A major downstream target of the synergistic effect of PI3K/Akt and Ras/MEK/Erk activation in T cells is the cell cycle machinery. Primary T lymphocytes naturally reside in the G0 phase and lack expression of cyclins, which are required to interact with cyclin-dependent kinases (Cdks) to form cyclin–Cdk holoenzyme complexes that drive cell cycle progression (91–93). p27^kip1, a member of the Kip/Cip family of Cdk inhibitors, interacts with Cdk2 and is abundantly present in T cells. Ubiquitin-dependent degradation of p27^kip1 is required to initiate cell cycle progression and entry to S phase by allowing activation of Cdk2. This event is predominantly mediated by Skp1-Cullin-F-box (SCF) family ubiquitin ligase, SCF^skp2 (94). TCR/CD3 and CD28 costimulation regulates the transcriptional induction of Skp2, a substrateSCF^skp2 ubiquitin ligase, and this process requires simultaneous activation of PI3K/Akt and Ras/MEK/Erk pathways (95). Ligation of PD-1 during the T cell stimulation causes abrogated expression of Skp2, resulting in increased p27^kip1 level and Cdk2 inhibition (83). The impaired Cdk2 activity inhibits Rb phosphorylation, impacting its interaction with chromatin remodeling proteins. Inhibited Cdk2 also fails to phosphorylate the checkpoint inhibitor Smad3, upregulating its transcriptional activity (96) and resulting in the increased abundance of the G1 phase Cdk inhibitor, p15^INK4B, and the loss of the Cdk-activating phosphatase Cdc25A (83, 97).

One major consequence of PD-1-mediated Cdk2 inhibition and subsequent reprograming of Smad3 transcriptional events is the conversion of naive T cells into induced T regulatory cells. Regulatory T cell populations are critical for the maintenance of peripheral tolerance, are potent inhibitors of immune responses and play an important role in the prevention of graft rejection (98, 99). Foxp3⁺ T_Regs can be divided into two subsets: natural T_Regs (nT_Regs) and induced T_Regs (iT_Regs). CD4+Foxp3+nT_Regs arise as committed regulatory cells from thymus (100), while iT_Regs (or adaptive T_Regs) develop in the periphery from CD4⁺Foxp3⁻ naive T cells in a TGF-β- and IL-2-dependent fashion (101–107). PD-1 regulates the function of Smad3 and synergizes with TGF-β-mediated signals (83). This synergizing effect on naive T cells promotes the differentiation of T_Reg cells (108), thereby suppressing generation and function of T effector cells (T_EFF) via a cell extrinsic mechanism. In addition, generation and function of T_Reg cells requires the aLb2 (LFA-1) integrin, whose activity is dependent on small GTPase Rap1 (109–112). Importantly, PD-1 does not inhibit Rap1 activation (83), indicating that PD-1 also supports the pathways required for T_Regs to perform their immunosuppressive functions. Experiments with PD-L1-deficient APCs resulted in minimal conversion of naive CD4⁺ T cells to iT_Regs. PD-L1-Ig has also been shown to increase Foxp3 expression and suppressive function of established iT_Regs through attenuation of Akt-mTOR signaling and concomitant upregulation of PTEN signaling events that are known to drive generation of iT_Regs (108, 113–115).

Upon activation, signals from the CD28 costimulatory pathway and the γ-chain signaling cytokines promote naïve T cells to switch their metabolism from oxidative phosphorylation to glycolysis, which is required to support their growth, proliferation, and effector functions (116–119). Divergence in the metabolic reprograming is critical for imprinting distinct T cell fates. Namely, preferential switching to glycolysis accompanies effector T cell differentiation (120) and switching to fatty acid beta-oxidation (FAO) causes the conversion of T effector to T memory cells (121). Furthermore, imposing FAO by pharmacologic means boosts the generation of T_Reg cells (122). Studies investigating metabolism profile of T cells receiving PD-1 signals have shown that PD-1 ligation disengaged them from glycolysis, glutaminolysis, or metabolism of branched chain amino acids, but induced increased rate of FAO (123). While PD-1 ligation inhibited the expression of receptors and enzymes involved in glycolysis and glutaminolysis, it increased the expression of carnitine palmitoyl transferase (CPT1A), the rate-limiting enzyme of FAO. T cell activation causes an increase in extracellular acidification rate (ECAR), an indicator of glycolysis, and in oxygen consumption rate (OCR), an indicator of oxidative phosphorylation. PD-1 engagement results in lower ECAR and OCR, but higher OCR/ECAR ratio compared with T cells stimulated without PD-1 ligation (Figure 1). By altering the metabolic programs of T cells, PD-1 ligation seems to generate a more oxidative environment (123, 124).

