Dendritic cells in cancer: the role revisited

Plasmacytoid DCs

pDCs is a multifunctional population of BM derived DCs [24, 25] specializing in the production and secretion of type I interferons (IFNs). In mice, pDCs express Siglec-H, B220, Ly6c, and low amount of CD11c along with variable amounts of CD8α and CD4. In the periphery, mouse pDCs express CC-chemokine receptor 9 (CCR9), LY49Q and SCA1 [26–28]. Human pDCs exhibit plasma cells morphology and express CD4, HLA-DR, CD123, and blood derived cell antigen-2 (BDCA-2), as well as Toll-like receptor (TLR) 7 and 9 within endosomal compartments but not CD11c [26, 28, 29]. Under homeostatic conditions, pDCs are found in small numbers in T cell areas of LNs and spleen, mucosal-associated tissues, thymus and liver. Upon TLR7/9 triggering, pDCs secrete high amount of type I IFN and produce interleukin-12 (IL-12), IL-6, tumor necrosis factor α (TNF-α), and other pro-inflammatory chemokines. pDCs can act as antigen presenting cells, but they are much less efficient than cDCs and depending on the context, antigen presentation by pDCs can induce immunogenic responses or tolerance [28].

pDCs represent small population of DCs and there is rather limited information about their involvement in antitumor responses. In mouse B16 melanoma, pDCs stimulated with TLR agonists were able to mediate tumor killing by the expression of TNF-related apoptosis-inducing ligand (TRAIL) and granzyme B and C and further exert their antitumor effects via production of type I IFN and subsequent activation of cytotoxic T and NK cells [30–32]. The initiation of immune responses leading to melanoma regression could be linked to the presence of pDC and their production of IFN-α [32]. In an orthotropic murine mammary tumor model, the administration of TLR7 ligands resulted in pDC activation and a potent antitumor effect [33]. The administration of activated pDCs loaded with tumor associated antigens to melanoma patients induced the specific CD4⁺ and CD8⁺ T cells responses [34]

Conventional DCs

In mice, cDCs can be divided into two main subpopulations: CD11b⁻ and CD11b⁺ cells. CD11b⁻DCs include lymphoid tissue CD8α⁺CD11b⁻ DCs and non-lymphoid tissue CD103⁺CD11b⁻ DC. Lymphoid-tissue resident CD11c⁺CD8α⁺Clec9a/DNGR-1⁺XCR1⁺ DCs and migratory CD11c⁺CD103⁺Clec9a/DNGR-1⁺XCR1⁺DCs are considered the most effective cross-presenting DCs in vivo [35]. Contrary to other subsets, Baft3 dependent CD103⁺ and CD8⁺ DCs have specific properties favoring cross-presentation. They limit the antigen degradation by maintaining an alkaline pH in their phagosomes through the production of reactive oxygen species (ROS). A recent study showed that the lectin family member Siglec-G negatively controlled ROS production by inhibiting NOX2 on DC phagosomes. This resulted in an excessive hydrolysis of exogenous antigens which led to a decreased formation of MHC class I-complexes for cross-presentation. Cross presenting CD8⁺ DCs showed lower expression of Siglec-G than CD8⁻ DCs, and Siglec-G deficient mice generated stronger cytotoxic T cells (CTL) than wild type mice [36]. CD11b⁺DCs are an IRF4 dependent subset of lymphoid resident DCs characterized by the expression of CD11c, CD11b, CD172a. CD11b⁺DCs have a dominant role in presenting antigens on MHC class II to CD4⁺ T cells. CD11b⁺DCs, contrary to CD103⁺DCs, induced Th2 cell priming in lung during allergic airway inflammation [37]. However, recently, a subset of migratory CD11b⁺ DCs from lung was also shown to cross-present soluble antigens in vivo and to induce cytotoxic T cells in the presence of TLR7 ligand [38]. Contrary to baft3 dependent DCs that cross-present efficiently even in the absence of activation, CD11b⁺ DCs need specific activation to induce cross-presentation.

In human, an equivalent of Baft3 dependent DC subset is characterized by the expression of CD11c, CD141 (BDCA3), Clec9a/DNGR1, and XCR1 [39], and an equivalent of Irf4 dependent DCs is characterized by the expression of CD11c, CD11b, CD1c (BDCA1), and CD172a [40]. These DCs have been described in different human cancer including breast tumors, metastatic melanoma and head neck squamous cell carcinoma. The presence of CD141 DCs correlates with better outcomes in patients with tumors [41].

cDCs are known to infiltrate different tumor tissues and there is evidence that in addition to lymphoid organs the presentation of tumor antigens by DCs can also occur in the tumors. This suggests that DCs in tumor microenvironment (TME) can substantially influence the functions of antitumor T cells [42–45]. Tumor infiltrating DCs capture antigens released from tumor cells and cross-present antigens to CD8⁺ T cells, driving the expansion of tumor specific CTLs [46–48] (Fig. 1). Baft3^−/− mice showed a profound reduction in the numbers cross-presenting DC and an impaired antitumor immunity [11, 49] indicating that only Batf3⁺ DCs are able to cross-present antigens. Baft3 independent DC subsets could mediate the antitumor responses under activating conditions [50]. In those experiments Baft3-deficient mice were treated with IL-12 to control tumor growth and IL-12 induced a Baft3-independent development of CD8⁺DCs. (Fig. 1)

