Immune Checkpoints and the T cell Amanda Flies, Ph.D. Cell Sciences® Inc., Newburyport, Massachusetts, USA.
[email protected]
Abstract
Immune checkpoints are stimulatory or inhibitory signals that play a major role in regulation of the immune system’s function. Interactions between surface molecules on immune cells lead to activation, differentiation and effector function, or inhibition of these activities. T cells, an important member of the adaptive branch of the immune system, are greatly affected by interactions between immune checkpoint molecules, including CD28 ↔ B7-1/B7-2 ↔ CTLA4 interactions, PD-1 ↔ PD-L1/PD-L2, LAG-3 ↔ MHC, and TIM-3 ↔ galectin-9 interactions. Proliferation, clonal expansion, differentiation into T cell subsets, and effector functions all depend on stimulation or inhibition through these surface molecules. Thus, regulation of T cells by way of blocking or stimulating the immune checkpoints can have significant effects on the function of the immune system. Recent advances in immunotherapy targeting immune checkpoint molecules have demonstrated efficacy in modulating immune reactions to self-tissues in both cancer immunity and autoimmunity. Continued research into this growing field of study is critical for advancing immunotherapeutic treatments against human diseases using immune checkpoint molecules. The immune system’s function depends upon many complex interactions between various cell types. These interactions are important for development of the cells from their immature states into mature cells, for activation of naïve cells and differentiation into various effector populations, for performance of effector functions, and for regulation of the cells at each of these milestones. Much of the communication between immune cells involves surface molecule interactions necessary to advance from one stage of the cell’s development to the next, termed “immune checkpoints.” Manipulation of these surface molecules to either stimulate or inhibit the cells at the critical checkpoint can have a profound effect on the individual cell’s, and the immune system’s, ability to perform effector functions, which may range from being able to fight off infections and cancer to causing devastating autoimmune disease. The ability to alter these immune states is a critical feature of immunotherapy. One cell type that is involved in many immune functions, and has been the study of much research in immunotherapeutics, is the T cell. T cells play a critical role in the immune system, providing adaptive responses to a wide variety of antigens, and are responsible for protecting the body from threats, both foreign and self-originating. As members of the adaptive cellular arm of the immune system, T cells have various roles, including killer functions, cytokine-production “helper” functions, regulation, memory, and others. Critical features of T cell-based immunity are the ability of T cells to be activated by their cognate antigen, to undergo clonal expansion, and to differentiate into effector T cells. In order to control these powerful immune cells and prevent them from targeting normal “self ” cells and tissues, there must be regulation of T cells, and immune checkpoint molecules are critical to that regulation.
APC MHC CD8
B7-1
B7-2
Ag
TCR
CD4
CD28
T cell Signal 1
Signal 2
Figure 1: T cell activation results from TCR recognition of the cognate antigen in the context of MHC (signal 1), in combination with CD4 or CD8, and co-stimulation (signal 2), e.g., CD28 interaction with B7-1 (CD80) or B7-2 (CD86).
Regulation of activation, differentiation, and function occurs in many immune cell types, but in T cells it typically involves an antigen presenting cell (APC). T cells are unable to recognize their cognate antigen, the peptide for which a given T cell is specific, without it being presented in the context of the particular set of surface molecules known as the major histocompatibility complex (MHC). For the T cell to receive its initial activation signal, an APC must present the antigen on the MHC and the 1
T cell must recognize this antigen-MHC complex via its T cell receptor (TCR) in combination with CD4 or CD8 (depending on the type of T cell) (Figure 1). However, this recognition of antigen by the T cell (often referred to as “signal 1”) is not enough to fully activate the cell (1). To become fully activated, T cells must not only receive signal 1, but must also receive co-stimulatory signals (sometimes referred to as “signal 2”). Historically, signal 2 has been most studied as the interaction between the surface glycoprotein CD28 on the T cell, and its ligands on the APC, CD80 (B7-1) or CD86 (B7-2) (Figure 1) (2). Without co-stimulation, a naïve T cell that receives signal 1 will be unable to become fully activated and will instead become anergic (unable to respond to the antigen/MHC stimulation) (1). Thus, the CD28-CD80/86 interaction is one checkpoint by which T cell functions are regulated. Without co-stimulation, anergy of an individual T cell to its cognate antigen produces tolerance to that antigen, and immune evasion of the targets bearing the antigen. More recently, other co-stimulatory, and also co-inhibitory immune checkpoint members have been characterized. Immune checkpoint proteins are considered as therapeutic targets for manipulating T cell immunity, most notably for the purpose of cancer immunotherapy. The immune system’s tolerance toward cancer cells, which originate as part of the larger organism, or “self ” tissues or cells, is a hurdle which many investigators have been trying to overcome in order to induce effective and durable immune responses to cancer. Indeed, the tolerance of the immune system toward self is critical to the function of the organism. Much of the normal regulation of the immune system, and of T cells, is therefore devoted to preventing attack of healthy tissue (autoimmunity). Breaking self-tolerance in an organism is a balancing act between targeting the malignant selftissue and sparing the healthy bystander tissue and cells, and as such, research into the detailed functions of immune checkpoint molecules is critical. Immune checkpoint molecules which are currently being studied as targets to advance immunotherapy include CTLA-4, PD-1, the PD-1 ligands PD-L1 and PD-L2, LAG-3, and TIM-3. CTLA-4 CTLA-4 (Cytotoxic T Lymphocyte Associated protein 4, also known as CD152) is a member of the immunoglobulin superfamily, expressed either on the surface of the T cell, or as a soluble protein (3). CTLA-4 acts as an immune checkpoint by two distinct mechanisms. First, it competitively inhibits the costimulatory signal transmitted through CD28 (Figure 2); CTLA4 has a higher affinity and avidity for the co-stimulatory ligands B7-1 and B7-2 on the APC (4). Secondly, CTLA-4 itself transmits an inhibitory signal to the T cell (Figure 2), reducing proliferation and cytokine production (2). CTLA-4 is expressed on CD8+ T cells, CD4+ “helper” T cells, and on regulatory T cells. Indeed, CTLA-4 can also be expressed on cancer cells themselves (5).