Programed cell death 1 has unique regulatory roles in the control of virus-specific immune responses, and these regulatory functions have been studied extensively during chronic viral infections. The CD8⁺ effector T cells behave differently in acute and chronic viral infections. During acute infection, naïve antigen-specific CD8⁺ T cells get activated, proliferate, and differentiate into effector CD8⁺ T cells and efficiently clear the virus. Most of these virus-specific effector CD8⁺ T cells then become apoptotic and a very small number (5–10%) of long-lived memory cell population arises, which is protective against secondary infection (125). However, during chronic viral infection, sustained antigenic stimulation engenders the loss of effector T cells and their failure to develop into memory CD8⁺ T cells (Figure 2A). Under these conditions, T cells become unresponsive to viral antigens and persist in a non-functional, exhausted state (T_EX), in which they are unable to clear virus effectively (126).

During the development of exhaustion, loss of effector functions happens in a hierarchical manner: IL-2 production, high proliferative capacity and in vitro cytolytic activity are lost first, followed by impairment in the production of TNF-α, IFN-γ, and degranulation (127, 128). Although incapable of functioning as effector or memory, T_EX cells are not functionally dormant. Instead, they commit to the containment of chronic infections, because depleting CD8⁺ T cells including T_EX during simian immunodeficiency virus (SIV) infection results in rapid increase in viral titers and progression to AIDS (129, 130), suggesting an important role for the residual function of SIV-specific T_EX in maintaining a host–pathogen equilibrium and contributing to the containment of the chronic infection. T_EX cells often retain the capacity to produce low levels of IFN-γ and/or beta chemokines and express high levels of granzyme B. In addition, one subset of T_EX retains some residual cytotoxicity (127, 131, 132). High granzyme B expression is an interesting feature of T_EX, given that the ex vivo killing capacity of these cells is impaired compared with T_EFF (131, 132). Thus, while T_EX cells exhibit impaired effector functions, some residual functionality remains, and this may be important in a host–pathogen equilibrium. In addition to PD-1, T_EX cells expressed higher levels of other inhibitory receptors (e.g., Tim-3, Lag-3, and CD160) as well (131). However, blockade of PD-1 is sufficient to induce reinvigoration of a significant fraction of this cell population, which selectively expresses T-bet^Hi Eomes^Lo PD-1^int and has the ability to proliferate after PD-1 blockade. In contrast, T_EX cells exhibiting Eomes^Hi PD-1^Hi are unable to respond after PD-1 blockade (132). Similar subsets of T_EX defined by reciprocal patterns of T-bet, Eomes, and/or PD-1 expression have been found in human patients with HCV and HIV infection (133, 134). In these patient populations, PD-1 blockade resulted in augmentation of pathogen-specific T cells and decrease of viral load (46, 47).

PD-L1 is expressed on a wide variety of non-hematopoietic cells and plays a key role for the maintenance of self-tolerance (Figure 2B). PD-1 and PD-L1 levels increase after heart allotransplantation and their levels correlate with the likelihood of rejection, while the use of a PD-L1-Ig fusion protein decreased rejection (135). This finding was of great interest as PD-L1 is expressed in endothelial cells, which are located between the graft cells and the immune cells and suggests a potential target to decrease the rates of graft rejection. Similarly, GvHD occurring after bone marrow transplantation (BMT) has been associated with expression of PD-1 in the infiltrating cells. However, deficiency of the PD-1 pathway has also been related to higher mortality resulting from GvHD (136, 137).

