Keywords

1 Introduction

It is evident that both the innate and adaptive immune systems participate in the recognition and clearance of tumor cells by a process known as cancer immunosurveillance. In particular, tumor-reactive CD8+ cytotoxic T-lymphocytes (CTL) are major effectors in the immune response against cancer cells. However, despite the powerful aspects of CTL-mediated immune reactions, too often tumor cells are able to evade immune recognition and destruction. Tumor cells exploit several mechanisms to escape from CTL-mediated immunity, such as disruption of antigen presentation, down-regulation of HLA molecules, recruitment of regulatory T-cells (TREG) and myeloid-derived suppressor cells, as well as secretion of immune suppressive cytokines [1]. In the last decade, another powerful immune suppressive mechanism came into the limelight: the repressive action of co-inhibitory receptors [2].

2 CTL Activation

Activation of T-cells to become CTL effectors, initially requires two signals [3–5]. Firstly, the TCR-CD3 complex needs to interact with the cognate peptide presented in HLA molecules on dendritic cells (DC). However, whether or not the T-cell becomes activated, is predominantly dependent on signaling of either co-stimulatory molecules (CSM) or co-inhibitory molecules (CIM) upon ligation with their corresponding ligands expressed by the APC [6]. The balance between these positive and negative co-signals determines the functionality of T-cells during immunity and tolerance. The stimulatory signal is generally provided by CD28, expressed on the T-cell, interacting with its ligands CD80 and CD86 on the DC. In the absence of co-stimulation the T-cell will become functionally anergic, and thereby tolerant to the antigen, which is one of the physiological mechanisms involved in the elimination of self-reactive T-cells. In addition, ligation of CIM to their corresponding ligands on APC results in T-cell inhibition, and via this natural feedback loop, sustained T-cell activation is prevented and the effector T-cell response resolves. Therefore, the balance in positive and negative co-signals determines the activation state of the T cells during immunity and tolerance.

The kinetics and differentiation of CTL that constitute anti-tumor responses are divided in several stages [7]. First, the CD62L+CCR7+CD45RA+ naive T-cells (TN) encounter the antigen presented by DC. Due to the expression of the selectin CD62L and the chemokine receptor CCR7 these cells home to the secondary lymphoid organs. However, upon this stimulation by the DC, these T-cells clonally expand and loose the expression of CD62L, CCR7, CD28 and CD45RA, while upregulating activation markers such as CD45RO, CD69 and CD25. These effector T-cells (TEFF) subsequently migrate to the target tissues, where they eradicate tumor cells. After the peak of the response, upon which most or all target cells have been destroyed, the contraction phase commences, and most tumor-reactive T-cells will undergo apoptosis. However, a minority of the T-cells will survive to become long-lived memory cells, either effector (TEM) or central memory (TCM). While the TEM reside in the periphery and upon recall show a strong effector response, the TCM have a strong proliferative property, regain expression of CD62L and CCR7, and migrate to the lymph nodes and BM, where they convey a lifelong memory against the antigen of their specificity. During all these activation and differentiation events, signaling through CSM and CIM has a great influence on the functional capacity and differentiation status of CTL.

3 Tumor-Associated Antigens

The immune system can harness a powerful attack against cancer cells. This can be done by cells from the innate immune system, such as NK cells that can attack tumor cells without prior sensitization [8]. Furthermore, the adaptive T-cell immune system has a great potential of recognizing and lysing tumor cells. This is mainly done by CTL, which recognize tumor-associated antigens (TAA) presented by the cancer cells. TAA are overexpressed, or ideally, solely expressed by tumor cells and consequently recognized as foreign, and an effective CD8+ T-cell immune response can be constituted against these antigens. Different classes of TAA exist. One class of TAA is highly overexpressed differentiation genes, such as tyrosinase and gp100 in melanoma, proteins which are differentially expressed at low levels in healthy melanocytes [9].

Another class is the oncofetal antigens, like carcinoembryonic antigen (CEA), which are usually expressed only in the fetal stage, and therefore no immune tolerance against these TAA exists [10]. Furthermore, cancer-testis antigens, including MAGE-A, NY-ESO-1, ACRBP and CTAG1B, can be aberrantly expressed by tumor cells [11, 12]. Since these are also only expressed by fetal tissues and the immune-privileged testes, a prominent immune response can be observed against these TAA. Finally, new antigens caused by de novo mutations in the cancer cells can occur in any gene. Since these mutations result in a true novel epitope, a very strong CTL response can be elicited. Especially in cancers with a high mutation rate, such as melanoma and lung cancer, these novel TAA occur frequently [13].

Altogether, these TAA are the major target in natural CD8+ T-cell tumor surveillance, and form an attractive field for immunotherapy, such as tumor infiltrating lymphocyte (TIL) infusion or DC vaccination loaded with TAA [14]. Vaccination with the most potent APC, i.e. DC, provides a great option for antigen-specific stimulation of tumor-reactive CTL [15]. Currently, DC vaccination is being performed in phase 3 clinical trials against four malignancies, including melanoma, prostate cancer, glioma and renal cell carcinomas [16]. In prostate cancer, vaccination is being performed against the prostate cancer-antigen prostate acid phosphatase (PAP) with the sipuleucel-T treatment [17]. In addition, vaccination with melanoma-antigens has reached promising results [18]. Several parameters of DC vaccination still need to be optimized, such as DC culture, choice of DC subpopulation, the method of loading of tumor antigens, choice of maturation stimuli, and method of administration to the patient [19]. However, in the majority of studies an increase in the median overall survival has been documented, underlining the potential of this therapy [16].

