Immune System Signaling Pathways

Introduction

Recall that both the innate and adaptive cells of the immune system are involved in anti-tumor activity. Factors of the immune system can either promote or reduce immunity. Activating, or stimulatory, factors promote or stimulate the immune response. Inhibitory factors reduce the ability of the immune system to mount response or block the ability of the immune system to function at full capacity. Many activating and inhibitory pathways have been identified in both effector and non-effector immune cells. Balance between activating and inhibitory pathways is necessary to maintain proper immune function. Cancer immunotherapy is an artificial stimulation of the immune system enhancing its natural ability to fight cancer.

T Cell Signaling

T lymphocytes, specifically CD4+ T cells, are considered the leaders of the adaptive immune response to protein antigens. These naive, undifferentiated T cells live in the lymphoid tissues (e.g., lymph nodes, spleen) and must produce effector T cells that may be “summoned” to the tumor site. T-cell receptors (TCRs) are formed when DNA recombines during T cell development and are expressed on the surface of naive T cells. These TCRs enable naive T cells to identify peptide antigens that are bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). This is the first of two required signals for T cell activation. The second signal comes from the recruitment of co-stimulators. A common co-stimulator is the CD28 receptor and B7 molecules. Other TCRs include CTLA-4, ICOS and PD-1 with ligands homologous to the B7 proteins. After initial T cell activation, expression of costimulatory and coinhibitory molecules will positively or negatively control the growth, differentiation and function of T cell response. Immune checkpoints, including CTLA-4 and PD-1, are regulators that maintain homeostasis, prevent autoimmunity and have the potential to aid in tumor immune evasion.

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Natural Killer Cell Signaling

Natural killer (NK) cells are lymphocytes critical in the early innate immune response to foreign cells, pathogens and stressed immune cells, such as those affected during tumor transformation. NK cell activation, like T cells, is controlled by a dynamic balance between stimulatory and inhibitory signaling pathways. Upon activation, the surface receptors of NK cells trigger cytotoxic activity and initiate secretion of cytokines and chemokines that, in return, regulate other immune cells. Studies have shown that NK cells may potentially provide a link between the innate and adaptive immune system due to their ability to develop antigen-specific long-term memory during viral infection. Additionally, NK cells inhibit adaptive immune response by decreasing tumor-specific CD8+ cell response and antigen presentation in tumor-associated lymph nodes.

B7 Family of Ligands

The B7 ligand family are cell-surface protein ligands that aid in regulation of the immune response via their interactions with receptors on lymphocytes. They augment the immune response through costimulatory signaling, as is seen during CD28 activation of B and T lymphocytes. On the reverse side, some members of this ligand family attenuate immune responses via coinhibitory pathways, for instance, during inhibition of the CD28 pathway by CTLA-4 and B7-2. There are currently seven known members of this family of ligands. The most well-known are B7-1 (CD80) and B7-2 (CD86), which interact with CD28 and CTLA-4 to regulate T cell signaling. B7 molecules are only expressed on the surface of dendritic cells (DC), macrophages and B cells in the presence of an infection. Manipulation of B7 ligand signals has shown promise as treatment for autoimmune and inflammatory disease and cancer.

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

CD28 is the only B7 receptor on the surface of naive T cells. While both B7-1 and B7-2 bind CD28 and co-stimulate T-cell growth, B7-1 is believed to be more involved in sustaining and regulating the activation process after the initial activation signal, while B7-2, which is constitutively expressed on dendritic cells, is believed to be more involved in the initiation of the early immune response. Cancer cells, apart from certain lymphomas, do not express B7-1 molecules and thus are one mechanism used by tumor cells to evade the immune system.

B7-H1 (CD274; PD-L1)

B7-H1, in addition being constitutively expressed on macrophages and DC, can also be induced on activated B and T cells as well as endothelial and epithelial cells. B7-H1, while similar to B7-1 and B7-2, does not bind to CD28, CTLA-4 or ICOS. Instead, it interacts with PD-1 on activated T cells. Cytokine production — specifically IL-2, which is upregulated during T cell activation — is essential for B7-H1 co-stimulation.