Programed cell death 1 appears to be of major clinical relevance in autoimmune diseases, such as diabetes mellitus type I (DM I) and systemic lupus erythematosus (SLE). PD-L1 is expressed in pancreatic beta islet cells and limits the activation and harmful cytotoxic function of self-reactive T-cells against islet cells, thereby protecting from autoimune damage. Treatment of non-obese mice with PD-1- and PD-L1-blocking antibodies caused faster development of DM I, while treatment with PD-L2 blocking antibody had no effect (138, 139). In SLE, data that associates polymorphisms of the PD-1 gene with susceptibility to the disease in humans (140) are in line with evidence that mice deficient in PD-1 develop manifestations that resemble SLE, including glomerulonephritis and arthritis (7). Involvement of the PD-1 pathway in other autoimmune diseases, namely multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease, is also suggested by studies in animals and attributed to either absence or non-functionality of regulatory T-cells (115).

The expression of PD-L1 and PD-L2 on APC after exposure to IFN-γ and the expression of PD-L1 in cancer cell lines (15) and primary cancer cells (141) led to the hypothesis that blockade of the PD-1:PD-L1/2 inhibitory pathway might induce antitumor immunity. The hypothesis that engagement of PD-1:PD-L1 pathway might dampen immune responses for tumors was confirmed by the observation that overexpression of PD-L1 on a mouse mastocytoma cell line inhibits CD8⁺ T cell cytolytic activity through PD-1 ligation, which intensifies tumor growth and invasiveness (142). Studies in various types of human cancers have confirmed that tumors exploit PD-1-mediated immune suppression to escape immune surveillance. A wide variety of solid tumors, including urothelial, ovarian, breast, cervical, colon, pancreatic, gastric, melanoma, glioblastoma, non-small cell lung cancer (NSCLC), and hematologic malignancies have been found to express PD-L1 and to a lesser extent PD-L2, which correlate with adverse prognosis (143–149). Importantly, the presence of PD-L1 within the tumor microenvironment (TME) also correlates with a better clinical response to PD-1/PD-L1 checkpoint blockade therapy (17, 18, 150). In addition to cancer cells, PD-L1 and PD-L2 are also expressed in other cellular components of the TME including macrophages (mostly M2), myeloid DCs, myeloid suppressor cells (MDSC), stromal fibroblasts, and endothelial cells (Figure 2C). Similarly to cancer cell-specific expression, PD-L1 expression on tumor-infiltrating immune cells correlates with clinical responses to PD-1:PD-L1 blockade therapy. Conversely, lack of PD-L1 upregulation in tumor cells or tumor-infiltrating immune cells correlates with lack of therapeutic response and disease progression (151).

PD-L1 expression on cancer cells can be mediated by cell intrinsic mechanisms activated by oncogenic mutations (152). PD-L1 expression on cancer cells and tumor-infiltrating immune cells can also be induced by local inflammation, i.e., type I/II IFN-gamma released by activated T cells, a condition termed “adaptive immune resistance” (Figure 2C) (153). It should be noted that reported studies use a different cutoff of PD-L1 expression level to define positivity and variable approaches regarding evaluation of PD-L1 expression only on cancer cells or also on tumor-infiltrating immune cells (17, 18, 150). Use of different antibodies for histopathological assessment of PD-L1 expression may also lead to variable conclusions. Regardless of these confounding factors, there is an unequivocal conclusion that the degree of PD-L1 expression in the TME positively correlates with clinical response.

When the PD-1/PD-L1 pathway is active in the TME, it promotes survival of cancer cells via antiapoptotic signals mediated via PD-L1 (141, 154) and inhibits the activation of signaling pathways, which are critical for survival, expansion, and differentiation of T cells that recognize tumor antigens. The imbalanced activation of signaling events in T cells results in tumor tolerance by inhibiting T effector and memory cell generation and promoting the differentiation of T_EX and T_Reg cells (Figure 3, left side). Importantly, high expression level of PD-1 has been detected on tumor-infiltrating T cells, compared with T cells in normal tissues and peripheral blood from the same patients and healthy donors, and correlate with an exhausted phenotype and an impaired effector function (155). Blocking the PD-1/PD-L1 pathway by anti-PD-1 or anti-PD-L1 antibodies suppresses cancer cell survival, reverses the effects of PD-1 on T cell signaling, and promotes the generation of T effector and memory cells while preventing the differentiation of T_EX and T_Reg cells. Together, these cell signaling and functional programs enhance antitumor T cell responses, leading to tumor regression and rejection (Figure 3, right side). It remains to be deciphered whether the therapeutic outcome of PD-1 blockade is different between patients with oncogenic PD-L1 expression versus immunogenic PD-L1 expression, in which PD-L1 is expressed on cancer cells and immune cells, respectively.