4 Allogeneic Stem Cell Transplantation

Another cancer immunotherapy, based on CTL recognition of antigens expressed on tumor cells, is allogeneic stem cell transplantation (alloSCT). This procedure can still be regarded as one of the most powerful cell-based immunotherapy to date, due to potent graft-versus-tumor (GVT) responses constituted by alloreactive T-cells [20]. These alloreactive CD8+ T-cell responses eradicate the malignant cells upon recognition of polymorphic HLA-presented peptides, known as minor histocompatibility antigens (MiHA). AlloSCT greatly enhanced the cure rate for aggressive hematologic cancers, although many patients still fail to launch effective immune responses and develop relapsed disease. Moreover, a major drawback of alloSCT is the occurrence of graft-versus-host disease (GVHD), a potentially life-threatening complication predominantly caused by alloreactive T-cells recognizing healthy tissues, notably the skin, liver and gastrointestinal tract. Since hemato-restricted MiHA are solely expressed by the redundant patient hematopoietic system including the malignant counterparts, they hold the key to separate GVT from GVHD [21]. In fact, these MiHA are equally immunogenic as de novo TAA or viral epitopes, since the antigens are completely foreign to the donor immune system and immune tolerance has not been initiated. Therefore, alloSCT can be a very powerful and curative cancer immunotherapy.

5 Adoptive T-Cell Transfer

Adoptive transfer of CTL is an appealing means to prevent or treat relapse of the tumor cells, and so far various strategies have been exploited. Nevertheless, specificity is crucial to avoid systemic toxicity. One method to obtain sufficient numbers of T-cells reactive against a TAA or MiHA is via isolation of these cells from the effector repertoire of patients present, followed by a fast expansion protocol [22, 23]. Already in the 1980s, the first studies with tumor-reactive T-cells in mice were performed by the isolation of TIL and subsequent culture and administration, which resulted in remission of cancer [24]. This led to clinical trials in humans, and a response rate near 50 % or more has made TIL administration an established treatment option [25]. A different technique is the isolation and expansion of naive tumor-reactive T-cells from a healthy donor by ex vivo stimulation with peptide-presenting DC [26, 27]. However, this can be a time-consuming and laborious process, especially for overexpressed TAA. The feasibility of both approaches has been demonstrated by several groups [22, 23, 26–28]. Importantly, one phase I trial reported in five out of seven patients with relapsed leukemia a complete, but transient, remission upon adoptive transfer of MiHA-specific T-cells expanded from post-transplant recipient PBMC [28]. Unfortunately, the infused T-cells failed to persist in vivo, which might be due to their terminal TEFF/EM differentiation stage and, consequently, rapid exhaustion of these cells as a result of the extensive in vitro culture protocol. To prevent the exhaustion of these T-cells, a search for the T-cell type with the highest proliferative potential has led to the identification of the stem cell memory T-cell (TSCM) [29, 30]. Although TSCM have experienced antigen-stimulation, they resemble TN in their expression of CD62L and their ability to differentiate into all other T-cell differentiation states. In addition, it was found that by inhibition of either the Akt or Wnt pathway in vitro, it is possible to generate high numbers of tumor-reactive TSCM [30, 31]. Together with their proven excellent anti-tumor effects in murine models, this subtype of CTL holds great promise for future therapies [32].

A third way to efficiently generate high numbers of tumor-reactive CTL with high-affinity TCR is by gene transfer of the antigen-TCR α and β chains into donor T-cells [22, 33]. To prevent the induction of GVHD in patients treated with alloSCT, the TCR genes should preferentially be transferred into donor T-cells with a known specificity that does not recognize and target GVHD-tissues, such as virus-specific T-cells [34]. Another potential complication might be mispairing of the introduced and native TCR chains, thereby generating a new potentially harmful specificity [35]. Efforts are being made to prevent this mispairing, amongst which is the transfer of TCR α and β chains into γδ T-cells. Successful TCR gene transfer and resultant cytolytic competence has been demonstrated for both TAA and MiHA [36–39]. Importantly, with TCR gene transfer the complete MiHA-TCR is introduced into the donor T-cells, therefore matching of the HLA-restriction allele between recipient and donor is no longer required.

A novel therapeutic approach utilizing the power of CTL is chimeric antigen receptor (CAR) T-cells. These CAR consist of an antibody fragment recognizing a tumor antigen expressed on the surface of these T-cells. Ingeniously, to enable T-cell activation, this antibody fragment is coupled to the CD3 ξ-chain, leading to an intracellular activation cascaded upon recognition of the antigen [40]. This chimeric receptor combines the high avidity and specificity of antibodies with the activation of CTL, resulting in highly effective CTL responses. Second and third generation CAR have been engineered to express motives of CSM in the intracellular domain, such as CD28, 4-1BB and OX40. Thereby, in addition to the cytolytic capacity of CAR, also proliferation and survival are sustained. Impressive results have been obtained in clinical trials. Especially CAR recognizing CD19 developed by the June lab, have been able to efficiently lyse cancer cells in patients with high tumor burdens, and have resulted in cure of leukemia patients [41]. After this pioneering work in leukemia, other target antigens are currently being explored in different malignancies, making CAR therapy hold great promise for the future.

