DENDRITIC CELLS: ARE THEY CLINICALLY RELEVANT?

Abstract

Cancer vaccines have undergone a renaissance due to recent clinical trials showing promising immunological data and some clinical benefit to patients. Current trials exploiting dendritic cells (DCs) as vaccines have shown durable tumor regressions in a fraction of patients. Clinical efficacy of current vaccines is hampered by myeloid-derived suppressor cells, inflammatory type 2 T cells and regulatory T cells (Tregs), all of which prevent the generation of effector cells. To improve the clinical efficacy of DC vaccines, we need to design novel and improved strategies that can boost adaptive immunity to cancer, help overcome Tregs and allow the breakdown of the immunosuppressive tumor microenvironment. This can be achieved by exploiting the fast increasing knowledge about the DC system, including the existence of distinct DC subsets. Critical to the design of better vaccines is the concept of distinct DC subsets and distinct DC activation pathways, all contributing to the generation of unique adaptive immune responses. Such novel DC vaccines will be used as monotherapy in patients with resected disease and in combination with antibodies and/or drugs targeting suppressor pathways and modulation of the tumor environment in patients with metastatic disease.

INTRODUCTION

Molecular identification of human cancer antigens has ushered in a new era of antigen specific cancer immunotherapy specifically targeting these antigens. One strategy is the transfusion of T cells, also called adoptive T cell therapy (reviewed in (1)). There, antigen specific T cells are expanded ex vivo and transfused to patients. Adoptive T cell therapy has been shown to be an effective treatment for viral infections (2) and has induced regression of cancer in early-stage clinical trials (3, 4). Another strategy is to expand T cells in vivo by means of vaccination. Initial attempts at vaccination (e.g. peptides, DNA vaccines, viral vectors and first generation DC-based vaccines) have thus far met with a limited success in the clinic. However, cancer vaccines are undergoing a renaissance due to increased knowledge on the regulation of immune responses as well as recent clinical trials showing promising immunological data and some clinical benefit to the patients. For example, an active immunotherapy product, sipuleucel-T (APC8015) based on antigen-loaded and GM-CSF activated PBMCs, appears to contribute to prolonged median survival in phase III trials in patients with prostate cancer (5). Sipuleucel-T has been recently approved by the FDA for treatment of metastatic prostate cancer thereby paving the regulatory path for the next generation of active immunotherapy products. A randomized phase II trial of a poxviral-based vaccine targeting PSA (PROSTVAC) in men with metastatic castration-resistant prostate cancer showed improved overall survival in patients who received PROSTVAC compared to patients receiving control vectors (6). While these first generation positive randomized phase II/III clinical trials need further analysis and mechanistic studies, they underline the therapeutic potential of the immune system that can be tapped into. Vaccines act through DCs which induce, regulate and maintain T cell immunity. Here we summarize our recent studies aimed at a better understanding of the DC system to unravel the pathophysiology of cancer and to design novel DC-based cancer vaccines with enhanced clinical relevance.

DENDRITIC CELLS

T cell priming is under the control of DCs. In the steady state, non-activated (immature) DCs present self-antigens to T cells, which leads to tolerance (7, 8). DCs induce immune tolerance in a number of ways including i) T cell deletion (9-11); ii) induction of T cell unresponsiveness (12); and iii) activation of regulatory T cells (Tregs) (13-16). Once activated (mature), antigen-loaded DCs are geared towards the launching of antigen-specific immunity (17, 18) leading to the T cell proliferation and differentiation into helper and effector cells (Figure 1). DCs are also important in launching humoral immunity partly due to their capacity to directly interact with B cells (19, 20) and to present unprocessed antigens (21-24).

Figure 1. Dendritic cells.

DCs reside in the tissue where they are poised to capture antigens (109). During inflammation, circulating precursor DC enter tissues as immature DC (109). DCs can encounter pathogens (e.g.: viruses) directly, which induce secretion of cytokines (e.g.: IFN-□); or indirectly through the pathogen effect on stromal cells. Cytokines secreted by DCs in turn activate effector cells of innate immunity such as eosinophils, macrophages and NK cells. Microbe activation triggers DCs migration towards secondary lymphoid organs and simultaneous activation (maturation). These activated migratory DCs that enter lymphoid organs display antigens in the context of classical MHC class I and class II or non-classical CD1 molecules, which allow selection of rare circulating antigen-specific T lymphocytes. Activated T cells help drive DCs toward their terminal maturation, which allows lymphocyte expansion and differentiation. Activated T lymphocytes traverse inflamed epithelia and reach the injured tissue, where they eliminate microbes and/or microbe-infected cells. B cells, activated by DCs and T cells, migrate into various areas where they mature into plasma cells that produce antibodies that neutralize the initial pathogen. Antigen can also reach draining lymph nodes without involvement of peripheral tissue DCs and be captured and presented by lymph node resident DCs (110). The quality of antibody responses is determined by intDCs while the quality of CTL responses is dictated by LCs.

