Immune checkpoints identified: Purpose, mechanism and detection

Immune Checkpoints

Immune checkpoints identified: purpose, mechanism and detection

Immune checkpoint mini-review

Immune checkpoint mini-review

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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

Checkpoint Receptor

Checkpoint Ligand

Regulation

CTLA-4 (CD152)

B7-1 (CD80) and B7-2 (CD86)

Negative

PD-1 (CD279)

PDL-1 (CD274) and PDL-2 (CD273)

Negative

LAG-3 (CD223) and KIR (CD158)

MHC class II

Negative

TIM-3

Galectin

Negative

TIGIT

Poliovirus receptor (CD155)

Negative

4-1BB (CD137)

4-1BBL (CD137L)

Positive

GITR (CD357)

GITRL

Positive

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:

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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

Marker

Synonyms

Human Antibodies

Mouse Antibodies

Rat Antibodies

CTLA-4

CD152

MCA1724

MCA4636

MCA2092

B7-1

CD80

MCA2071, MCA2813C, VMA00138

MCA2462

MCA2873

B7-2

CD86

MCA1118

MCA2463F

MCA2121GA, MCA2874

CD28

 

MCA709GA, VPA00258

MCA2473F, MCA2587EL, MCA1363

 

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

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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

Marker

Synonyms

Human Antibodies

Mouse Antibodies

PD-1

CD279

AHP1705, AHP1706, MCA2628, MCA4624GA

MCA2464, MCA2470

PDL-1

B7-H1, CD274

MCA2627, AHP2128, AHP1703

MCA2626

PDL-2

B7-DC, CD273

MCA2630, AHP1704

MCA2465

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

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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.

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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

Marker

Synonyms

Human Antibodies

Mouse Antibodies

Rat Antibodies

4-1BB

CD137

AHP1863B, MCA2653

MCA4660

 

4-1BBL

CD137L

AHP1743B

MCA2286

 

B7-1

CD80

MCA2071, MCA2813C, VMA00138

MCA2462

MCA2873

B7-2

CD86

MCA1118

MCA2463F

MCA2121GA, MCA2874

CD27

 

MCA755, MCA2990Z

MCA4701

 

CD27L

CD70

MCA2302A488

MCA1765G

 

CD28

 

MCA709GA, VPA00258

MCA2473F, MCA2587EL, MCA1363

 

CD40

 

MCA1590, MCA2788GA

MCA1143

 

CD40L

CD154

 

 

 

CD122

IL-2Rβ

 

MCA2363

 

CD270

HVEM

MCA4826

 

 

CD278

ICOS

MCA4718

GITR

CD357

 

MCA2416

 

GITRL

 

 

MCA2417

 

OX40

CD134

 

MCA1420

MCA730GA

OX40L

CD134L, CD252

 

MCA1881

MCA4634

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Table 5: Negative regulation immune checkpoint markers and their ligands

Marker

Synonyms

Human Antibodies

Mouse Antibodies

Rat Antibodies

B7-1

CD80

MCA2071, MCA2813C, VMA00138

MCA2462

MCA2873

B7-2

CD86

MCA1118

MCA2463F

MCA2121GA, MCA2874

B7-H3

CD276

HCA143, MCA2633

MCA5646

MCA5647

B7-H4

VTCN1

MCA2632

 

 

CD272

BTLA

MCA2631

 

 

CTLA-4

CD152

MCA1724

MCA4636

MCA2092

Galectin 9

TIM-3 ligand

VMA00212

 

IDO1

 

MCA5433Z, MCA5434Z, AHP833, OBT2037G

 

 

KIR

CD158

MCA2243

 

 

KIRL

CD158b

MCA2340, MCA1584

 

 

LAG-3

CD223

 

MCA2386

 

PD-1

CD279

AHP1705, AHP1706, MCA2628, MCA4624GA

MCA2464, MCA2470

 

PDL-1

B7-H1, CD274

MCA2627

MCA2626

 

PDL-2

B7-DC, CD273

MCA2630, AHP1704

MCA2465

 

TIM-3

HAVCR2

AHP1773

MCA5790GA

 

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Table 6: Associated immune checkpoint markers

Marker

Human Antibodies

Mouse Antibodies

Rat Antibodies

CD3

MCA463, MCA2184, MCA1477, VMA00015

MCA500, MCA2690

MCA772

CD4

MCA1267, MCA6002MCA484G

MCA2691, MCA4635, MCA1107, MCA1767

MCA55G, MCA153GA, MCA372G, MCA1022R

CD8

 

MCA1768

 

CD8α

MCA1226, MCA351G, MCA2511, MCA1817, MCA4609T

MCA2694, MCA609, MCA1108

MCA48

CD8β

 

MCA2805

 

CD25

MCA2127, MCA2808C

MCA1260G

MCA273GA, MCA494G

CD44

MCA89, MCA2726VMA00034

MCA1014, MCA4703

MCA643, MCA930F

CD45R/ B220 in mouse

 

MCA1258

MCA1326

CD45.1

 

 

MCA415F

CD45.2

 

 

MCA473S

CD47

MCA2514, MCA911

 

MCA1997

CD62L

MCA1076G

MCA1259

 

CD69

MCA2806

MCA1580R

 

CD127

HCA145, HCA144

 

 

CD155

MCA4644

 

 

KLRG1

 

MCA4659

 

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


Resources for immuno-oncology research

 

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More about custom generation of anti-biotherapeutic antibodies


Learn more about the immune response against cancer

 


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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.

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    Further Reading

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

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