Nevertheless, despite the curative potential of the cellular therapies described afore, numerous studies have demonstrated that tumor cells explore immune suppressive mechanisms to dampen tumor-reactive CTL responses, resulting in sub-optimal clinical efficacy. One of the pivotal mechanisms exploited by tumor cells is manipulation of CTL activation, either by enhancing CSM or interfering with CIM signaling. Tumor cells can evade immune control by down-regulating CSM such as CD80 and CD86, and up-regulating various co-inhibitory ligands, thereby limiting the therapeutic potential of current immunotherapies against cancer. This chapter will address the role of CIM in tumor immune evasion from CTL attack, and discuss options to prevent T-cell inhibition without severe adverse effects. We will discuss the role of separate CIM involved in tumor escape from CTL, and subsequently elaborate on combinations of CIM in the tumor setting. Finally, the incorporation of CIM interference in near future anti-cancer immunotherapy will be discussed.

6 Co-inhibitory Molecules in Cancer

A variety of CIM have been implicated in cancer immune escape. Here, we discuss the CIM prominently involved in suppressing anti-tumor immunity.

6.1 PD-1

6.1.1 Expression and Function of PD-1

Programmed death 1 (PD-1; CD279) is an immunoreceptor and member of the B7/CD28 family [42]. In 1992, PD-1 was identified on hybridoma T-cells undergoing apoptosis and was believed to be a programmed cell death-induced gene [43]. Further characterization demonstrated that PD-1 is inducibly expressed on stimulated CD8+ T-cells, CD4+ T-cells, B cells and monocytes [44]. PD-1 binds two ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273) [45]. While PD-L1 is expressed on various non-lymphoid tissues, PD-L2 expression is mainly restricted to APC, like DC and macrophages [46]. Furthermore, multiple tumor types express PD-L1 and its expression is elevated upon IFN-γ exposure [47]. PD-L1 molecules on tumor cells can deliver negative signals towards PD-1-expressing tumor-reactive CTL, thereby inhibiting anti-tumor immunity [48]. Indeed, PD-L1 expression has been associated with poor prognosis in solid tumors [47].

It has been demonstrated that PD-1 plays a crucial role in T-cell regulation in various immune responses such as peripheral tolerance, autoimmunity, infection and anti-tumor immunity [46]. High PD-1 expression on viral antigen-specific CTL in chronic viral infections was recognized as a hallmark for T-cell dysfunction upon antigen re-encounter [49]. This phenomenon, known as exhaustion, is characterized by the sequential loss of the ability to proliferate, secrete cytokines and kill target cells. Especially in HIV infection, T-cell impairment could be relieved by PD-1 blockade both in vitro and in animal models [50, 51]. It has also been shown that PD-1 strongly attenuates the downstream signaling of the TCR [52]. In an elegant model system, the influence of PD-1 ligation on T-cell triggering was investigated [53]. Engagement of PD-1 raises the threshold of T-cell stimulation by increasing the number of TCR/peptide-MHC complexes needed for activation. It has been reported that exhausted T-cells have elevated expression of multiple CIM and a distinct gene signature, different from anergic cells, resulting in changes in TCR and cytokine signaling pathways [54]. The importance of downstream PD-1 signaling was nicely demonstrated by the identification of an exhaustion-specific gene signature in HIV-specific T-cells [55].

6.1.2 PD-1 in Cancer

PD-1 has been shown to have a prominent suppressive function in anti-cancer immunity. Expression of its ligand PD-L1 on tumor cells conveys a negative signal to tumor-reactive T-cells [56]. In addition, PD-1-expressing TIL present in breast cancer tissue are associated with a poor prognosis [57]. Moreover, in melanoma was shown that PD-1+ TIL were functionally impaired as compared to their PD-1-negative counterparts [58].

The involvement of PD-1 in alloSCT has been investigated both in mouse models and in the human setting. In a study investigating chronic myeloid leukemia (CML), using a retrovirus-induced CML model, it was demonstrated that tumor-specific T-cells can become exhausted [59]. In this model, consisting of PD-1+ tumor-reactive T-cells and PD-L1+ CML cells, exhaustion could be overcome by the administration of either PD-L1 antagonistic antibody or PD-1 deficient T-cells. In accordance, it was shown that the bulk T-cell population from CML patients exhibited increased expression of PD-1 [59]. Also, in the alloSCT setting, high PD-1 expression was observed on alloreactive CTL that specifically recognize hemato-restricted MiHA in myeloid leukemia patients [60]. Furthermore, proliferation of these PD-1+ MiHA-specific CTL by stimulation with MiHA-loaded DC ex vivo was suboptimal, indicating dysfunctional CTL due to PD-1 signaling. Importantly, upon treatment with anti-PD-1 or anti-PD-L1 blocking antibodies ex vivo proliferation of the MiHA-specific CTL was reinvigorated.