To allow resistance to infection and tolerance to self, DCs are endowed with two critical features: subsets and functional plasticity (25). The two major subsets are the myeloid DCs (mDCs) and the plasmacytoid DCs (pDCs). The best studied human mDC subsets are those from skin, where three subsets can be identified. The epidermis hosts only Langerhans Cells (LCs) while the dermis displays two mDC subsets, CD1a⁺ DCs and CD14⁺ DCs, as well as macrophages (26-29). pDCs are considered as the front line in anti-viral immunity owning to their capacity to rapidly produce high amounts of type I interferon (30, 31). Similarly to mDCs (as discussed in detail below), pDCs display a remarkable functional plasticity. Thus, pDCs exposed to viruses, such as live influenza virus, are able to launch memory responses by inducing the expansion and differentiation of antigen-specific memory B and T lymphocytes into plasma cells (32), and CTLs (33, 34), respectively. On the contrary, pDCs activated with CpG or IL-3/CD40L induce in vitro IL-10-secreting regulatory CD4⁺ T cells (35) as well as suppressor CD8⁺ T cells through the expression of ICOS ligand (36). Their role in active immunotherapy of cancer is largely undefined. Hereunder, we will discuss in a greater detail the recent advances in our understanding of the biology of mDCs as it applies to vaccination.

Dermal DCs, antibody responses and IL-12

In the mid 90's, we observed that CD14⁺ DCs derived from CD34⁺ hematopoietic progenitor cells (HPCs) induce CD40-activated naïve B cells to differentiate into IgM-producing plasma cells through the secretion of IL-6 and IL-12 (37). A decade later, we found that CD14⁺ DCs, but not LCs, induce naïve CD4⁺ T cells to differentiate into cells with properties of T follicular helper cells (Tfh) (27), a CD4⁺ T cell subset specialized in B cell help (38, 39). There, CD4⁺ T cells primed by CD14⁺ DCs help naïve B cells to produce large amounts of IgM, and switch isotypes towards IgG and IgA. Our recent studies in human indicate that acquisition of Tfh phenotype and function depends on IL-12p70 (40).

Thus, IL-12 appears to contribute to humoral immunity in humans through a direct path in DC-B interaction, and an indirect path in DC-T cell interaction and induction of Tfh cells. These findings might explain the modest clinical efficacy of systemic IL-12 administration in cancer patients (41, 42). Furthermore, the injection of IL-12 into tumor sites of head and neck cancer patients resulted in the activation of B cells in the draining lymph nodes, which was associated with their infiltration into tumor sites and tumor regression (43).

LCs and CD8⁺ T cell responses

LCs induce a robust proliferation of naïve allogeneic CD8⁺ T cells when compared to CD14⁺ DCs (27). Furthermore, when pulsed with MHC class I peptides derived from tumor or viral antigens, LCs are far more efficient than CD14⁺ DCs in the priming of antigen-specific CD8⁺ T cells. LCs are also efficient in cross-presenting peptides from protein antigens to CD8⁺ T cells. CD8⁺ T cells primed by LCs show high avidity in tetramer binding assays and express higher levels of cytotoxic molecules, such as granzymes and perforin. Accordingly, they are remarkably more efficient in killing target cells; in particular tumor cells that express low level of peptide/HLA complexes (27). IL-15 might explain the remarkable effects of LCs on the development of Cytotoxic T Lymphocyte (CTL) responses (44-46). Thus, the two different arms of adaptive immunity, i.e., humoral and cellular arms, might be differentially regulated by the two skin mDC subsets (Figure 1). Such framework might be of capital importance for the understanding of the immune alteration in malignancy and for development of novel and improved vaccination strategies against cancer, as well as chronic infections.

DENDRITIC CELLS IN VACCINATION AGAINST CANCER

Outcomes of current DC vaccination trials

Ex vivo-generated DCs have been used as therapeutic vaccines in patients with metastatic cancer for over a decade and early studies have been discussed in detail elsewhere (47). While a fraction of patients can experience durable tumor regressions (48), the most common outcome of the current DC vaccination protocols is a demonstration of expanded antigen-specific immunity, most often using IFN-γ ELISPOT, but no durable objective tumor regression.