The use of antibodies that block immune checkpoints in the treatment of solid tumors was officially established in the armory of anticancer therapies in 2010 when ipilimumab, a CTLA-4 inhibitor, showed to improve survival in metastatic melanoma and led to the FDA approval of ipilimumab for the treatment of melanoma (175). Based on the fact that – similar to CTLA-4 – PD-1 is a coinhibitory receptor, antibodies have been developed with the goal to inhibit the PD-1:PD-L1 pathway (Table 1). These antibodies have generated remarkable responses in a wide spectrum of cancers (Table 2) and have shown better clinical benefit and better toxicity profile than CTLA4-blocking antibodies (158, 170).

Nivolumab, an IgG4 PD-1 antibody, binds to PD-1 with high affinity and specificity and was the first PD-1 blocking agent to demonstrate clinical activity in several different types of cancers, including melanoma, RCC, and NSCLC in a phase I/II trial completed in 2012 (18). In a phase Ib dose escalation study, 32% of the patients with advanced melanoma developed durable remission, which correlated with expression of PD-L1 in the tumor cells defined as positive at a minimum level of 5% (18, 168). Subsequently, in a phase III study, which compared nivolumab with dacarbazine in patients with melanoma without B-Raf mutation, nivolumab was associated with a survival benefit (73 versus 42%) and higher objective response rate (40 versus 14%). The response rate of patients with PD-L1-positive tumors (defined as PD-L1-positive tumor cells >5%) was also better than in patients with PD-L1-negative/intermediate tumors (53 versus 33%) (150). In the second phase III trial, nivolumab was compared with chemotherapy in patients with advanced melanoma non-responsive to ipilimumab (or ipililumab and BRAF inhibitor in BRAF-mutant tumors). A response rate of 32% was noted in the nivolumab treatment group versus 11% in the chemotherapy treatment group. This study also found PD-L1 as a response-predictive biomarker with 44% response rate in PD-L1 positive versus 20% in the PD-L1 negative tumors (171). Based on these outcomes, FDA approved nivolumab on December 22, 2014 for the treatment of patients with melanoma, whose disease state has progressed after prior treatment.

Pembrolizumab is a very high affinity humanized IgG4 antibody directed against human PD-1. Randomized trials have been performed in both ipilimumab naïve (NCT01866319) and previously treated patients (NCT01704287). Promising results in preliminary studies (165, 166) led to a phase II dose escalation trial comparing two dose levels of pembrolizumab to chemotherapy in ipilimumab-refractory melanoma patients (176). This study showed clear benefit for both groups that received pembrolizumab with 6-month progression-free survival (PFS) of 34 and 38% compared to the PFS of 16% of the chemotherapy group. A subsequent phase III trial compared the treatment outcome of two different administration schedules of pembrolizumab to ipilimumab in patients with advanced melanoma and provided evidence of improved survival rate in both pembrolizumab treatment groups compared to the ipilimumab group (74 and 68 versus 58%) (158). On September 4, 2014 pembrolizumab was approved by FDA for the treatment of advanced melanoma in patients previously treated with ipilimumab or a BRAF inhibitor in BRAF V600 mutation positive patients. To date, both nivolumab and pembrolizumab have also been approved by FDA and used for treatment in NSCLC, head and neck cancer, RCC, and Hodgkin lymphoma.

