To increase (tumor) vaccine efficacy, there is an urgent need for phenotypic and functional characterization of human dendritic cell (DC) subsets residing in lymphoid tissues. In this study we identified and functionally tested 4 human conventional DC (cDC) subsets within skin-draining sentinel lymph nodes (SLNs) from early-stage melanoma patients. These SLNs were all tumor negative and were removed on average 44 days after excision of the primary melanoma. As such, they were considered representative of steady-state conditions. On comparison with skin-migrated cDC, 2 CD1a ؉ subsets were identified as most likely skin-derived CD11c int Langerhans cells (LC) with intracellular langerin and E-cadherin expression or as CD11c hi dermal DCs with variable expression of langerin. Two other CD1a ؊ LNresiding cDC subsets were characterized as CD14 ؊ BDCA3 hi CD103 ؊ and CD14 ؉ BDCA3 lo CD103 ؉ , respectively. Whereas the CD1a ؉ skin-derived subsets displayed greater levels of phenotypic maturation, they were associated with lower levels of inflammatory cytokine release and were inferior in terms of allogeneic T-cell priming and IFN␥ induction. Thus, despite their higher maturation state, skin-derived cDCs (and LCs in particular) proved inferior T-cell activators compared with the CD1a ؊ cDC subsets residing in melanoma-draining LNs. These observations should be considered in the design of DC-targeting immunotherapies. (Blood. 2011;118(9):2502-2510) IntroductionDendritic cells (DCs) are the most powerful APCs and play critical roles in keeping the balance between immune tolerance and activation. DCs are therefore also important for starting an efficient antitumor immune response and are seen as promising targeting candidates for tumor immunotherapy strategies. 1,2 Current DCbased immunotherapies generally use ex vivo-generated autologous monocyte-derived or CD34 ϩ hematopoietic precursorderived DCs. 2,3 Despite occasionally observed clinical benefits from DC-based vaccination, there is a large gap between the actual and expected efficacy of such trials on the basis of in vivo animal experiments. 4 Many questions remain as to which DC type to use, how to stimulate them, or where best to administer the DCs to achieve vaccination with mature, migratory, and Th1-inducing DCs that provoke an efficient antitumor immune response. [5][6][7] An ever-increasing insight in specialized functions of murine DC subsets is sadly mirrored by a lack of knowledge on how human DCs relate to mouse DCs and whether subsets that have been identified in mice have a (phenotypically different, but functionally equivalent) counterpart in humans. 4,[8][9][10][11] In particular the interrelationship between nonplasmacytoid, conventional DC (cDC) subsets has been obscure, in large part because of their plasticity and dynamic changes in their differentiation and maturation state, which is accompanied by shifts in associated phenotypic markers. 12 In mice, extensive DC-subset analyses have been performed through the use of transgenic models, the ability ...

Melanoma-induced suppression of dendritic cells

ABC transporters were identified originally for their contribution to clinical MDR as a result of their capacity to extrude various unrelated cytotoxic drugs. More recent reports have shown that ABC transporters can play important roles in the development, differentiation, and maturation of immune cells and are involved in migration of immune effector cells to sites of inflammation. Many of the currently identified, endogenous ABC transporter substrates have immunostimulating effects. Increasing the expression of ABC transporters on immune cells and thereby enhancing immune cell development or functionality may be beneficial to immunotherapy in the field of oncology. On the contrary, in the treatment of autoimmune diseases, blockade of these transporters may prove beneficial, as it could dampen disease activity by compromising immune effector cell functions. This review will focus on the expression, regulation, and substrate specificity of ABC transporters in relation to functional activities of immune effector cells and discusses implications for the treatment of cancer on the one hand and autoimmune diseases on the other.

Review of in vivo targeting of tumor antigens to lectin receptors on antigen-presenting cells using antibodies or ligands may improve the antitumor efficacy of vaccines.

Dendritic cells (DC) express the ATP-binding cassette (ABC) transporters P-glycoprotein (ABCB1) and multidrug resistance protein 1 (MRP1; ABCC1). Functionally, both these transporters have been described to be required for efficient DC and T cell migration. In this study, we report that MRP1 activity is also crucial for differentiation of DC. Inhibition of MRP1, but not P-glycoprotein, transporter activity with specific antagonists during in vitro DC differentiation interfered with early DC development. Impaired interstitial and Langerhans DC differentiation was characterized by 1) morphological changes, reflected by dropped side scatter levels in flow cytometric analysis and 2) phenotypic changes illustrated by maintained expression of the monocytic marker CD14, lower expression levels of CD40, CD86, HLA-DR, and a significant decrease in the amount of cells expressing CD1a, CD1c, and Langerin. Defective DC differentiation also resulted in their reduced ability to stimulate allogeneic T cells. We identified the endogenous CD1 ligands sulfatide and monosialoganglioside GM1 as MRP1 substrates, but exogenous addition of these substrates could not restore the defects caused by blocking MRP1 activity during DC differentiation. Although leukotriene C4 was reported to restore migration of murine Mrp1-deficient DC, the effects of MRP1 inhibition on DC differentiation appeared to be independent of the leukotriene pathway. Though MRP1 transporter activity is important for DC differentiation, the relevant MRP1 substrate, which is required for DC differentiation, remains to be identified. Altogether, MRP1 seems to fulfill an important physiological role in DC development and DC functions.

