Dendritic cells and their role in tumor immunosurveillance

Introduction

Numerous studies have demonstrated that the immune system plays a significant role in the control of tumor development and progression (reviewed in Vesely et al. ¹ and Schreiber et al. ² ). The components of both innate and adaptive immunity are involved in fighting tumor cells; however, current data suggest that the pivotal role in antitumor immune response belongs to T cell-mediated responses.^3–6 It is well known that naïve and central memory T cells can be appropriately activated only when they receive at least two activation signals.^7,8 The first is delivered through the interaction of TCR with the peptide-loaded MHC molecules (i.e. MHC-I for CD8⁺ T cells and MHC-II for CD4⁺ T cells)^7,8 or (glyco)lipid-loaded non-polymorphic CD1 family molecules, mainly CD1d [for the activation of natural killer T (NKT) cells]. ⁹ The second (co-stimulatory) signal is delivered through the interaction of various other molecules; the best described (but in no way the only) co-stimulus essential for adequate immunogenic T cell activation involves the interaction of CD28 molecules on the surface of a T cell with co-stimulatory molecules CD80 (B7.1) and CD86 (B7.2) on the surface of professional APC (pAPC).^7–9 In the absence of co-stimulation, T cell activation induced through the engagement of TCR alone will, in the majority of cases, lead to T cell tolerance,^10,11 which may manifest as: (i) T cell anergy (a long-term, hyporesponsive state characterized by an active inhibition of TCR signaling, and expression of IL-2 and inflammatory cytokines);^10,12,13 (ii) peripheral T cell clonal deletion, which is more characteristic of CD8⁺ T cells, particularly if the TCR stimulus is weak;^14,15 and (iii) generation of induced regulatory T (iTreg) cells, especially in the presence of immunosuppressive cytokines, such as TGF-β ¹⁶ and IL-10. ¹⁷

The essential co-stimulatory molecules are expressed mainly on the surface of pAPCs, including dendritic cells (DCs), monocytes/macrophages and B cells. However, more than 30 years ago it was found that DCs are at least two orders of magnitude more potent than other pAPCs in presenting Ags to T cells in order to elicit Ag-specific T cell responses. ¹⁸ It is generally accepted that only DCs are able to initiate primary immune responses, i.e. to activate naïve T cells efficiently, whilst macrophages and B cells function as APCs in the expansion of pre-activated T cells that are already responding to Ag stimulation; they are also involved in activation of memory T cells.^19,20

The delivery of Ag-specific signal 1 and co-stimulatory signal 2 by DCs ensures optimal activation and clonal expansion of naïve CD4⁺ and CD8⁺ T cells; however, these two signals are not sufficient for the induction of functional polarization of the activated CD4⁺ Th0 cells, i.e. their differentiation into distinct Th1, Th2, Th17, or Th9 effectors, which are needed for fighting particular pathology (e.g. fungal infection versus cancer), which requires different defense mechanisms.^21–23 Current data reveal that a third signal is obligatory for driving the selective Th-cell response. Only mature DCs may provide a full-scale polarizing signal 3 which is delivered both by cytokines (such as IL-12p70) and DC surface molecules (such as Delta and Jagged which are ligands of Notch receptors on T cells).^21,22,24 It was shown that signal 3 is also required for the generation of functionally efficient CD8⁺ T lymphocytes (CTLs).^25–27 The nature of the third signal depends highly on the nature and combination of danger signals, their duration, and the order of delivery to DCs.^21,22,24,28 Moreover, there is evidence that only directly activated DCs are able to produce signal 3 molecules, i.e. DCs should receive a direct signal from a pathogen or tumor cell through triggering various pattern recognition receptors (PRRs) rather than be indirectly activated by pro-inflammatory factors secreted by other cells.^26,29 Furthermore, to be effective the signal 3 must be provided to the T cell by the presenting DC rather than by various cytokines secreted by non-presenting DCs or other cells in the microenvironment.^26,29,30

In relatively rare cases both MHC-I and MHC-II-class molecules, as well as co-stimulatory molecules (such as CD80, CD86), may be expressed on tumor cells, ³¹ enabling them to activate T cells directly. ³² However, tumor cells would hardly be able to deliver the crucial polarizing signal 3 to T cells. Therefore, generally, tumor cells should be uptaken and processed (loaded onto MHC-I and MHC-II class or CD1 family molecules) by immature DCs in the periphery. Following Ag uptake, immature DCs should undergo maturation and migrate through the afferent lymphatics to the draining lymph nodes where they “find” and activate cognate naïve and central memory T cells (reviewed in Petersen et al. ³³ ).