B7-H1 has dual functions: co-stimulation of early, naive T cell differentiation and inhibition of activated-effector T cell differentiation, survival and cytokine production to prevent over activation. Additionally, B7-H1 is shown to be upregulated on the surface of a variety of tumor cells, including lung, ovarian and colon tumor cells, as a means of evading immunosurveillance. Higher B7-H1 expression has been shown to correlate with worse prognosis in renal cell carcinoma, esophageal and gastric cancer.

B7-H3 pathway

B7-H3 is induced on APCs, dendritic cells, monocytes, NK cells and B cells. While its exact receptors are still unknown, it is believed to co-stimulate the early innate immune system by enhancing secretion of proinflammatory cytokines. It has also been shown to play a role in the later immune response as an inhibitor of effector T cell proliferation and cytokine production (IFN-Ƴ and IL-2). B7-H3 is highly expressed in a range of human solid cancers. In fact, its expression has been reported in 60% to 93% of tumors compared with very limited expression seen in normal healthy tissue. Due to its enhanced expression in tumor cells compared with healthy cells, and the fact its expression directly correlates with poor prognosis and clinical outcomes in a variety of cancers, B7-H3 has the potential to be a powerful biomarker and attractive target for immunotherapy.

Stimulatory Signaling Pathways

Costimulatory pathways occur via interactions between naive and/or activated T cells and their ligands on APCs. The costimulatory signals amplify and modify the original TCR activation signal and, in turn, activate and regulate T cell expansion and/or differentiation to translate the signal into an immune response or immune tolerance.

CD28 + CD80 (B7-1)

The second but primary signal of the costimulatory pathway involved in T cell activation occurs when CD28 on the T-cell surface binds to one or both of the B7 molecules, B7-1 (CD80) and B7-2 (CD86), on the APC. The combination of intracellular signals generated by the antigen receptor, coreceptor (CD4+ or CD8+) and costimulatory receptor CD28, initiates naive antigen-specific T cell activation and proliferation, as well as enhances production and secretion of inflammatory cytokines, including IL-2, IL-4 and IL-10. Increased expression of inflammatory cytokines, in turn, stimulates T cell differentiation into appropriate effector T cells based on the immunological insult. The CD28 signaling pathway also plays an important role in inhibiting T cell anergy, or absence of normal immune response, via cytokine-mediated enhancement of T cell proliferation. However, tumor evasion of the CD28/B7 stimulatory pathway has been shown to result in enhanced immunosuppression and poorer prognosis in patients with cancer.

CD28 co-stimulation is not only a key player in activation of T cells, but also T cell effector responses, differentiation, survival and migration to areas of infection and inflammation. They also play a vital role in the development and proliferation of regulatory T cells (Tregs) through the production of IL-2. Lastly, co-stimulation with B7 molecules, CD80/CD86, triggers NK cell-mediated cytotoxicity and is thought to play a role in NK cell activation, proliferation and cytokine secretion.

ICOS + CD275 (ICOS-L)

Inducible T cell co-stimulator (ICOS), a member of the CD28 family of immune receptors, is a stimulatory receptor expressed on the surface of activated CD4+ and CD8+ T cells and NK cells upon initiation of CD28 signaling. Its ligand, ICOSL, is expressed on APCs, including dendritic cells and macrophages. Following T cell activation, ICOS upregulation perpetuates the activation, proliferation, function and survival of cytotoxic T cells and the survival of memory T cells. Additionally, ICOS signaling plays a significant role in T cell-dependent B cell maturation. Upon recognition of specific pathogen, increases in helper T cells leads to enhanced inflammatory cytokine production, which, in turn, initiates B cell differentiation. The ICOS/ ICOS-L pathway has also been suggested to enhance the function of activated NK cells during the innate immune response. Recently, ICOS expression has been investigated as a useful predictive biomarker of response to checkpoint inhibitor treatments for cancer and could be an attractive target for combination immunotherapy. Research has shown combination immunotherapy that provides artificial stimulation of the ICOS pathway, along with inhibition of the CTLA-4 inhibitory pathway, enhances the immune response.