Altogether, three outcomes emerge from our studies:

1) No immune response. Patients of this group usually progress quickly. These patients mount immune responses to control antigens such as KLH or viral peptides (Flu-M1 or CMV). In vitro experiments indicated that T cells of several patients can be primed to differentiate into CTLs with specificity for multiple melanoma antigens (49). Thus, tumor antigen-specific CD8⁺ T cells are kept anergic rather than deleted. This inability to mount immune responses to tumor antigens in vivo might be at least partly related to the presence of tumor antigen-specific Tregs (50, 51). Tregs limit the onset of protective immunity through several mechanisms, for example by eliminating DCs in lymph nodes (52). As discussed later, the control of Tregs becomes a key target to address for the coming vaccination trials. 2) Immune response without clinical response. The most common outcome of current DC vaccination protocols is the induction of immune responses in the absence of clinical responses. This might in part be explained by the quality of the elicited T cells including their capacity to migrate into tumors and penetrate tumor stroma (53). Improved immunomonitoring is expected to provide insights into the mechanisms of immune efficacy as discussed hereunder (54, 55). 3) Immune response and clinical response. Vaccination with DCs can elicit therapeutic immunity. These patients represent a formidable opportunity for the development of cancer immunotherapy. The challenge is two-fold. First, to establish the immunological mechanism that allowed tumor eradication. Second, we need to find ways to increase the fraction of patients experiencing durable tumor regression and/or prolonged survival.

The quality of elicited antigen-specific immune responses

Establishing causative links in clinical studies is a difficult task which often requires large patient cohorts. The current data suggest an association between the tumor-specific CD8⁺ T cell responses and clinical outcomes. In our view, four critical components will determine whether the induced immune response will be therapeutic: 1) the quality of elicited CTLs; 2) the quality of induced CD4⁺ helper T cells; 3) the elimination and/or non-activation of Tregs; and 4) the breakdown of immunosuppressive tumor microenvironment.

Indeed, the immune responses elicited by the first generation DC vaccines might not be of the quality required to allow the rejection of bulky tumors. For example, the induced T cells might not migrate into the tumor lesions (56, 57). Furthermore, low avidity T cells might be unable to recognize peptide-MHC class I complexes on tumor cells and/or to kill them (56). Finally, the tumor micro-environment might inhibit effector T cell functions, for example by action of myeloid derived suppressor cells and Tregs as summarized in recent reviews, respectively (58, 59).

The recent progresses in immunomonitoring of specific immune responses in the blood and at the tumor site should help us address these questions (48, 50, 54, 55, 60). Modern approaches including polychromatic flow cytometry rather than the analysis of a single cytokine (e.g., IFN-□ ELISPOT) and/or frequency of tetramer positive cells will contribute to a better assessment of the quality of the immune responses elicited in the patients (61, 62). Indeed, several studies, mostly performed in the context of HIV vaccines, have led to the conclusion that a mere measurement of the frequency of IFN-γ secreting CD8⁺ T cells is insufficient to evaluate the quality of vaccine-elicited immunity (56, 63, 64).

BUILDING ON DENDRITIC CELL SUBSETS TO IMPROVE CANCER VACCINES

Optimal DCs

The results summarized above prompted us to hypothesize that DCs with the properties of LCs might prove to be the best ones for the generation of strong cellular immunity (Figure 1). In line with this, the combination of cytokines used to differentiate monocytes into DCs play a critical role in determining the quality of the elicited T cell responses. For example, DCs generated with GM-CSF and IL-15 display the phenotype and characteristics of LCs. In particular, they are more efficient in priming melanoma-antigen specific CD8⁺ T cells in vitro than DCs derived with GM-CSF and IL-4 (44, 45). Thus, vaccination with IL15-DCs might elicit stronger CD8⁺ T cell responses that might lead to improved clinical responses. We are currently initiating such a clinical trial in patients with malignant melanoma. The selection of methods for activating DCs also represents a critical parameter in the design of DC vaccines. First, immature (non-activated) DCs induce antigen specific IL-10 producing T cells (65, 66). Second, IL-4 DCs activated with a cocktail of IFN-α, polyI:C, IL-1β, TNF, and IFN-γ induce up to 40 times more melanoma-specific CTLs in vitro than DCs matured with the “standard” cocktail of IL-1 □/TNF/IL-6/prostaglandin E₂ (PGE₂)(67-69).