These and many more studies have led to clinical trials exploring the potency of PD-1 blocking antibodies, putting the PD-1 pathway in the forefront of anti-tumor therapy. Three antagonistic anti-PD-1 antibodies are currently in advanced clinical trials, i.e. pidilizumab, nivolumab and pembrolizumab (formerly lambrolizumab) (Table 2.1). Furthermore, three anti-PD-L1 antibodies, BMS-936,559, MEDI4736, MPDL3280A and MSB0010718C, are being investigated inclinical trials (Table 2.1). In 2012, exciting reports on the use of the anti-PD-1 nivolumab and the anti-PD-L1 blocking antibody BMS-936,559 in patients with advanced malignancies were published [61, 62]. Response rates upon administration of anti-PD-1 to patients with solid tumors ranged from 18 % to 28 %, depending on tumor type. Importantly, responses were durable, with the majority of patients having responses for over a year. Notably, the therapy was relatively well tolerated and only for one patient a serious adverse event, inflammatory colitis, was reported [63]. Interestingly, also blocking the ligand, PD-L1, could induce durable tumor regression with an objective response rate of 6–17 %, with prolonged responses of over a year. The lack of strong toxic effects in this study provided promise that the PD-1 blockade might have a more subtle effect than the CTLA-4 blockade. This rendered anti-PD-1 antibodies as interesting candidates for cancer therapy and gave rise to more extensive trials. In 2013, the results were reported for the anti-PD-1 antibody pembrolizumab in melanoma [64]. In this more homogenous patient group, a response rate of 38 %, and even 52 % in the highest dose was obtained. As in the previous studies, most responses were durable. These exciting results have encouraged registration if these PD-1 blockers. On July 4th 2014, Ono Pharmaceutical and partner Bristol-Myers Squibb (BMS), gained approval in Japan for nivolumab [65]. Furthermore, Merck aims to receive the First US approval for pembrolizumab in melanoma on October 28th 2014. These approvals open up endless possibilities of using PD-1 antagonists against various malignancies, as well as combining anti-PD-1 antibodies with other treatment modalities.

Table 2.1 Blocking antibodies and fusion proteins targeting CIM in clinical trials
Full size table

6.2 CTLA-4

6.2.1 Expression and Function of CTLA-4

Cytotoxic T lymphocyte associated antigen-4 (CTLA-4; CD152), was the first identified CIM, and is partly similar to the co-signaling molecule CD28 [66]. While CD28 is constitutively expressed on the membrane of naïve T-cells, CTLA-4 is primarily localized in intracellular compartments and quickly translocates to the cell membrane upon T-cell activation. The inhibitory function of CTLA-4 was revealed in knockout mice, which showed multi-organ T-cell infiltration leading to lethal lymphoproliferative disease [67]. Like CD28, CTLA-4 has an extracellular domain containing the MYPPPY binding motif, enabling both receptors to interact with CD80 (B7-1) and CD86 (B7-2) expressed by APC. However, the binding affinity of CTLA-4 for these ligands is higher by a factor 10–100, thus outcompeting CD28 and thereby promoting immune inhibition [68].

As CTLA-4 is up-regulated upon TCR ligation, it plays an important role in attenuating effector T-cell activation and maintaining immune homeostasis and central tolerance. In addition, CTLA-4 signaling in immunosuppressive TREG mediates the control of auto-reactive T-cells, as in vivo interference with CTLA-4 on these T-cells elicited pathological autoimmunity [69]. The effect of CTLA-4 interference could either be due to depletion and/or inhibition of TREG. It was shown that TREG-specific CTLA-4 deficiency resulted in down-regulation of CD80/CD86 on APC [70]. This can be explained by endocytosis of APC-derived CD80 and CD86 by TREG [71]. Subsequently, the APC acquires a less stimulatory phenotype, resulting in a lasting inhibitory effect after CTLA-4 ligation. This concept has been investigated further in vivo. Here it was found that TREG can reduce CD80/CD86 expression after encounter with a DC. When CTL are subsequently activated with these hypostimulatory DC, the effector T-cells display enhanced levels of T-cell immunoglobulin and mucin domain 3 (Tim-3) and PD-1. Via this mechanism, CTL function is indirectly attenuated via CTLA-4 signaling [72].

CTLA-4 as such is not a marker of exhausted cells, but elevated levels on viral antigen-specific T-cells correlated with their dysfunction in patients with chronic viral infections, which in turn could be restored by CTLA-4 blockade [73]. Also in metastatic melanoma, high expression of CTLA-4 was correlated to antigen-specific T-cell dysfunction [74]. Moreover, in various CD80 and CD86-positive tumor models, monotherapy with CTLA-4 blocking antibody resulted in elimination of established tumors and long-lasting antitumor immunity [75]. Interestingly, CTLA-4 also has an influence on the motility of CTL. After addition of a CTLA-4 antagonist in a mouse model, it was shown that CTL exhibited increased motility, indicating that CIM blockade does not only restore cytolytic activity, cytokine secretion and proliferation, but could also enhance CTL migration [76]. Although anti-CTLA-4 treatment works in vivo, CTLA-4 blockade in vitro has not been successful in reversing T-cell dysfunction. This can be due to limitations of the in vitro models, as CTLA-4 blockade may exert it’s in vivo action via multiple immune mediators (e.g. effector T-cells, antibody responses, TREG) [77].