APC MHC B7-1
B7-2
Ag
CD28
TCR
Signal 1
CTLA-4
T cell
Figure 2: CTLA-4 prevents T cell activation by competitively inhibiting the co-stimulatory signal through CD28, as CD28’s ligands (B7-1 and B7-2) have a higher affinity/avidity for CTLA4. CTLA-4 also transmits an inhibitory signal intracellularly, which reduces proliferation and cytokine production.
The inhibitory nature of the CTLA-4 molecule, preventing activation and proliferation of T cells, has been widely studied with regards to immunotherapy in general, and cancer immunotherapy specifically (6). A correlation between high levels of CTLA-4 on T cells and worsening disease states has been demonstrated for some cancers, including non-small cell lung cancer and breast cancer. Inhibiting CTLA-4 signaling improved patient survival in many cases, particularly melanoma patients (7). CTLA-4 was the initial target for therapeutic antibodies against checkpoint molecules: ipilimumab, a blocking antibody against CTLA-4, was approved by the FDA in 2011 for treatment of metastatic melanoma (8). On the other side of the coin, however, treatment with anti-CTLA-4 can lead to loss of immune tolerance to normal self-tissue, as inhibition of this molecule affects many specificities of T cells, not just those that may attack cancer cells, leading to “adverse autoimmune events” upon immunotherapy treatment (8). The antagonistic anti-CTLA-4 antibody ipilimumab blocks the ability of CTLA-4 to bind to, and thereby out-compete, CD28 for the ligands B7-1 and B7-2. By blocking inhibition, activation of the T cell is allowed to proceed, potentially resulting in many cancer-specific T cells being activated instead of anergic. More recently, the therapeutic CTLA-4-Ig fusion protein abatacept (the CTLA-4 active domain linked to an immunoglobulin Fc fragment) has been used to treat the opposite problem: inappropriate T cell activation, or autoimmunity. In this case, the soluble CTLA-42
Ig binds CD28 ligands preventing T cell activation in patients with a T cell response against self-antigens, such as auto-immune rheumatoid arthritis (9). Cell Sciences offers recombinant human and mouse CTLA4 fusion proteins, which bind the CTLA-4 ligands CD80/B7-1 and CD86/B7-2 for use in research on this important checkpoint molecule. Catalog No
Description
CRH030
Recombinant Human CTLA-4/CD152:Fc Chimera
CRH031
Recombinant Human CTLA-4/CD152:Fc Chimera Non-Lytic
CRH032
Recombinant Human CTLA-4/CD152:Mouse Fc Chimera
CRM035
Recombinant Mouse CTLA-4/CD152:Fc Chimera
CRM036
Recombinant Mouse CTLA-4/CD152:Fc Chimera Non-Lytic
PD-1 - PD-L1/2 PD-1 (Programmed Death 1 receptor, also known as CD279) is found on the surface of T cells, and upregulated upon activation (7). PD-1 is also expressed on B cells, dendritic cells and other immune cell types (6). It is a member of the immunoglobulin superfamily (10) which transmits an inhibitory signal blocking TCR-mediated activation upon ligation with PD-L1 or PD-L2 (Figure 3). PD-1 ligation is also important for regulatory T cell
APC MHC Ag
TCR
PD-L1/2
PD-1
T cell T cell “exhaustion” Figure 3: PD-1 transmits an inhibitory signal upon ligation with PD-L1 or PD-L2, which causes the T cell to adopt an “exhausted” phenotype, typified by down-regulation of proliferation, cytokine production and effector activities.