Additional studies will be necessary to establish the therapeutic value of the newer generation DC vaccines in patients. Besides the quality of CD8⁺ T cells, the quality of CD4⁺ T cells will become one of the key parameters of immune efficacy. These studies are critical to the understanding of the human immune system because they permit us to assess in vivo the type of immune responses elicited by human DCs generated in different cytokine environments.

This in turn is essential for building a novel approach to vaccination that is based on the delivery of antigens directly to DCs in vivo using chimeric proteins that are made of an anti-DC receptor antibody molecularly fused to a selected antigen (DC targeting). Studies in mice demonstrate that the specific targeting of antigen to DCs in vivo results in considerable potentiation of antigen-specific CD4⁺ and CD8⁺ T cell immunity. The induction of immunity is observed only when the DC maturation signal is provided (7, 70, 71), and otherwise, tolerance ensues (7). Thus, selection of appropriate adjuvant is also a critical parameter for the induction of the immunity of the desired type. Although TLR-ligands are widely considered to promote protective immunity against infectious agents, selecting the appropriate ligand will be critical. For instance, TLR2 ligation, which promotes the induction of Tregs rather than Th1 or Th17 cells (72), does not appear to be a preferred option for cancer vaccines.

These pioneering studies have been already extended to demonstrate the targeting of tumor antigens to DCs (73) and Langerhans cells (LCs) in animal models (74, 75) and the generation of anti-tumor immunity (76). The therapeutic success of these vaccines will build on the recent knowledge and progress in our understanding of the biology of human DC subsets, cutaneous myeloid DCs (mDCs) in particular.

Optimal antigen loading

While much attention has been paid to CD8⁺ T cells, CD4⁺ T cells have long been known to be involved in anti-tumor immunity (77). A number of recent studies in murine models of cancer suggests the role of tumor antigen-specific CD4⁺ T cells in anti-tumor immunity, which appear to act through different mechanisms including i) help in the expansion of tumor antigen-specific CTLs (78), ii) activation of macrophages at tumor sites (79, 80), and iii) active killing of tumor cells (81, 82). Furthermore, it is now well established that antigen-specific CD4⁺ T cells are fundamental for the induction of long-term memory CD8⁺ T cells (83). Aiming at the induction of tumor antigen-specific CD4⁺ T cell responses, DC vaccines loaded with tumor antigen-derived HLA class II peptides (84), or transduced with a vector encoding a defined tumor antigen (85) have been tested in metastatic melanoma, and shown to induce tumor antigen-specific CD4⁺ T cell responses. Whether vaccination results in the development of durable memory CD4⁺ T cell response remains to be established.

Intimately linked to the quality of CD4⁺ T cells responses is the nature of the antigen and the way to load tumor antigen on DC vaccines. As illustrated in Figure 2, in our own studies we have started with loading DC vaccines with short tumor antigen-derived peptides that can be presented by MHC class I. These were combined with KLH as a foreign helper antigen with the idea to provide CD4⁺ T cell help via generation of new CD4⁺ T cell responses. Both antigens elicited immune responses and we found long-lived melanoma specific CD8⁺ T cells in some patients (86-88). Loading DCs with exogenous peptides that bind MHC class I molecules can generate a limited set of high quality antigen-specific CD8⁺ T cells. Peptides are however limited to certain HLA types and to known tumor antigens. Furthermore, there is no evidence that the currently used melanoma-derived tumor antigens might be the best targets for vaccination. Foreign helper antigens (such as KLH) can be used to prime helper T cells and potentially avoid reactivation of tumor-antigen specific T regs. However, human tumor antigen-specific CD4⁺ T cells might prove more efficient in their helper function for tumor antigen-specific CD8⁺ T cells. An alternative strategy to generate broad immune responses is to load DCs with complex antigen preparations such as tumor-derived RNA (89) or killed tumor cells (90).

Figure 2. Loading antigen on ex vivo DC vaccines.

A number of ways by which the antigen can be delivered to DCs have been identified. The advantages and disadvantages are discussed. Currently, we are developing the strategy to load DCs with long peptides representing defined tumor antigens and providing both CD8⁺ and CD4⁺ T cell epitopes.