All these preclinical findings have stimulated clinical exploration of anti-CTLA-4 blocking antibodies. At the moment, two blocking antibodies exist, ipilimumab and tremelimumab (Table 2.1). Most studies have been performed with ipilimumab in melanoma, and impressively, in these patients the median overall survival almost doubled [78]. In follow-up studies, the effects of CTLA-4 blockade were consistent [79] and in 2011, the FDA and EMA approved ipilimumab treatment for advanced melanoma, thereby paving the way for further exploration of therapies targeting CIM in cancer. Unfortunately, not all studies involving tremelimumab displayed a positive effect on overall survival [80]. Also, for both blocking antibodies, not all patients gained clinical benefit and individual responses are hard to predict. Furthermore, the occurrence of adverse toxic effects upon CTLA-4 blockade were a problem, even leading to death in some cases [80]. However, these were the pioneering studies involving CIM blockade, and by increased clinical awareness and protocols to tackle these immune related complications, severe adverse events have been decreased.

6.2.2 CTLA-4 in Stem Cell Transplantation

Experimental and clinical studies have demonstrated that co-inhibitory molecules hamper T-cell immunity against hematologic cancers in both the autologous and allogeneic settings. This might be due to native expression of CD80 and CD86 on hematologic tumor cells. CTLA-4:CD80/86 interactions also take place between T-cells and hematologic tumor cells. In multiple myeloma (MM) patients, CD86 but not CD80 was expressed by tumor cells, while CTLA-4 was up-regulated on T-cells, which led to anergy of tumor-specific T-cells [81]. Similar to these results, T-cells from chronic lymphocytic leukemia (CLL) patients responded to anti-CD3 activation by a decrease in CD28 and an increase in CTLA-4 expression, resulting in an inhibitory phenotype [82]. In addition to MM, also in acute myeloid leukemia (AML) cells tumor cells were demonstrated to have heterogeneous CD86 expression, but CD80 levels were generally low or absent [60, 83].

The alloreactive T-cell function after alloSCT is also strongly influenced by CIM. The importance of CTLA-4 in modulating allogeneic immune responses has been confirmed by association of certain CTLA-4 genotypes with overall survival and the incidence of leukemia relapse after alloSCT. It was demonstrated that CTLA-4 blockade shortly after alloSCT increased GVHD [84]. However, when anti-CTLA-4 was administered at later time-points after alloSCT, the GVT effect was boosted without signs of GVHD. In patients, ipilimumab administration at late time-points after alloSCT has been explored in one phase I trial [85]. Following a single infusion of the ipilimumab in 29 alloSCT patients with a recurrent or progressive hematological malignancy, three clinical responses were observed. Importantly, no induction or exacerbation of clinical GVHD was reported, although similar to other CTLA-4 blockade trials 14 % of the patients showed organ-specific immune-related adverse events. The lack of GVHD induction is likely attributed to the median interval of 1 year between the last donor T-cell infusion and ipilimumab administration. This provides a window for anti-tumor immunotherapy in the post-alloSCT setting and emphasizes the importance of appropriate timing of CIM blockade.

7 Combining PD-1 and CTLA-4 Blockade

It has been recognized that CTLA-4 and PD-1 exert their role in attenuating T-cell activation at different physiological locations and moments of the immune response. CTLA-4 is mostly involved in the inhibition of CTL priming in the lymph node, while PD-1 seems to limit T-cell proliferation and function in lymphoid tissues as well as in the periphery, i.e. at the tumor site. Therefore, the effects of concurrent PD-1 and CTLA-4 blockade are of great interest. In a mouse tumor model, it was demonstrated that double-positive CD8+ TIL was more dysfunctional than either single PD-1 or CTLA-4 positive CD8+ T-cells. In addition, double PD-1/CTLA-4 blockade led to reversal of TIL dysfunction and subsequent tumor rejection in the majority of mice [86]. Two studies which strengthen the idea of CTLA-4’s role in T-cell priming versus PD-1’s role in peripheral tolerance investigated the TCR repertoire [87, 88]. In patient who had received CTLA-4 blocking antibodies, an increased repertoire of TCR was observed. This indicates that at least part of the effect of CTLA-4 blockade is by an increase in T-cell priming. In contrast, in patients who had been treated with PD-1 blockade, this extended TCR repertoire was not observed, indicating that the clinical efficacy of this treatment is more likely due to reinvigoration of existing CTL responses.

These distinct roles of PD-1 and CTLA-4 warranted combined clinical trials to investigate whether administration of blocking both CIM would have an additive or a synergistic clinical effect. In the first study testing this hypothesis, in an impressive number of 65 % of patients clinical activity was observed, while these were patients with a very poor prognosis [89]. Also at the maximum dosages, 53 % of the patients fulfilled the criteria for the stringent objective responses, all with tumor reduction of 80 % or more. These impressive clinical responses were associated with in grade 3/4 adverse events in 53 % of the patients, which generally were reversible and were not more severe than observed with monotherapy. Also in follow-up data, it was shown that a group of 17 patients who received the optimum combination dose of anti-PD-1 and anti-CTLA-4 showed an overall survival rate of 94 % at 1 year and of 88 % at 2 years, exceeding by far the suboptimal responses typically observed in patients treated with either antibody alone [65]. These results are very promising for the future as optimal combinations of CIM blockade can yield very impressive clinical responses.