(Treg) induction and function (7). The PD-1 ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) are expressed on a variety of immune and non-immune cell types, including, in the case of PD-L1, on tumor cells. PD-L1 can be upregulated by various stimuli, including cytokines, particularly interferons, which are often used in various treatments (6). PD-1 signaling on activated T cells results in inhibition of the antigen-specific signal from the TCR, and down-regulation of the T cell’s proliferation, cytokine production, and effector activities such as cytotoxicity (in the case of CD8+ T cells) (4). This inhibition of the normal function of activated T cells results in an “exhausted” phenotype after prolonged antigen stimulation (6) (7). Blockade of the PD-1 checkpoint has been studied alone and in combination with other checkpoint modulators for various cancers including melanoma (1) (11), pancreatic cancer (4), lung cancer (12) and others. PD-1 blockade is considered to have less risk of adverse immune events than CTLA-4 blockade (11). Whether the effect of PD-1 blockade is direct, allowing the activated T cells to remain functional, or indirect, by reducing the activity of regulatory T cells in the tumor micro-environment (12), or a combination of these and other effects, is still under investigation. Indeed, while immunotherapy involving blockade of PD-1 and CTLA-4, alone or in combination, is known to be effective against several malignancies (11), further study into the mechanisms of immune checkpoint blockade, both singly and in combination, is critical to unraveling the complex interactions of the immune system and cancer. Cell Sciences offers several products for use in research on these important checkpoint molecules, including: recombinant human and mouse PD-1 fusion proteins, which bind the PD-1 ligands PD-L1 and PD-L2; recombinant human and mouse PD-L1 and PD-L2 fusion proteins, which bind PD-1. Catalog No
Description
CRH046
Recombinant Human PD-1/CD279:Fc Chimera
CRH047
Recombinant Human PD-1/CD279:Fc Chimera Non-Lytic
CRH036
Recombinant Human B7-H1/PD-L1/CD274:Fc Chimera
CRH037
Recombinant Human B7-H1/PD-L1/CD274:Fc Chimera Non-Lytic
CRH033
Recombinant Human PD-L2/CD273:Fc Chimera
CRH034
Recombinant Human PD-L2/CD273:Fc Chimera Non-Lytic
CRH035
Recombinant Human PD-L2/CD273:Mouse Fc Chimera
CRM044
Recombinant Mouse PD-1/CD279:Fc Chimera
CRM038
Recombinant Mouse B7-H1/PD-L1/CD274:Fc Chimera
CRM039
Recombinant Mouse B7-H1/PD-L1/CD274:Fc Chimera Non-Lytic
CRM037
Recombinant Mouse PD-L2/CD273:Fc Chimera
3
LAG-3 LAG-3 (Lymphocyte Activation Gene-3, also known as CD223) is a molecule expressed on activated T cells, as well as other immune cells. It is similar to CD4, and contains four immunoglobulin-like domains. LAG-3 binds to MHC II, as does CD4, but with higher affinity (13). Upon ligand binding, LAG-3 negatively regulates signal transduction through the T cell receptor complex by inhibition of TCR-induced calcium flux (14), resulting in down-regulated proliferation, function and survival of the T cell (Figure 4) (15). The resulting “exhausted” phenotype of the T cells has been shown to result in immune escape of tumor cells in cases such as Hodgkin’s lymphoma and prostate cancer (15), as well as progression of disease in persistently HIV-infected individuals (13). In the case of autoimmunity, studies in mouse models have shown the importance of LAG-3 for maintenance of tolerance to self-antigens, as in diabetes, where the absence of the LAG-3 molecule leads to substantially accelerated disease (16). As with other immune checkpoint molecules, LAG-3 has complex interactions depending upon the cell type and disease state, and has the potential to be an important target in immunotherapy for many diseases. Cell Sciences offers antibody clones which bind and block human LAG-3, as well as recombinant human and mouse LAG3 fusion proteins, for use in studying this immune checkpoint pathway.