Loading DCs with killed tumor cells builds upon the unique capacity of DCs to present internalized antigens via MHC class I leading to cross-priming of naïve CD8⁺ T cells. A major advantage is that it is not limited to a selected haplotype such as HLA-A*0201. We have shown that DCs loaded with killed allogeneic tumor cells from melanoma (49, 91), prostate cancer (92) and breast cancer (93, 94) can cross-prime naive HLA-A*0201⁺ CD8⁺ T cells to differentiate into CTLs specific for defined tumor antigens. It also has the potential to allow presentation of tumor antigens on MHC class II molecules for tumor specific help. Type 1 CD8⁺ T cell immunity to MART-1 was found in three out of 13 patients with metastatic melanoma who received eight DC vaccines loaded with killed allogeneic melanoma cells (48). Two of these patients showed improved immunity in response to vaccination with DCs. Indeed, increased frequency of the specific CD8⁺ T cells and/or their proliferation in response to MART-1 derived peptides indicated in vivo cross-presentation of melanoma antigens by DC vaccine. In one patient, vaccination led to elicitation of IFN-γ producing CD8⁺ T cells specific to a MART-1 peptide to which no response could be detected at the onset. This suggests that in-vivo DC vaccines loaded with killed allogeneic melanoma cells are capable of cross-priming.

However, a potential limitation of such strategy is the expansion of tumor antigen-specific regulatory/suppressor cells. Furthermore, the assessment of immune responses in such trials represents a challenge as both antigens and their T cell epitopes as well as their HLA restriction elements are largely unknown. Recently we begun to assess the immune and clinical efficacy of DC vaccines loaded with long peptides derived from selected tumor antigens so as to allow the presentation of epitopes for both CD8⁺ and CD4⁺ T cells (Figure 2).

Combining DC vaccines with other therapies

In view of the remarkable diversity of regulatory/suppressive pathways present in patients with metastatic cancer, any durable clinical response elicited by vaccination with DCs is already a remarkable achievement. However, to improve the outcomes in metastatic disease, DC vaccines need to be combined with other therapies that offset the suppressive environment created by -tumors (95). Such combination regimens will involve several drugs that target different pathways (Figure 3).

Figure 3. DC vaccines in combination therapies.

Current active immunotherapy trials have shown durable tumor regressions in a fraction of patients. However, clinical efficacy of current approaches is limited, possibly because tumors invade the immune system by means of myeloid-derived suppressor cells, inflammatory type 2 T cells and regulatory T cells (Tregs). To improve the clinical efficacy of immunotherapies, we need to design novel and improved strategies that can boost adaptive immunity to cancer, help overcome Tregs and allow the breakdown of an immunosuppressive tumor microenvironment. This can be achieved by developing combination therapies targeting these three major components.

In particular, blocking antibodies or soluble receptors can be exploited for the blockade of suppressive cytokines in the tumor microenvironment such as IL-10 (96), IL-13 (97), TGF-β (98, 99) and VEGF (100, 101). Such strategies can be used to block immune-inhibitory signals in lymphocytes as illustrated by anti-CTLA-4 (102, 103) and/or anti-PD1 (104-106), or to block their ligands expressed on tumors or DCs (for example anti-PD-L1).In contrast, agonistic antibodies (100, 101) might further promote co-stimulation of effector T cells as for example with anti-CD137 (107), a ligand for 4-1BB (108). Just as different tumors are treated with different combinations of cytostatic drugs and targeted therapies, we foresee development of clinical protocols combining DC vaccines with individualized adjunct therapies.

CONCLUDING REMARKS

The considerable progress made in the knowledge of DC biology as well as effector/regulatory T cell biology has clearly opened avenues for development of vastly improved clinical protocols. These, optimized DC vaccines eliciting strong and long-lived antigen-specific CD8⁺ and CD4⁺ T cell immunity will be offered to patients with early stage disease. For patients with late stage disease, strategies that combine novel highly immunogenic DC-based vaccines and immunomodulatory antibodies will have high impact on enhancing therapeutic immunity in cancer by simultaneously enhancing the potency of beneficial immune arms and offsetting immunoregulatory pathways (Figure 3). These optimized therapeutic strategies will be tailored to a single patient level, including strategies to break suppressive pathways (Figure 4).

Figure 4. Approaches to DC-based immune intervention in cancer.

1) Vaccines based on antigen with or without adjuvant that target DCs randomly. That might result in vaccine antigens being taken up by a “wrong” type of DCs in the periphery which might lead to “unwanted” type of immune response. Vaccine antigens could also flow to draining lymph nodes where they can be captured by resident DCs; 2) Vaccines based on ex-vivo generated tumor antigen-loaded DCs that are injected back into patients; and 3) specific in vivo DC targeting with anti-DC antibodies fused with antigens and with DC activators. 4) Next generation clinical trials will test optimized DC vaccines combined with patient-adjusted approaches to block Tregs and to breakdown the tumor environment. These therapies will be tested in pre-selected patients thereby leading to personalized therapy.