8 BTLA

8.1 Expression and Function of BTLA

B and T lymphocyte attenuator (BTLA), (CD272), is an inhibitory receptor with structural similarities to CTLA-4 and PD-1 [90]. BTLA is mainly expressed by immune cells, including T- and B-cells, DC and myeloid cells [91, 92]. In contrast to other B7/CD28 family members, BTLA binds a member of the tumor necrosis factor receptor (TNFR) superfamily, namely herpes virus entry mediator (HVEM) [93]. HVEM is part of an intricate signaling network as it has at least four additional binding partners that distinctively mediate T-cell responses: i.e. CD160, LIGHT (for lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), lymphotoxin-α (LT-α) and herpes simplex virus glycoprotein D (gD) [94]. BTLA or CD160 signaling upon HVEM binding results in T-cell inhibition [93, 95]. Interestingly, naïve T-cells express both HVEM and BTLA, and these molecules form a T-cell intrinsic heterodimer complex [96]. Due to formation of this complex, HVEM is unavailable for extrinsic ligands, and no co-stimulatory signal is transduced. In humans, persistent expression of BTLA was observed on EBV- and CMV-specific CD8+ T-cells, which negatively affected T-cell function [97, 98]. Furthermore, high BTLA expression correlated with impaired tumor-reactive T-cell function in melanoma patients [74, 97]. These tumor-specific T-cell responses could be restored in vitro by interference with the BTLA-HVEM pathway in combination with vaccination therapy. In addition, co-expression of BTLA, PD-1 and Tim-3 rendered melanoma-specific CD8+ T-cells highly dysfunctional, which could be reversed by combined blockade of all three CIM [99]. In a mouse tumor vaccination model, blockade of the BTLA/CD160/HVEM pathway caused regression of large tumor masses [100]. These results show that in the right setting BTLA blockade can be of great significance, warranting evaluation of clinical effectiveness. In addition, the effect of a BTLA blocking antibody has been investigated on MiHA-specific T-cell functionality in samples from alloSCT patients [91]. As shown for PD-1, BTLA was also highly expressed on MiHA-specific CTL. Moreover, in the majority of the patients BTLA blockade resulted in increased outgrowth of MiHA-specific CD8+ T-cells. Interestingly, in three patients BTLA blockade effects were more prominent than those of PD-1, indicating that BTLA has a non-redundant function to PD-1, and therefore it holds promise in cancer immunotherapies.

8.2 Tim-3

The co-signaling receptor Tim-3 is expressed on Th1 CD4+ and CD8+ T-cells, and is involved in co-inhibition. In mice, the interaction of Tim-3 with its ligand galectin-9 was demonstrated to prevent in autoimmune diseases and promote malignancies [101]. Furthermore, in HIV [102] and melanoma patients [103], dysfunctional CD8+ T-cells have been shown to co-express Tim-3. In this regard interference with Tim-3 signaling is an interesting treatment option, and enhanced tumor vaccine efficacy has been observed by Tim-3 blockade [104]. Interestingly, both Tim-3 and PD-1 were expressed on a subset of exhausted CD8+ T-cells in a murine AML model, and expression levels increased [105] during tumor progression [106]. While either Tim-3 or PD-L1 blockade alone was not sufficient to improve survival, the combination of the two antagonistic antibodies significantly decreased tumor burden and enhanced survival. Also, in a melanoma vaccination model, the vast majority of vaccination-induced CTL upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by CTL at the time of vaccine administration correlated inversely with their expansion potential in vivo. Importantly, dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CTL in vitro [107]. Also, combining Tim-3 blockade with activation of CD137, a co-stimulatory receptor, conveyed long term protection against ovarian carcinoma in a mouse model [108]. Finally, blocking of Tim-3 and its family member Tim-4 resulted in a better anti-tumor effect against murine melanoma. All these studies show that, although always in conjunction with another co-signaling molecule, Tim-3 can be involved in tumor evasion, making it an attractive partner in combinatorial blockade. In contrast, a stimulatory role for TIM-3 and galectin-9 has been reported in the interaction of CD8+ T-cells and DC [109]. This discrepancy might be explained by the findings that Tim-3 signaling enhances TCR stimulation [96]. T-cell exhaustion may be caused by prolonged TCR signaling via Tim-3, thereby prolonging the effector phase of T-cell activation at the expense of T-cell memory [110]. Therefore, depending on the setting, Tim-3 may act as either a co-stimulatory or a co-inhibitory receptor.

8.3 LAG3

Lymphocyte-activation gene 3 (LAG3; CD223) is a co-inhibitory receptor highly similar to CD4 and therefore also binds HLA class II molecules [111, 112]. LAG3 seems to be non-redundant from PD-1, as both are expressed on distinct populations CTL [113]. Recently, it was demonstrated that PD-1 and LAG3 act synergistically in the onset of autoimmune diseases and tumor escape in mice [114, 115]. In a leukemia model, PD-1 and CTLA-4 were blocked to reverse CTL tolerance. However, also blockade of LAG3 was necessary to fully restore CTL function [116]. Altogether, these results indicate that LAG3, like Tim-3, is a good candidate as an additive blocking target. At the moment, clinical trials are being performed with a blocking antibody and a soluble LAG3 fusion molecule (Table 2.1), and these studies have the potential to add LAG3 to the list of targets in cancer immunotherapy [117].