The success of PD-1 blocking antibodies in NSCLC has made headlines since checkpoint blockade was thought to be responsive solely in immunogenic tumors like melanoma and RCC. A phase I dose-evaluating study of nivolumab has shown responses in patients with squamous and non-squamous histology, with or without EGFR or KRAS mutations, with or without tumor PD-L1 expression, and across different dose levels (18). On the basis of these preliminary results, two randomized phase III studies were undertaken. One compared nivolumab to docetaxel in advanced squamous NSCLC and found an improved median overall survival (OS) (9.2 versus 6.0 months) (172). In this study, tumor expression of PD-L1 determined at three different expression cutoff levels (1, 5, and 10%) had neither prognostic nor predictive of treatment benefit. The second study followed the same design and studied responses of patients with non-squamous NSCLC. In this patient group, nivolumab also resulted in improved median OS benefit compared to docetaxel (12.2 versus 9.4 months) while the OS at 1 year was 51% in the nivolumab group versus 39% in the docetaxel group (177). Correlations between therapeutic benefit and PD-L1 expression on tumor cells at the same cutoff levels (1, 5, and 10%) was also studied. In contrast to the observations in patients with advanced squamous NSCLC (172), patients with PD-L1-positive non-squamous NSCLC tumors had therapeutic benefit over those with PD-L1-negative tumors and this was observed across all three PD-L1 expression levels. Nivolumab was approved by FDA in March, 2015 for treatment of squamous NSCLC, and eventually for all patients with advanced NSCLC progressing after platinum-based chemotherapy on October 9, 2015. Almost in parallel, FDA also approved Pembrolizumab on October 2, 2015 for PD-L1-positive NSCLC, based on a large clinical trial, which assessed efficacy and safety of pembrolizumab in patients with advanced NSCLC (17). As in previous studies, therapeutic benefit was correlated with tumor PD-L1 positivity, which in this study was defined at the >50% cutoff. Importantly, this study also provided evidence that a striking survival benefit was observed in patients who received pembrolizumab without prior treatment with chemotherapy.

More than 100 trials are currently investigating the use of PD-1 blockade agents as monotherapy or in combination with chemotherapeutic agents, targeted therapies, or alternate immunotherapy modalities for multiple tumor types (http://clinicaltrials.gov).

For RCC, immunotherapy has always been considered as a primary therapeutic strategy because of its immunogenic nature. The rationale for treatment with PD-1:PD-L1 blockade in RCC was further supported by the excessive PD-L1 expression in inflamed and cancerous kidney tissues (178). A randomized phase II trial comparing different doses of nivolumab in advanced RCC patients has shown a long-lasting objective response in about 22% of the patients (179). Currently, a combinatorial treatment regimen of nivolumab with either sunitinib or pazopanib is being developed, which has shown better efficacy, but higher toxicity (180). The efficacy of pembrolizumab is also currently being evaluated in a phase I/II trial in treatment naïve metastatic RCC patients in combination with pazopanib or axitinib.

One of the most impressive responses has been observed in HL, in which PD-1 blockade with nivolumab resulted in response rate of 87% (19, 181, 182). This outcome is based on the molecular upregulation of the PD-1:PD-L1 pathway through amplification of 9p24.1, which increases the gene dosage of PD-L1 and PD-L2 together with Jak2 in nodular sclerosing HL (183).

Currently, ongoing clinical trials are investigating PD-L1 blocking antibodies. Such antibodies, specifically MPDL3280A (Atezolizumab) and MEDI4736 (Durvalumab) are being evaluated in metastatic melanoma. Interestingly, Atezolizumab was associated with good responses and less pulmonary toxicity compared to PD-1 antibodies (184). In a randomized phase II trial, Atezolizumab is being compared with platinum-based chemotherapy and docetaxel after platinum failure in NSCLC. Durvalumab in combination with an EGFR inhibitor is being compared to chemoradiation in stage III NSCLC, where an objective response rate of 14% has been noticed across all histologies (185, 186). Atezolizumab is also currently being investigated as monotherapy or in combination with bevacizumab in comparison to a control group of sunitinib in treatment-naïve locally advanced or metastatic RCC. In a recent study, one complete and two partial responses were observed in patients with recurrent or metastatic triple negative breast cancer who are PD-L1 positive. PD-L1 blockade therapy also appears to be effective in bladder cancer. In a phase I study of atezolizumab in advanced bladder cancer, an objective response rate of 43% has been observed in tumors expressing high levels of PD-L1 (160).