General characteristics of DCs

DCs are a heterogenous group of cells of innate immunity that originate from bone marrow CD34⁺ hematopoietic precursors and are the most potent pAPCs that play a central role in the delicate regulation (active immunity versus immune tolerance) of naïve and memory adaptive immune responses. ³⁴

DCs are defined by their distinct (but not entirely unique) phenotype.

DCs lack expression of lineage-specific markers (CD3, CD19, CD20, CD14, CD16, CD56) and are defined as lineage-negative (Lin^-). There are some exceptions, such as a subset of human dermal DCs, which express CD14, ³⁵ or a subset of human circulating DCs expressing CD16. ³⁶ These DC subsets are briefly described below.

MHC-I and MHC-II class and various co-stimulatory molecules are also expressed on the surface of monocytes/macrophages and B lymphocytes, which are also defined as pAPCs. However, with their unique capability to initiate effective primary immune responses DCs are the most potent APCs, as discussed above.

It should be noted that DCs are also involved in Ag presentation to B cells and their activation.^35,40 During processing of Ags, engulfed as immune complexes through the inhibitory FcγRIIB (CD32b) receptor, DCs can direct these Ags to non-degradative recycling compartments thus preserving them from proteolytic degradation and enabling their presentation in intact (three-dimensional) form to B cells, whose receptor (BCR) recognizes unprocessed Ags. ⁴⁰ Moreover, human dermal DCs expressing CD14 can induce naïve Th0 cells to differentiate into T-follicular helper (Tfh)-like cells, which are specialized in B-cell help. ³⁵

Endothelial cells (ECs) also express both MHC-I and MHC-II class molecules, as well as co-stimulatory molecules, and secrete various cytokines after activation with inflammatory cytokines. However, ECs are not migratory (i.e. they lack a very important feature of pAPCs) and they are also not able to induce primary immune responses (although they can activate circulating memory T cells and induce secondary immune responses); therefore, they are termed non-professional APCs (reviewed in Choi et al. ⁴¹ ).

Types and origin of DCs

There are several subtypes of DCs which differ in their phenotype, localization, migration pathways, and function, i.e. they may differ in effects on innate and adaptive immunity. ⁴² All these subtypes can be grouped into two major DC subsets: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs), with the former being more prevalent.^43,44

Immature and mature DCs

DCs exist in two stages—immature and mature—which differ in their morphology, phenotype, and functional activity. In the steady state immature DCs constantly screen their environment, like sentinels. Upon encountering an Ag, they capture it by phagocytosis, macropinocytosis, or receptor-mediated (clathrin-dependent or clathrin-independent) endocytosis. ⁸¹ DCs may also uptake tumor Ags by “nibbling” ⁸² or trogocytosis, ⁸³ or they may engulf tumor-derived exosomes. ⁸⁴ High endocytic activity (Ag uptake and sequestration) is a hallmark of immature DCs. ⁸⁵

Once inside the DC, protein Ags are processed into peptides which are complexed with both MHC-I and MHC-II class molecules and presented on the surface of DC. ⁸⁵ It is well known that exogenous protein Ags are presented mainly with MHC-II class molecules by pAPCs, whereas MHC-I class molecules present mainly intracellular Ags. However, some DC subsets (e.g. human LCs ³⁵ or circulating CD141⁺ DCs ⁵³ ), but generally not monocytes/macrophages and B cells, are capable of presenting exogenous protein Ags with MHC-I class molecules through an alternate process called cross-presentation. ⁸⁶

MHC-loaded antigenic peptides interact with TCR; however, as was mentioned above, various co-stimulatory signals are needed for the optimal activation of naïve and central memory T cells, and initiation of an adaptive immune response. Immature DCs express only low or intermediate levels of these molecules on their surface.^37,85 Therefore, after encountering an Ag DCs should upregulate the expression of co-stimulatory molecules, apart from MHC-I and MHC-II molecules, through the process called maturation.