CD40 + CD40L

CD40, a member of the tumor necrosis factor (TNF) receptor family, is expressed by APCs and B cells. Its ligand, CD40L, is primarily expressed on helper T cells and other immune effector cells shortly following T cell activation. CD40-CD40L binding on activated T cells stimulates cytokine secretion from B cells, which subsequently enhances T cell activation and anti-tumor immunity. T cell activation then signals macrophages to increase expression of MHC II protein and B7, thereby further stimulating proliferation of helper T cells.

CD40 is often expressed in advanced tumors, and anti-CD40 antibodies have been shown to have promising effects in the treatment of myelomas and leukemia. CD40L expression is believed to indirectly effect anti-tumor activity by augmenting the anti-tumor response of B and T cells.

OX40L + OX40

OX40, another member of the TNF receptor family, is expressed on activated cytotoxic and Treg cells. Upon binding with its ligand (OX40L) on APCs, OX40 both activates and amplifies T cell responses by promoting T cell proliferation, function, survival and cytokine production. OX40 also aids in T cell differentiation into memory T cells. OX40 has been shown to inhibit the reproduction, immunosuppressive effects and suppressive function of Tregs on T cell activation, thereby aiding in the attenuation of autoimmune reactions. In addition to T cells, the OX40/OX40L pathway is also involved in NK cell activation, cytokine production, cytotoxicity and enhancement of NK’s cell lysis function. Studies have shown that OX40 plays a vital role in creating a more favorable antitumor immune response during the tumor microenvironment, thus explaining its utility in enhancing tumor free survival in cancer patients.

CD137L + CD137

CD137 binding to its ligand CD137L on CD8+ T cells leads to markedly enhanced activation, proliferation and survival of cytotoxic T lymphocytes. Stimulation of CD137 expression has been shown to play an important role in inhibition of autoimmune and inflammatory diseases mediated by CD4+ T cells. CD137L/CD137’s role in the inflammatory response is mediated through NK cells and its effects on myeloid cell differentiation and function as well as the induction of B cell proliferation and immunoglobulin production. Further understanding of CD137L/CD137’s roles in the inflammatory response and T cell immunity are key to its use in immunotherapy. Current therapies have shown inhibition of CD137 activates cytotoxic T cells and increases cytokine production, which are known to be critical for effective activation and regulation of the immune response.

SLAMF7 + SLAMF7 and activating adaptor protein

Signaling lymphocytic activation molecule family member 7 (SLAMF7) is an activating receptor expressed on NK cells and other immune cells, apart from solid tissues and hematopoietic stem cells. Activation of the SLAMF7 pathway is involved in the activation of NK cells’ natural cytotoxicity and rapid response against cancer and other diseases. SLAMF7 is known to stimulate both the innate and adaptive immune responses. Upon B cell activation, SLAMF7 expression is upregulated, resulting in B cell proliferation and production of autocrine cytokines (i.e. IL-14). SLAMF7 is a target for multiple myeloma immunotherapy due to its high expression pattern in multiple myeloma cells and lack of expression on healthy tissue, primary tumors and hematologic and nonhematologic cells. Moreover, SLAMF7 has the potential to serve as biomarker for multiple myeloma progression because its expression correlates with disease stage.

Inhibitory Signaling Pathways

Coinhibitory receptors serve as immune checkpoints against unrestrained T cell activation by limiting the costimulatory signaling and ligation pathways. These receptors also play an important role during active infection and inflammation by maintaining peripheral tolerance and immune system homeostasis.