The combination of checkpoint blockade was first tested in advanced melanoma patients treated with nivolumab and ipilimumab and the resulting clinical activity was phenomenal (187). In a phase II study, the objective response rate for nivolumab plus ipilimumab was 59% in comparison to 11% with ipilimumab alone (188). Most recently, a phase III study of nivolumab plus ipilimumab versus nivolumab versus ipilimumab was performed in treatment naïve advanced melanoma patients (170). Again, the response rate was 57.6% for the combination therapy in comparison to 43.7% for nivolumab and 19% for ipilimumab monotherapies. The improved outcomes of the combination therapy over ipilimumab alone appear to be sustained within the 2-year follow-up of patients with combination therapy (189). The combination approach was also tested in patients with metastatic RCC. In this patient group, ipilimumab plus nivolumab in two different dose levels gave a response rate of 43 and 48%, respectively (190). Comparison between the combination checkpoint immunotherapy and sunitinib in advanced RCC is under investigation.

In NSCLC, a phase III study of nivolumab plus ipilimumab versus nivolumab monotherapy versus chemotherapy is currently undergoing (NCT02477826). Promising results were also reported from a phase I study of combination of Durvalumab and tremelimumab in NSCLC. Also, another phase III study in untreated, advanced NSCLC has recently begun with durvalumab plus tremelimumab versus durvalumab versus chemotherapy (NCT02453282). The combinatorial studies examining efficacy and safety of these drugs are also been undertaken in several other malignancies, including SCLC (191), gastric, and bladder cancer (NCT01928394). Because of different cellular expression/localization of PD-1 and PD-L1 in normal tissues, the tolerability of combination of PD-1 plus PD-L1 is also being investigated (NCT02118337).

In order to understand how PD-1 blockade imparts tumor rejection, it is critical to identify the cell population(s) that are targeted and altered during antibody treatment (192). The presence of PD-L1 within TME in more than 1% of tumor cells has been shown to correlate with a better clinical response to PD-1/PD-L1 checkpoint blockade therapy. In contrast, lack of PD-L1 upregulation in tumor cells or lack of tumor-infiltrating immune cells has been observed in most progressing patients (18, 151, 193). However, studies in RCC have determined that detectable tumor expression of PD-L1 can be documented only in a small fraction of patients (20–30%), yet, a higher number of patients with PD-L1 negative RCC responded to PD-1 blockade (194).

As mentioned above, in addition to tumors, PD-L1 expression on tumor-infiltrating immune cells, mainly myeloid APC (macrophage and myeloid DCs) correlates with clinical responses to PD-1:PD-L1 blockade therapy (151). Based on these findings, it is possible that therapeutic PD-1 blockade might work more effectively if the tumors have already been identified by the host immune system and PD-L1 expression in cancer and innate immune cells is the consequence of local IFN-γ production by tumor-activated T cells (151, 193). Thus, one key approach to understand which cell types are important for tumor rejection is to determine location, density, and phenotype of the immune cells inside the TME and their spatiotemporal expression of PD-1 and PD-L1. Techniques to achieve this goal include, but not limited to, slide-based quantitative immunehistochemistry (IHC) and quantitative multiplexed IHC in situ gene expression assay (193, 195–197). The use of different anti PD-1 and anti PD-L1 antibodies, the different cutoff points to measure expression, the different cell types in which expression is being evaluated and the different scoring systems used by various pathology laboratories has caused difficulty in harmonizing the IHC readouts. The Cancer Immunotherapy Trials Network has started to review the immunodynamic effects of checkpoint inhibitors with the goal to identify and define immune assessment modalities and sites, both systemic and intratumoral, which are critical to the therapeutic success (198). Refining immune endpoints will provide the tools for the design of improved clinical trials, for selection of appropriate candidate patients for PD-1-based immunotherapy, and for assessment of induction and maintenance of therapeutic response.