Tolerogenic/regulatory DCs

It is important to note that immature DCs are capable of presenting Ags to naïve T cells. However, due to the lack of co-stimulation and low expression of MHC-I and MHC-II class molecules they induce suboptimal T cell priming resulting in T cell tolerance which is characterized by anergy or depletion of Ag-specific T cells, or by their conversion into iTreg subsets. ¹⁰⁰ In fact, all immature DCs, irrespective of their tissue origin are tolerogenic per se. ¹⁰¹ In addition, some immature DCs may exhibit immunosuppressive/regulatory phenotype and activity. For example, immature tumor-infiltrating myeloid DCs (mTIDCs) co-expressing programmed death-1 (PD-1) and PD-L1 (B7-H1, CD274) were recently indentified in mouse ovarian carcinoma. ¹⁰² These mTIDCs show low expression of co-stimulatory molecules, low secretion of inflammatory cytokines, and poor response to danger signals. Accumulation of these cells at the tumor site is associated with both a decreased number of tumor-infiltrating T cells and suppression of tumor Ag-specific effector activity. ¹⁰²

It should be emphasized that during the maturation process DCs may undergo an alternative maturation program and establish a stable semi-mature or “mature tolerogenic” state, which is characterized by:^103–108 (i) relatively high (intermediate) expression of CD83, co-stimulatory,and MHC-II class molecules (“mature-like” phenotype), i.e. their expression is higher than on immature DCs but still lower than on fully mature DCs; (ii) lower production of pro-inflammatory cytokines, such as IL-12p70; (iii) higher secretion of immunosuppressive cytokines, such as IL-10 and TGF-β; and/or (iv) increased expression of inhibitory molecules, such as immunoglobulin-like transcript ILT3 and ILT4 on the surface of the DC.

The generation of mature immunogenic versus tolerogenic myeloid and plasmacytoid DCs depends on the microenvironment (in vitro and in vivo) in which the maturation takes place, both in health and disease.^100,109,110 For example, the generation of semimature tolerogenic DCs was induced by culturing immature DCs from healthy donors with the plasma of ductal pancreatic adenocarcinoma patients ¹⁰⁷ or supernatants of various tumor cells lines. ¹⁰⁵

It should be also noted that there are DC subsets with intrinsic “natural” tolerogenic potential. Gregori et al. indentified a stable mDC-10 subset which comprises only 0.2% of human PBMC and is characterized by a mature phenotype (CD83⁺, CD86⁺, HLA-DR⁺); expression of CD14, CD16, and the tolerogenic molecules HLA-G and ILT4; spontaneous secretion of immunosuppressive cytokine IL-10; low secretion of pro-inflammatory cytokine IL-12; and generation of induced regulatory Tr1 cells. ¹¹¹

Recognition of tumor cells—a challenging task for DCs

pAPCs easily recognize various microbial PAMPs, which have distinct evolutionary conserve structure and are inherently foreign for humans. ¹¹² By contrast, tumor cells are transformed own cells and the majority of Ags expressed on their surface are normal. Thus, they are recognized as “self” and not “worthy” of inducing an immune response.

It is known that owing to genetic instability and a high mutation rate in tumor cells, various altered (mutant) protein molecules may harbor potentially immunogenic peptides (epitopes). ¹¹³ Although it was demonstrated that immature DCs are able to recognize various tumor glycoprotein Ags, for example, aberrantly glycosylated carcinoembryonic Ag (CEA) on colorectal cancer cells, ¹¹⁴ it should be borne in mind that even such highly altered tumor cell surface glycoproteins are distributed between abundant unaltered normal self-Ags, which act like a camouflage on the cancer cell surface. Furthermore, various tumor glycoprotein Ags expressed on the surface of cancer cells may have very subtle structural changes, i.e. only slightly altered glycosylation. Therefore, they may be weakly “non-self” ⁹⁰ and not easily recognized by the components of non-specific innate immunity, including pAPCs. In general, it appears that the recognition of live tumor cells and their subsequent phagocytosis or “nibbling” of plasma membrane and cytosol is really a very difficult task for DCs.