9 Other Co-inhibitory Players

In addition to the afore discussed molecules, other co-inhibitory players are being studied to characterize their contribution to functional suppression of tumor-reactive T-cell immunity.

In 2011, a new CIM highly similar to PD-1 was discovered by two groups: PD-1H (PD-1 homolog) or VISTA (V-domain Ig suppressor of T-cell activation) [105, 118]. This molecule is broadly expressed on hematopoietic cells and expression levels are further up-regulated on T-cells and APCs following activation. In in vitro studies the interaction with soluble VISTA-Ig fusion protein or VISTA+ APC mediated the suppression of T-cell cytokine production and proliferation, which could be alleviated by blocking antibody treatment [118, 119]. Recently, it was also demonstrated that VISTA can enhance the conversion of naïve T-cells into FoxP3+ T-cells [119]. In vivo, VISTA overexpression on tumor cells strongly hampered protective tumor-reactive T-cell responses. Importantly, VISTA blockade impaired the suppressive function and emergence of TREG, as well as modulated the suppressive tumor micro-environment, thereby, promoting tumor-reactive T-cell immunity [120]. Interestingly, treatment with PD-1H blocking antibody prevented the induction of GVHD in murine alloSCT models, although the mechanism of action has not been elucidated [105]. These data illustrate that PD-1H/VISTA exerts both an immunoregulatory function in the tumor micro-environment, as well as a direct immunosuppressive action on anti-tumor T-cell responses, making it an interesting therapeutic target.

Killer cell lectin-like receptor G1 (KLRG1), also known as CLEC15A or MAFA, is an inhibitory receptor expressed on NK cells and subsets of CD4+ and CD8+ T-cells [121]. It has been demonstrated that interaction with its ligand E-cadherin results in the functional inhibition of KLRG1+ NK cells, thereby preventing effective killing of tumor cells [122, 123]. In T-cells, KLRG1 expression has been mostly studied as a marker of terminal differentiation. KLRG1+ antigen-experienced TEFF/EM cells exhibited preserved capacity to secrete cytokines upon antigen reencounter, but were incapable of proliferation [121]. Importantly, one study demonstrated that interference with KLRG1 signaling, by targeting of E-cadherin with a blocking antibody, results in enhanced TCR-induced proliferation of highly differentiated CD28−CD27− CTL [124]. More studies are warranted to characterize the involvement of KLRG1 signaling in tumor immune escape and the potential of KLRG1 blockade for cancer immunotherapy.

2B4 (i.e. CD244, SLAMf4) is a member of the CD2 subset of the immunoglobulin superfamily, and is expressed on NK cells, monocytes, basophils and eosinophils. Furthermore, 2B4 expression is up-regulated on a subset of CD8+ T-cells following activation [125, 126]. Its binding partner is CD48 [127]. Most functional studies have been performed with NK cells, where 2B4 was demonstrated to have both activating and inhibitory functions [128]. Interestingly, in a murine transplantation model, 2B4 expression was up-regulated on allograft-reactive CD8+ T-cells, but not CD4+ T-cells, following selective CD28 blockade [129]. Preservation of inhibitory signaling via CTLA-4 was required for the up-regulation of 2B4. Subsequent inhibitory signaling via 2B4 reduced expression levels of the co-stimulatory molecule ICOS, and mediated enhanced allograft survival. These results indicate that 2B4 is involved in the control of antigen-specific CTL functionality.

CD200 Receptor (CD200R) is an inhibitory receptor expressed by cells of myeloid and lymphoid origins, including NK cells and T-cells following activation [130]. Its ligand CD200 (OX2) is expressed by diverse cell types, including immune cells, neurons and epithelia. Importantly, overexpression of CD200 by tumor cells has been associated with progression of various solid and hematologic cancers [131]. In a murine leukemia model, CD200Fc suppressed CD4+ and CD8+ T-cell functionality, resulting in loss of protection from tumor growth [132]. Ex vivo studies with human CLL demonstrated that CD200 is involved in the functional suppression of CTL-mediated tumor killing and CD4-mediated suppression of CTL functionality, which could be reverted with CD200 blocking antibody or CD200 siRNA treatment [133]. In a murine model with CD200+ human B-CLL, administration of CD200 blocking antibody resulted in restored T-cell proliferation and tumor control [134]. Moreover, patients with CD200+ AML were found to have reduced numbers of functional NK cells [135], had significantly compromised Th1 memory and CTL memory responses [136], and showed increased numbers of FoxP3+ TREG [137]. CD200 blockade in vitro could recover NK cell and T-cell functionality [135, 136], and is therefore an attractive target for therapy. Interestingly, the first clinical results have already been reported about an anti-CD200 blocking antibody (Table 2.1) [138]. Although it was in a small cohort, promising results were obtained in a study with B-CLL and MM patients: 36 % of patients experienced at least a 10 % reduction in bulky disease and notably, one patient experienced a partial response with a maximum of 71 % reduction in bulky disease.