CTLA-4 + CD86 (B7-2)

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is expressed exclusively on the surface of activated T cells and counteracts the activity of costimulatory receptor CD28 upon antigen recognition. Upregulation of CTLA-4 dampens the downstream signaling of TCR and CD28 by competing with CD28 for its ligands CD80/86 (B7-1/B7-2). Due to its greater affinity for the B7 ligands, it outcompetes CD28, resulting in inhibition of both T cell activation and proliferation. Competition between CD28 and CTLA-4 for binding to B7 ligands ensures the balance of the immune system is preserved and inhibits overactivation of the immune response and enhanced autoimmunity. Additionally, CTLA-4 is constitutively expressed on Treg cells, enhancing Treg cell’s suppression of the T cell anti-tumor immune response.

Tumor cells utilize the CTLA-4 pathway to suppress initiation of the immune response, leading to decreased T cell activation and proliferation into memory T cells, which are key to mounting an effective long-term immune response against tumor antigens. Therefore, blocking the CTLA-4 inhibitory pathway using immunotherapy can help restore the immune anti-tumor response by increasing accumulation, function and survival of T cells and memory T cells, while inhibiting Treg activity. Inhibition of effector T cell differentiation, memory generation and enhancement of effector T cell exhaustion highlights another prime reason CTLA-4 is a target for immunotherapy.

PD-1 + CD274 (PD-L1; B7-1)

PD-1 plays a vital role in maintaining physiologically-appropriate T cell responses and limiting the activity of effector T cells in peripheral organs to prevent immune-mediated tissue damage. PD-1 is expressed in early stages of T cell activation on the surface of CD4+, CD8+ and regulatory T cells, NK cells, B cells and activated monocytes. PD-1 can interact with either of two coinhibitory ligands PD-L1 (CD274; B7-H1) or PD-L2 (CD273). Co-stimulation of PD-1 with PD-L1 leads to increased T cell apoptosis, inhibition of both proliferation and cytokine production in order to prevent autoimmune diseases, and inflammation. PD-L1’s ability to compete with CD28 and CTLA-4 for binding CD80 provides a bi-directional inhibitory immune response. PD-1/PD-L2 binding inhibits T cell proliferation, cytokine production and apoptosis by blocking cell cycle progression.

Several cancers use this pathway to evade immune destruction. Thus, blocking PD-1/B7-H1 has the potential to protect T cells from exhaustion and enhance immunity against cancer. Deficiencies in PD-1 are associated with various autoimmune diseases. Uncontrolled PD-1 expression, as is seen in the cancer environment, results in enhanced T cell exhaustion, thereby decreasing the anti-tumor response of T cells. Over time, the exhausted T cells become disabled and lose their ability to function, reproduce and fight tumor cells. PD-L1 and PD-L2 are expressed in multiple solid tumors, including renal cancer, melanoma and non-small cell lung cancer. Research has shown that enhanced expression of PD-L1 is a potential biomarker for poor survival in patients with advanced tumor stages. Decreased PD-L2 expression has been associated with longer median survival times in certain cancers. Inhibiting PD-1 binding to both PD-L1 and PD-L2 has shown to increase response rates in patients with metastatic cancer. Combination immunotherapy inducing a co-blockade of PD-1 and CTLA-4 could enhance therapeutic efficacy by mitigating their suppression of the immune response and reinvigorating exhausted T cells. Additionally, stimulation of these pathways could also be of interest in downregulation of immune responses during transplant rejection and autoimmune diseases.

LAG3 pathway: MHC + LAG3

Lymphocyte-activation gene 3 (LAG3) is an immune checkpoint receptor involved in downregulation of T cell-mediated immune response and is expressed on activated cytotoxic T cells, Tregs and NK cells. Upon binding to MHC, LAG3 signaling downregulates T cell proliferation and development of memory T cells. T cells that co-express LAG3 and PD-1 are thought to increase T cell exhaustion to a greater degree than either signal alone. Repeated exposure to a tumor antigen increases the expression and activity of LAG3 leading to enhanced T cell exhaustion, which is often seen in a variety of cancers. Elevated LAG3 expression has also been associated with tumor-infiltrating Tregs in various cancer types. Dual blockage of LAG3 with other immune checkpoint receptors, including PD-1, hold promise as strong immunotherapeutic options by mitigating the effects of T cell exhaustion.