Tumors always contain apoptotic and necrotic cells as a result of hypoxia or nutrient deprivation, and these dying tumor cells are more easily recognized by DCs. ³³ There are also autophagic cells and tumor cells undergoing mitotic crisis (mitotic catastrophe, M2-type mortality) or pyroptosis, ¹¹⁵ which are not discussed here in detail. As reviewed by Kono and Rock ⁸⁸ , Green et al., ¹¹⁶ and Griffith and Ferguson ¹¹⁷ recognition and uptake of necrotic cells and unremoved apoptotic bodies undergoing rupture (secondary necrosis) generally leads to inflammation, because various DAMPs are abundantly released during necrotic cell death. These DAMPs and their induced inflammatory milieu may trigger DC maturation. The uptake of apoptotic cells, which is a normal process during natural tissue turnover, is performed mainly by macrophages, ¹¹⁸ and, to a lesser extent, by immature DCs and neighboring cells. Most importantly the uptake of apoptotic cells is an immunologically silent process; moreover, it induces pAPC tolerance rather than their activation in order to avoid autoimmunity. ^88,116,117 Indeed, it was shown in vitro that recognition of surface phosphatidylserine (PS), an early marker of apoptotic cell death, renders DCs resistant to activation stimuli, ¹¹⁹ for example by suppressing TLR signaling. ¹²⁰ Similarly, ingestion of apoptotic cells by macrophages and, most likely, by DCs induces secretion of immunosuppressive substances, such as TGF-β, prostaglandin E₂ (PGE₂), platelet-activating factor (PAF), IL-10, which in autocrine and paracrine action inhibit the release of pro-inflammatory cytokines, such as IL-12, TNF-α, or IL-1.^121–123 Finally, apoptotic cells themselves can secrete immunosuppressive cytokines, such as TGF-β. ¹²⁴ Therefore, in general, it appears that recognition of apoptotic tumor cells by innate immunity results in the secretion of various immunosuppressive cytokines and generation of tolerogenic DCs which, in turn, contribute to the establishment of the immunosuppressive microenvironment at the tumor site.

Obviously, the numbers of apoptotic (tolerogenic) and necrotic (potentially immunogenic) cells varies among tumors. Moreover, various immunosuppressive factors released by apoptotic tumor cells and pAPCs that have phagocytosed them, as well as by live tumor cells (as will be discussed below), may interfere with the activation and immunogenic maturation of DCs that have encountered tumor cells undergoing immunogenic necrotic cell death. Therefore, despite the fact that DCs more easily recognize dying tumor cells, this may not lead to the generation of immunogenic DCs capable of inducing an effective antitumor immune response.

It is important to note that under certain conditions tumor cells may undergo an immunogenic apoptotic cell death, a recently described subtype of apoptosis, which is characterized by:^{115,125–127} (i) translocation of calreticulin (CR) from the endoplasmic reticulum to the cell surface (in this ectopic position it acts as an “eat me” signal)—importantly, this translocation of CR occures at an early pre-apoptotic stage, prior to the exposure of phosphatydilserine (PS; an inhibitor of DC maturation) on the outer layer of the plasma membrane; and (ii) exposure of various other DAMPs, which may be released extracellularly (such as ATP, HMGB1, uric acid) or ectopically translocated to the cell surface (e.g. Hsp90). These DAMPs are pivotal for DC activation, as the sole relocation of CR to the cell surface is not sufficient, albeit absolutely required, for the induction of immunogenic DC maturation. Other additive tumor-derived factors that induce DC maturation during immunogenic cancer cell death are yet to be identified.^127,128

It should be emphasized that immunogenic apoptosis occurs only under specific cellular stress, including: (i) exposure to some anti-neoplastic agents, such as anthracyclines (e.g. doxorudicine, daunorubicine) and oxaliplatin, but not cisplatin or topoisomerase inhibitors, such as etoposide; (ii) ultraviolet C (UVC) radiation; (iii) γ-radiation;^125–128 (iv) effect of some targeted cancer therapy agents, such as epidermal growth factor receptor antagonists ¹²⁹ or the protease inhibitor, bortezomib; ¹³⁰ and (v) photodynamic therapy. ¹³¹ Therefore, immunogenic apoptosis of tumor cells does not occur naturally and is not involved in eliciting natural antitumor immune responses; however, it may play a critical role in determining the therapeutic activity of various chemotherapeutical and radiation therapy regimens.

