Immunotherapy has emerged as a promising strategy for boosting antitumoral immunity. Blockade of immune checkpoints (ICs), which regulate the activity of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells has proven clinical benefits. Antibodies targeting CTLA-4, PD-1, and PD-L1 are IC-blockade drugs approved for the treatment of various solid and hematological malignancies. However, a large subset of patients does not respond to current anti-IC immunotherapy. An integrative understanding of tumor-immune infiltrate, and IC expression and function in immune cell populations is fundamental to the design of effective therapies. The simultaneous blockade of newly identified ICs, as well as of previously described ICs, could improve antitumor response. We review the potential for novel combinatory blockade strategies as antitumoral therapy, and their effects on immune cells expressing the targeted ICs. Preclinical evidence and clinical trials involving the blockade of the various ICs are reported. We finally discuss the rationale of IC co-blockade strategy with respect to its downstream signaling in order to improve effective antitumoral immunity and prevent an increased risk of immune-related adverse events (irAEs).
Tumor growth involves a complex interplay between tumor, immune, and stromal cells, and extracellular matrix components. In the last decade, the relevance of the tumor-immune microenvironment and its direct impact on patients’ clinical outcome has become widely acknowledged. The host immune system is primed to identify and kill malignantly transformed cells to prevent tumorigenesis and tumor growth. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are immune cell populations responsible for immunosurveillance and, when required, for eliminating target cells. Tumor cells can be identified by CTLs as altered cells by the expression of neoantigens displayed by the major histocompatibility complex (MHC). Tumor cells expressing low levels of MHC molecules can become invisible to T cells and may escape T-cell immune control. In these cases, NK cells can identify and target cancer cells that lack MHC expression. However, tumor immune evasion, defined as the ability of tumor cells to evade the host’s immune response, happens during tumorigenesis and tumor growth. Multiple activating and inhibiting mechanisms tightly regulate the effector function of CTLs and NK cells to prevent autoimmune events and preserve tissue homeostasis. In this regard, immune checkpoints (ICs) are signaling pathways that modulate the immune response. CTLs and NK cells can express IC receptors that, when interacting with IC ligands, activate IC signaling pathways, blocking their cytotoxic activity. These IC ligands can be expressed by immunosuppressive cells, including M2-like macrophages, myeloid-derived suppressor cells (MDSCs), and T-regulatory (Treg) cells, as well as cancer cells. The continuous interaction between IC ligands and their respective IC receptors expressed by CTLs and NK, help produce a dysfunctional state in these immune cells known as exhaustion. Tumors avoid antitumoral immunity by upregulating the expression of IC ligands and recruiting immunosuppressive cells, which give rise to an immunosuppressive tumor microenvironment (TME). Tumors with a strong immunosuppressive TME have been associated with impaired immune cytotoxicity, are more aggressive, and have a poor prognosis.
Effector cells: cytotoxic T lymphocytes and natural killer cells
CTLs and NK cells are the two major immune populations that are able to eliminate malignant cells. CTLs participate in the adaptive immune response while NK cells are part of the innate immune system. Cytotoxicity arises by two pathways: Perforin/Granzyme B/Granulysin-related lysis, and death receptor-induced apoptosis. Although CTLs and NK cells act in a mechanistically similar fashion, the regulation of the activity of these immune cells, and the recognition of the targets differ. CTL cytotoxicity is acquired after T cell activation upon antigen presentation by antigen presenting cells (APCs) —mainly dendritic cells (DCs) — whereas NK cells lyse target cells without antigen presentation. When activated, CTLs and NK cells both secrete interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which stimulate a pro-inflammatory immune response. Antitumoral effects have been extensively ascribed to these two immune cell populations, highlighting the relevance of comprehensively understanding the activation and inhibition mechanisms that regulate their cytotoxic activity against cancer cells by pharmacological strategies.
CTLs are defined as activated effector CD3+ CD8+ T lymphocytes and recognize target cells via the interaction between polyclonally rearranged T-cell receptor (TCR) with a peptide/MHC class I complex. Naïve CD8+ T cells interact with APCs and, upon the correct presentation of the peptide-MHC class I complex, TCR signaling causes the formation of a stabilization complex between T cells and the APC. To become fully activated, the co-stimulatory receptor CD28 must interact with its ligands, CD80 (B7.1) and CD86 (B7.2). The activity of T cells is determined by the balance of positive and negative signals from co-activator and co-inhibitory receptors when they recognize their target. To eliminate target cells, CTLs produce a stabilization complex, after which, lytic granules are secreted. Perforin forms pores in the extracellular membrane of the target cells, allowing Granzyme B and Granulysin to enter the cytosol and induce apoptosis, while membrane-bound FasL binds to its receptor Fas, inducing apoptosis in an independent manner.
Human NK cells are phenotypically defined as CD3− CD56+ lymphocytes. Two functionally distinct subsets of NK cells have been defined on the basis of their levels of expression of CD56 and CD16 (also known as Fcγ receptor III). NK cells with high-density expression of CD56 (CD56bright) and CD16− are mostly found in lymph nodes and have a great ability to release immune-modulating cytokines such as IFN-γ and TNF-α. On the other hand, low-density expression of CD56 (CD56dim) CD16+ NK cells mostly occurs in peripheral blood, where it presents a more cytotoxic phenotype characterized by high levels of Perforin and Granzyme B expression. Cytotoxic NK cell activity is independent of foreign antigens presented by MHC molecules. The balance between activation and inhibition signals, which NK cells sense through multiple innate receptors, allows the cells to respond to alterations such as cellular stress, cellular transformation, and malignancy. When activated, NK cells form a stabilization complex similarly to CTLs and release cytotoxic granules.
CTL and NK-cell activity is tightly controlled to preserve antigenic self-tolerance. Autoreactive T-cell clones are eliminated in the thymus by a process known as central tolerance. Also, a peripheral regulation of the cytotoxic response is essential to avoid inappropriate responses to self-antigens. The release of immunosuppressive molecules by M2-like macrophages and Treg cells plays a key role in establishing immune self-tolerance. Activated CTLs and NK cells upregulate the expression of multiple coinhibitory receptors, known as ICs receptors, which downregulate their cytotoxic activity when binding to their ligands, ensuring the precise regulation of their effector function (Fig. 1). Although self-tolerance mechanisms are tightly regulated, T-cell exhaustion occurs and is often observed in tumors and chronic infections. NK cells can present a similar exhausted phenotype that is characterized by stronger expression of coinhibitory receptors and weaker expression of activating receptors. Tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating NK cells exhibit enhanced expression of IC receptors. This has boosted interest in understanding how these coinhibitory receptors function in order to therapeutically block them. The best characterized IC receptors are the cytotoxic T-lymphocyte-associated molecule 4 (CTLA-4) and the programmed cell death protein 1 (PD-1), but many other ICs play key central roles in controlling CTL and NK cell effector functions. (J Exp Clin Cancer Res. 2022; 41: 62.)