In cancer patients pervasive systemic suppression of Dendritic Cell (DC) differentiation and maturation can hinder vaccination efficacy. In this study we have extensively characterized migratory DC subsets from human skin and studied how their migration and T cell-stimulatory abilities were affected by conditioning of the dermal microenvironment through cancer-related suppressive cytokines. To assess effects in the context of a complex tissue structure, we made use of a near-physiological skin explant model. By 4-color flow cytometry, we identified migrated Langerhans Cells (LC) and five dermis-derived DC populations in differential states of maturation. From a panel of known tumor-associated suppressive cytokines, IL-10 showed a unique ability to induce predominant migration of an immature CD14+CD141+DC-SIGN+ DC subset with low levels of co-stimulatory molecules, up-regulated expression of the co-inhibitory molecule PD-L1 and the M2-associated macrophage marker CD163. A similarly immature subset composition was observed for DC migrating from explants taken from skin overlying breast tumors. Whereas predominant migration of mature CD1a+ subsets was associated with release of IL-12p70, efficient Th cell expansion with a Th1 profile, and expansion of functional MART-1-specific CD8+ T cells, migration of immature CD14+ DDC was accompanied by increased release of IL-10, poor expansion of CD4+ and CD8+ T cells, and skewing of Th responses to favor coordinated FoxP3 and IL-10 expression and regulatory T cell differentiation and outgrowth. Thus, high levels of IL-10 impact the composition of skin-emigrated DC subsets and appear to favor migration of M2-like immature DC with functional qualities conducive to T cell tolerance.

Targeting dendritic cells (DC) through the release of suppressive factors is an effective means for tumors to escape immune control. We assessed the involvement of downstream signaling through the JAK2/STAT3 and p38 MAPK pathways in tumor-induced suppression of human DC development. Whereas the JAK2/STAT3 pathway has been pinpointed in mouse studies as a key regulator of myeloid suppression, in human DC this is less well established. We studied the effects of STAT3 inhibition on the suppression of monocyte-derived DC differentiation mediated by a short-list of four predominant suppressive factors and found that pharmacological STAT3 inhibition could only counteract the effects of IL-6. Accordingly, in testing a panel of supernatants derived from 11 cell lines representing various types of solid tumors, STAT3 inhibition only modestly affected the suppressive effects of a minority of supernatants. Importantly, combined interference in the STAT3 and p38 pathways completely prevented inhibition of DC differentiation by all tested supernatants and effected superior DC function, evidenced by increased allogeneic T cell reactivity with elevated IL-12p70/IL-10 ratios and Th1 skewing. Combined STAT3 and p38 inhibition also afforded superior protection against the suppressive effects of primary glioma and melanoma supernatants and induced a shift from CD14 + cells to CD1a + cells in metastatic melanoma single-cell suspensions, indicating a potential for improved DC differentiation in the tumor microenvironment. We conclude that combined interference in the STAT3 and p38 MAPK signaling pathways is a promising approach to overcome tumor-induced inhibitory signaling in DC precursors and will likely support clinical immunotherapeutic strategies.

The capacity of dendritic cells (DCs) to migrate from peripheral organs to lymph nodes (LNs) is important in the initiation of a T cell-mediated immune response. The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; ABCB1) and the multidrug resistance protein 1 (MRP1; ABCC1) have been shown to play a role in both human and murine DC migration. Here we show that a more recently discovered family member, MRP4 (ABCC4), is expressed on both epidermal and dermal human skin DCs and contributes to the migratory capacity of DCs. Pharmacological inhibition of MRP4 activity or downregulation through RNAi in DCs resulted in reduced migration of DCs from human skin explants and of in vitro generated Langerhans cells. The responsible MRP4 substrate remains to be identified as exogenous addition of MRP4's known substrates prostaglandin E 2 , leukotriene B 4 and D 4 , or cyclic nucleotides (all previously implicated in DC migration) could not restore migration. This notwithstanding, our data show that MRP4 is an important protein, significantly contributing to human DC migration toward the draining lymph nodes, and therefore relevant for the initiation of an immune response and a possible target for immunotherapy. IntroductionThe ATP-binding cassette (ABC) transporters were initially identified by their roles in clinical multidrug resistance (MDR) against a broad range of functionally and structurally unrelated anticancer agents. 1 Over the past 10 years, it has become apparent that several of the ABC transporters transport not only cytostatic agents but also inflammatory mediators such as platelet activating factor, 2 leukotrienes, 3 or prostaglandins. 4 Unraveling the specific roles of ABC transporters in the functioning of the immune system may therefore reveal new links for future immunotherapeutic approaches.Dendritic cells (DCs) are key initiators of the immune response, sampling their surroundings for foreign antigens. 5 They are also seen as a promising tool for targeted immunotherapeutic strategies. 6 We have shown that dendritic cell differentiation is impaired when the transporter activity of Multidrug Resistance Protein 1 (MRP1; ABCC1) is inhibited. 7 Randolph and coworkers previously reported that both P-glycoprotein (P-gp; ABCB1) and MRP1 are required for optimal DC migration. 8,9 The ABC transporter MRP4 (ABCC4) [10][11][12] is an organic anion transporter and has been described to transport prostaglandins such as PGA 2 , PGE 1 , and PGE 2 . 4 Because PGE 2 is believed to be crucial for DC migration, [13][14][15] we explored the role of this ABC transporter in DC migration. Moreover, a recent study showed that MRP4, like MRP1, can transport leukotrienes (eg, LTB 4 and LTC 4 ). 16 Thus, when expressed on DCs, MRP4 could have contributed to the original observations made for the role of MRP1 in DC migration, 9 because the used antagonist MK-571 can inhibit both transporters. In this manuscript we show that MRP4 is abundantly expressed in epidermal human skin DCs and at lower levels in dermal human ski...