10 Future Prospects

Several therapeutic strategies are being developed to dampen the inhibitory signaling by CIM in order to optimize tumor-reactive CTL immunity (Fig. 2.1). The challenge of interference with immune checkpoints is to boost anti-tumor reactivity, while avoiding adverse events such as systemic toxicity. This can potentially be achieved by combining the alleviation of co-inhibition with other therapeutic options or optimal dosage and timing of antibody administration. Appealing combinations are the simultaneous targeting of multiple co-inhibitory receptors, co-stimulatory agonists in parallel with CIM antagonists, or incorporation in existing cellular therapies. For example, DC vaccination may be applied together with blocking antibodies against CIM to boost CTL-mediated anti-tumor immunity.

Fig. 2.1
figure 1

To boost tumor-reactive T cell immunity different immunotherapeutic strategies can be exploited as monotherapy or in combination. First, co-inhibitory signaling pathways can be blocked with antagonistic antibodies to prevent and/or alleviate the functional impairment of CTLs. Furthermore, agonistic antibodies targeting co-stimulatory molecules can be applied to further augment CTL functionality. In addition, dendritic cell vaccination can be applied to provide efficient antigen presentation and strong stimulatory signals to tumor-reactive CTLs. Another strategy is the alloSCT in hematological malignancies, which can elicit powerful MiHA-reactive CTL responses. Finally, by adoptive transfer of highly potent TCR-transduced or CAR-transduced (stem-cell like) T cells direct attack of tumor cells can be provoked. The power of these immunotherapeutic approaches can be further intensified by combination with antibodies to interfere with co-inhibitory signaling pathways

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CAR T-cells are a promising treatment modality in cancer therapy. Although in second and third generation CAR T-cell constructs a strong co-stimulatory signal is incorporated in the form of CD28, 4-1BB and OX40 intracellular signaling domains, this powerful therapy also seems to be dampened by CIM [139, 140]. In a murine tumor model with Her-2-specific CAR-T-cells, a significant increase in tumor growth inhibition was observed after PD-1 blockade [141]. In addition, the amount of immune suppressive myeloid derived suppressor cells was decreased upon PD-1 blockade, through a yet unknown mechanism. Therefore, CIM blockade in combination with CAR-T-cell therapy may improve the clinical efficacy of this novel therapy.

Although anti-CTLA-4 and anti-PD-1 monotherapies have shown very exciting results, toxic effects of blocking CIM may still be a problem. Approaches that concurrently deliver a tumor-antigen-specific stimulus may lead to less adverse events. These include combination therapies with treatment modalities such as immunomodulatory anti-cancer agents, vaccines, TREG depletion or nanoparticles. Recently, another treatment modality in which an antigen-specific stimulation is combined with an intervention for co-inhibition was explored. PD-L1/L2 silenced MiHA-loaded DC boosted the expansion of MiHA-specific T-cells ex vivo [142], and following these promising results, a clinical trial combining DLI with vaccination of PD-L1/L2 silenced donor DC loaded with hemato-restricted MiHA will start. All clinical studies provide a platform for incorporating blockade of CIM as adjuvant therapy of choice in cancer patients, with numerous options for combination therapies. Importantly, the risk of breaking tolerance systemically by blockade of one CIM could be prevented by using lower levels of multiple blocking antibodies targeting different CIM simultaneously, since together these may boost immune responses in a non-redundant manner. This is stressed by the fact that exhausted T-cells are known to display multiple co-inhibitory receptors [143]. Notably, the impressive results obtained by combining blocking antibodies against PD-1 and CTLA-4 is a perfect example of harnessing the power of these two non-redundant immune checkpoints, and many more combinations need to be investigated in the clinical setting.

After identification of the role of CIM in CD8+ T-cell functions, their significance on T-cell exhaustion was clearly established. However, with time, the notion of CIM as direct markers of dysfunction has been adjusted. Although their negative effect on T-cell functions is evident, expression as such does not qualify a T-cell as exhausted [144]. It has been shown that CIM, most notably PD-1, are also present on healthy cells [145] and that several CIM are up-regulated after T-cell activation [146, 147], while their expression had no direct effect on cytokine production by CTL. The activation-induced up-regulation indicated the physiological role of CIM as a negative feedback loop in CTL effector responses. Moreover, during T-cell differentiation most CIM are also up- or down-regulated [91, 146]. All these results indicate that, although on the whole T-cell population PD-1 expression can be an indicator for exhaustion, expression as such is not a marker of exhaustion on the individual T-cell level. Especially the fact that PD-1 can be an activation marker is demonstrated by a study investigating TIL in melanoma. Here it was shown that PD-1, LAG3 and Tim-3 are the identifying markers for tumor-reactive CTL [148]. The realization that not expression, but signaling via the CIM causes CTL dysfunction, has prompted the investigation of downstream signaling pathways and gene expression in impaired CTL. It was found that exhausted T-cells display a distinct gene signature, different from anergic cells, resulting in changes in TCR and cytokine signaling pathways [54]. Indeed, it was demonstrated that PD-1 downstream signaling results in an exhaustion gene signature in HIV-specific T-cells [55]. Further research into these mechanisms in CTL impairment in the tumor setting can yield novel targets to prevent or reverse exhaustion.

Altogether, CIM play a pivotal role in natural and therapeutic CTL-mediated immunity against cancers. With increasing knowledge of a growing number of CIM, novel mono- and combinatorial treatment options are becoming available. In the end, this can lead to optimized immunotherapy against cancers.