Recently, a rare subset of cross-presenting DCs, MHC⁺CD24⁺F4/80⁻CD103⁺ (DC2), has been identified in some mouse models, as a distinct population from macrophages and from a second subset of DCs (DC1): MHC⁺CD24⁺F4/80⁻CD11b⁺ [51]. Similar DC populations have been found in human tumors, including metastatic melanoma, breast tumors, and head neck squamous cell carcinoma [52]. Human cDCs are represented by CD45⁺ HLA-DR⁺CD14⁻CD11c⁺ and either BDCA1⁺ (counterpart of mouse CD11b⁺DCs) or BDCA3⁺ (counterpart of mouse CD103⁺ DCs). BDCA3⁺ DCs express X-C motifchemokine receptor 1 (XCR1), providing an alternative marker for their characterization. The presence of BDCA3⁺DCs within TME correlated with better clinical outcome [51, 52]. DC2 have unique antigen processing and presentation properties and are dependent on the transcription factors Irf8, Zbtb46 and Baft3 as well as Flt3l. DC1 depends on GM-CSF for their differentiation. DC2 are more potent CTL stimulators within TME than DC1. DC2s traffic tumor antigens in a CCR7-dependent manner to the dLN and this trafficking is critical for the effective anti-tumor CD8⁺ T cell priming [53]. Importantly, tumor infiltrating human CD141+DCs, the equivalent of mouse DC2, express detectable amount of CCR7, and the expression of CCR7 correlates with better patients’ outcomes. The importance of tumor infiltrating CD103⁺ DCs in regulating antitumor responses has also been demonstrated in two different models of mouse melanoma. These migratory DCs show low expression of lysosomal enzymes, which favor the transport of intact antigens to dLNs and the antigen cross-presentation [54]. Furthermore, efficacy of checkpoint inhibitors relies on the presence of cross-presenting migratory CD103⁺ DCs in TME [48]. The cross-priming DCs induced a measurable CTL response to tumor antigens, which was enhanced by anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) and anti-programmed death-1 (PD-1) mAb therapy [55]. Expanded and activated CD103⁺DCs by injecting FLT3L and poly I:C are shown to be required to enhance anti-tumor responses upon blockade of the checkpoint ligand PD-L1 and BRAF inhibition [54]. The production of type I IFNs within the TME is important for DC function [56]. Tumor-derived DNA can induce the production of type I IFNs by tumor infiltrating DCs via cytosolic DNA-sensing stimulator of IFN genes (STING) pathway [57]. Importantly, type I IFN signaling is particularly required within Baft3 dependent DCs and is critical for the spontaneous regression of immunogenic mouse transplantable tumors [46].

Monocyte-derived inflammatory DCs

Inflammatory DCs (inf-DCs) originate from monocytes [58], as a consequence of inflammation, cancer or infection and largely absent in steady state conditions. Circulating Ly6C^high monocytes are considered to be the direct precursors of inf-DCs [5]. In mice, inf-DCs are identified as MHC II⁺CD11b⁺CD11c⁺F4/80⁺Ly6c⁺, and express CD206 CD115/GM-CSFR, Mac-3/CD107b, FcεRI and CD64 as well as zbtb46. FcεRI is useful marker to distinguish inf-DCs from cDCs and macrophages. Several studies have been shown that inf-DCs can activate antigen specific CD4⁺ T cell responses ex vivo. Inf-DCs can also cross-present exogenous antigens in different models, including HSV-1 reactivation, EAE and allograft models.

Human inf-DCs have been described in several pathological conditions, including psoriasis and cancer. They express HLA-DR, CD11c, BDCA1, CD1a, FcεRI, CD206, CD172a, CD14 and CD11b. Similar to murine inf-DCs, they express M-CSFR and ZBTB46 [58, 59].

In tumors, Ly6C^high monocytes exist as highly suppressive monocytic myeloid-derived suppressor cells (M-MDSC)[60] and can differentiate into tumor associated macrophage and inf-DCs [61]. The cytokines and factors that control the differentiation of monocytes into inf-DCs are not well defined. Elevation of GM-CSF concentration induces accumulation of CD11b⁺MHCII⁺ DCs [62, 63]. Moreover GM-CSF producing B16 melanoma triggers the expansion of CD11b⁺Ly6c^hi-lo MHC II⁺ cells similar to CD11b+DCs [64]. A very recent study has shown that monocytes are heterogeneous and contain different precursors giving rise to macrophages and DCs. The differentiation of DCs from PU.1^hi Flt3⁺MHCII⁺ monocytes is depending on GM-CSF. Importantly this study supports the view that monocyte derived macrophages and monocyte derived DCs are ontogenically distinct populations [65]. Other key requirements appear to be T cell activation signals [66] and toll like receptor ligands (TLRs) [20].

The presence of inf-DCs correlated with CD8⁺ T cell activation and treatment success in several tumor models. A population of inf-DCs has been identified and characterized in untreated ovary and cancer patients. These inf-DCs have a unique phenotype and are potent stimulator of Th17 cells as compared to macrophages [59]. In a melanoma model, the local treatment with the immunostimulatory agent monosodium urate crystals and M. segmatis showed the critical role of inf-DCs in the successful treatment. Intra-tumoral CD11c⁺CD11b⁺Ly6c^high cells, that display some characteristics of inf-DC, were found important for the efficacy of anticancer chemotherapy [67–69]. These DCs were efficient in capturing and presenting tumor antigens, and once adoptively transferred into naïve mice they can protect against a challenge with tumor cells [70]. Very recently, it has been shown that a subset of inf-DCs producing TNFα and NO (TIP-DCs) are expanded and activated after adoptive T-cell cell transfer (ACT) [71]. TIP-DCs seem necessary for tumor growth control because of their ability to promote the expansion of anti-tumor T cells and antitumor effect of TNFα and NO. Importantly, antitumor effect of these cells required activation of the CD40-CD40L pathway [71] (Fig. 1). Moreover, tumor of colorectal cancer patients with prolonged disease free survival (DFS) shared a signature comprising activated DCs, CD8⁺T cells and as well as CD40L, NOS and TNF.