APC
Catalog No
Description
CMH003
Mouse Anti-Human LAG-3 Clone 17B4 Biotin mAb
CMH004
Mouse Anti-Human LAG-3 Clone 17B4 Blocking Azide Free mAb
CMH005
Mouse Anti-Human LAG-3 Clone 17B4 FITC mAb
CMH006
Mouse Anti-Human LAG-3 Clone 17B4 Blocking mAb
CMH007
Mouse Anti-Human LAG-3 Clone 11E3 Blocking mAb
CMH008
Mouse Anti-Human LAG-3 Clone 11E3 Blocking Azide Free mAb
CRH048
Recombinant Human LAG-3:Fc Chimera
CRM045
Recombinant Mouse LAG-3:Fc Chimera
TIM-3 TIM-3 (T-cell immunoglobulin and mucin-domain containing-3, also known as CD366) is a molecule expressed on the surface of activated T helper type 1 (Th1) cells, among others (17). It has a complex role in activated immune cells of many types, including T cells, dendritic cells, natural killer cells and macrophages (18) (19) (20). Primarily in activated Th1 cells, TIM-3’s role as an immune checkpoint molecule is inhibitory: upon interaction with its ligand, galectin-9, TIM-3 transmits a signal to the active T cell to stop proliferating, stop production of particular cytokines, and even to undergo apoptosis, all symptoms of “exhausted” T cells (Figure 5). The physiological role of this inhibition signal is to “put the brakes” on an ongoing inflammatory response, to calm
APC MHC LAG-3
MHC Ag
Galectin-9
Ag
CD4 TCR
TCR
Tim-3
T cell T cell “exhaustion” Figure 4: LAG-3 binds MHC II and inhibits the TCR signal, resulting in an “exhausted” phenotype.
T cell T cell “exhaustion” Figure 5: TIM-3, upon binding its ligand, galectin-9, transmits inhibitory signals to the T cell, including downregulation of proliferation and cytokine production, and initiation of apoptosis.
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T cell activity so as to prevent “bystander” tissue pathology (19). However, this inhibition has been shown to prematurely limit T cell anti-viral activity, reduce vaccine effectiveness, and is taken advantage of by various types of cancer which express galectin-9 in order to inhibit T cells in the tumor’s vicinity (20). Blocking TIM-3 function allows “exhausted” T cells to resume their antitumor or anti-viral activity (21) (22) (23) (24). On the opposite side of regulation, it has been shown that polymorphisms in TIM3, presumably affecting its inhibitive functions, correlate with autoimmunity such as rheumatoid arthritis (19). Thus, TIM-3 is a complex immune checkpoint molecule, and modulation of its activities in various immune cell types have potential implications in treatments for a variety of diseases, from infections, to cancer, to autoimmunity. Cell Sciences offers recombinant TIM-3 fusion proteins which bind galectin-9, to aid in research on this immune checkpoint molecule. Catalog No
Description
CRH050
Recombinant Human Tim-3:Fc Chimera
CRH051
Recombinant Human Tim-3:Mouse Fc Chimera
In addition to these negative regulators of T cell function, there are many other immune checkpoint molecules that have the potential for impressive effects on the treatments of diseases from cancer to autoimmunity to infection. The possibilities for increasing the basic understanding of complex immune interactions, as well as the therapies that may emerge from future studies on immune checkpoints, are exciting! For a full catalog of immune checkpoint modulators, please see the Cell Sciences website (www.cellsciences.com), or contact us at info@ cellsciences.com.
Additional Immune Checkpoint Reagents Catalog No
Description
CRH022
Recombinant Human Multimeric TNF-alpha
CRH023
Recombinant Human Multimeric FasL
CRH024
Recombinant Human Multimeric CD40L
CRH025
Recombinant Human Multimeric APRIL
CRH026
Recombinant Human Progranulin FLAG
CRH027
Recombinant Human Progranulin
CRH028
Recombinant Human B7-H4:Fc Chimera
CRH029
Recombinant Human B7-H4:Mouse Fc Chimera
CRH038
Recombinant Human B7-H2/CD275:Fc Chimera
CRH039
Recombinant Human B7-H2/CD275:Fc Chimera Non-Lytic
CRH040
Recombinant Human B7-H2/CD275:Mouse Fc Chimera
CRH041
Recombinant Human B7-H3/CD276:Fc Chimera
CRH042
Recombinant Human B7-H3/CD276:Mouse Fc Chimera
CRH043
Recombinant Human ICOS/CD278:Fc Chimera
CRH044
Recombinant Human ICOS/CD278:Fc Chimera Non-Lytic
CRH045
Recombinant Human ICOS/CD278:Mouse Fc Chimera
CRH049
Recombinant Human Tim-1:Fc Chimera
CRH051
Recombinant Human Tim-3:Mouse Fc Chimera
CRH052
Recombinant Human Tim-4:Fc Chimera
CRM030
Recombinant Mouse Multimeric CD40L
CRM031
Recombinant Mouse Multimeric OX40L
CRM032
Recombinant Mouse Multimeric APRIL
CRM033
Recombinant Mouse Progranulin
CRM034
Recombinant Mouse B7-H4:Fc Chimera
CRM040
Recombinant Mouse B7-H2/CD275:Fc Chimera
CRM041
Recombinant Mouse B7-H2/CD275:Fc Chimera Non-Lytic
CRM042
Recombinant Mouse B7-H3/CD276:Fc Chimera
CRM043
Recombinant Mouse ICOS/CD278:Fc Chimera
CRM046
Recombinant Mouse Tim-4:Human Fc Chimera
CRM047
Recombinant Mouse CD137:Human Fc Chimera
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