3 immunotherapy is in development.

6. T cell immunoreceptor with Ig and ITIM domains (TIGIT)

Negative regulation of immune response.

TIGIT can be found on a range of lymphocytes such as T and NK cells. When bound to its ligand, poliovirus receptor (CD155), present on immune cells such as macrophages and DCs, TIGIT can inhibit T and NK cell activation and stimulate TIM-3 expression. Blocking TIGIT binding has led to T cell activation, showing potential for therapeutics.

7. 4-1BB

Positive regulation of immune response.

4-1BB, also known as CD137, is a member of the TNF receptor family and is expressed on a number of immune cells including CD4⁺ and CD8⁺ T cells, activated NK cells, DCs and neutrophils. It binds to 4-1BB ligand found on macrophages, B cells and DCs. Being a co-stimulatory molecule, once bound, 4-1BB initiates pro-inflammatory signaling pathways such as c-jun, nuclear factor kappa B (NF-kB) and p38 to promote an immune response.

As 4-1BB stimulates a positive immune response in a range of different immune cells, it has become a potential immunotherapy target. Moreover, agonist antibodies to 4-1BB have been shown to enhance and maintain an immune response resulting in tumor regression.

8. Glucocorticoid-induced tumor necrosis factor receptor (GITR)

Positive regulation of immune response.

GITR, also known as CD357, is a cell surface receptor found on T regs, and CD4⁺ and CD8⁺ T cells, with its ligand, GITRL, located on APCs and various epithelial cells. The signaling pathway induced by the binding of GITR to its ligand probably increases the immune response by:

• Enhancement of T cell and effector T cell activity
• Reduction of T reg activity

Like 4-1BB, agonist antibodies generated to GITR have shown tumor regression and therefore show potential as a target for immunotherapy.

Key markers for the study of immune checkpoints
 

Table 4: Positive regulation immune checkpoint markers and their ligands

Table 5: Negative regulation immune checkpoint markers and their ligands

Table 6: Associated immune checkpoint markers

Applications include: ELISA, flow cytometry, functional assay, immunofluorescence/immunocytochemistry, immunofluorescence/immunocytochemistry, immunohistochemistry – frozen, immunohistochemistry – paraffin, immunoprecipitation and western blotting.

Resources for immuno-oncology research

Custom anti-idiotypic antibody generation

The Bio-Rad custom antibody team are experts in the generation of recombinant antibodies against monoclonal antibody drugs. Highly specific, high affinity anti-idiotypic antibodies can be generated against immune checkpoint inhibitor drugs, for use in the bioanalytical assays needed to support clinical studies.

These antibodies are made using the HuCAL recombinant monoclonal antibody library and a novel and proprietary method of phage display. Antibodies are generated in monovalent or bivalent Fab format in as little as 8 weeks, and are suitable for development of PK bridging ELISAs. They can also be converted to fully human immunoglobulins for use as reference standards in immunogenicity anti-drug-antibody assays.

More about custom generation of anti-biotherapeutic antibodies

Learn more about the immune response against cancer

Cell health products

It is essential to be able to determine the health of normal or cancerous cells.

• Assess the viability of cells
• Monitor cell proliferation
• Detect and measure apoptotic cell death

Immune checkpoint antibodies

Bio-Rad provides a comprehensive range of antibodies for immune checkpoint research, either search for these using the filter table below or by entering your marker in the search box at the top of the page.

Further Reading

1. Kim ES et al. (2016). Immune checkpoint modulators: An emerging antiglioma armamentarium. Jour Immunol Research article ID4683607.