DCs and the tumor microenvironment

There is some in vivo evidence that both tumor-infiltrating mDCs^132–134 and pDCs ¹³⁵ are able to recognize tumor Ags, fully mature in the tumor microenvironment, and act as effective pAPCs inducing specific antitumor immune response. However, tumor-infiltrating DCs (TIDCs), both myeloid and plasmacytoid, often have impaired functions, even if they recognize tumor Ags. For example, TIDCs may remain immature^136,137 or may acquire expression of various immunosuppressive molecules.^138–140 Therefore, they are inefficient Ag presenters, but potent inducers of T cell tolerance, T cell death or conversion into iTreg cells.^141,142 Plasmacytoid TIDCs may exhibit an altered secretion of type I IFNs, which are very important players in antitumor immune response. ¹⁴³ This alteration in pDC functional activity may result from tumor-induced decrease of TLR9 expression ¹⁴⁴ or active signaling through ILT7 whose ligand BST2 (bone marrow stromal cell Ag-2) may be expressed on tumor cells.^145,146 Collectively, these data suggest that TIDCs may act as inducers of immune tolerance to tumor Ags and promote tumor development rather than stimulate an antitumor immune response.

The immunogenic versus tolerogenic or tumor growth-facilitating potential of TIDCs seems to be very flexible, as DCs show high functional plasticity and their functional activity may be modulated by tumor microenvironment.^109,147 Tumor-derived factors that may influence tolerogenic versus immunogenic polarization of TIDCs include the lack of danger signals required for DC activation and maturation (as discussed above), and/or exposure of TIDCs to various local immunosuppressants, including cytokines (such as IL-6, IL-10, IL-13, TGFβ), enzymes (e.g. indoleamine 2,3-dioxygenase, arginase), growth factors [e.g. GM-CSF, vascular endothelial growth factor (VEGF)], and other factors (e.g. some liver X receptor ligands, gangliosides, PGE₂).^{109,147–149} These immunosuppressive factors may be secreted by a variety of tumors (their parenchima, stroma, or both), including tumor-infiltrating immune cells, in particular myeloid-derived suppressor cells (MDSCs) ¹⁵⁰ and tumor-associated macrophages. ¹⁵¹

Importantly, many studies demonstrated that the effect of the tumor on DC functional alterations is systemic rather than localized to tumor tissue and may be restored by either removing the tumor or by isolating DC precursors from tumor-bearing host and culturing them ex vivo under normal conditions, i.e. in the absence of tumor-associated changes in microenvironment.^107,109 However, even the removal of the tumor is not always associated with the restoration of DC function and this sustained negative effect may be explained, at least in part, by continual secretion of various suppressive inflammatory factors by tumor-adjacent tissues. ¹⁰⁷

Cancer may be associated not only with impaired functions of DCs, but also with their reduced numbers, especially in advanced disease stages.^{107,109,147,152} This situation may recover after removal of the tumor.^109,147,152 The decrease of DC numbers in cancer patients was shown to be a consequence of general tumor-induced immunosuppression rather than increased migration of DCs to the tumor site. ¹⁵²

Conclusion: DCs need help to elicit an efficient antitumor immune response

All the peculiarities discussed above indicate that the recognition of tumor Ags and induction of immunogenic antitumor T cell responses are rather complicated tasks for DCs. Therefore, various strategies facilitating the introduction of tumor Ags to DCs have been developed in order to stimulate a robust and long-lasting Ag-specific antitumor immune response for the reduction or elimination of tumor cells, or for prevention of recurrences and metastases. ¹⁶⁰ These strategies are defined as specific active tumor immunotherapy or therapeutic cancer vaccination and can be divided in two major approaches:^160–162

DCs may be targeted in vivo, e.g. as a result of administration of irradiated autologous or heterologous tumor cells (which may be additionally pre-treated with haptens to augment their immunogenicity or transfected with genes, coding immunostimulatory molecules), tumor Ag-derived peptides, intact tumor proteins, plasmid DNA encoding tumor Ags, etc.;

Although it is evident that DCs need help to induce an effective antitumor immune response in cancer patients, this help should be rational and well-designed. Indeed, current therapeutic cancer vaccination strategies still show relatively limited clinical efficacy. ¹⁶⁴ It is crucial to verify the most potent protocols for the generation of immunogenic DCs as there are data that some current DC manipulation protocols may result in the generation of cancer vaccines with low immunogenic, or even potentially, tolerogenic activity.^165,166 Furthermore, parameters of tumor microenvironment and antitumor immune response should be evaluated^4–6,167 and in certain cases manipulated (e.g. MDSCs and Tregs should be disarmed or depleted)^150,168 in order to achieve a full-scale clinical advantage of methods that resuscitate the function of DCs in cancer patients.