Constitutively active GSK3ß as a means to bolster dendritic cell functionality in the face of tumour-mediated immune suppression.

In patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivo vaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3ß (GSK3ß) as a pivotal kinase in both DC development and suppression. GSK3ß inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3ß induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivo melanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3ß activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.

Induction of dendritic cell maturation in the skin microenvironment by soluble factors derived from colon carcinoma.

Autologous tumor cell-based vaccines provide a wide range of tumor antigens and personalized neo-epitopes based on individual tumors' unique antigenic mutanome signatures. However, tumor-derived factors may hamper in situ maturation of dendritic cells (DC) and thus interfere with the generation of effective anti-tumor immunity. As the skin is a preferred site for tumor vaccine delivery, we investigated the influence of primary colon carcinoma-derived soluble factors on the maturation state of migrating DC in a human skin explant model. Primary tumor-derived supernatants (TDSN) enhanced the phenotypic maturation state of skin-emigrated DC, resulting in an increased T-cell stimulatory ability in an allogeneic mixed leukocyte response. In case of monocyte-derived DC a similar TDSN-induced maturation induction was found to entirely depend on cyclooxygenase (COX)-regulated prostaglandins. In contrast, the increase in skin-emigrated DC maturation was completely prostaglandin-independent, as evidenced by the inability of the COX inhibitor indomethacin to abrogate this TDSN-induced effect. Although TDSN conditioning affected a drop in IL-12p70 release by the skin-emigrated DC and induced a predominant Th17/Th22 transcriptional profile in subsequently stimulated T-cells, Th cell subset differentiation, as assessed by intracellular cytokine expression upon polyclonal priming and re-stimulation, was not affected. Comparative analysis of phenotypic and transcriptional profiles suggests that the observed maturational effects in skin-derived DC may have been induced by tumor-derived GM-CSF. In conclusion, soluble factors derived from whole-cell colon tumor vaccines will not negatively impact DC migration and maturation in human skin, but rather induce DC maturation that will facilitate the priming of a poly-functional Th cell response.

Dendritic Cell Plasticity in Tumor-Conditioned Skin: CD14(+) Cells at the Cross-Roads of Immune Activation and Suppression.

Tumors abuse myeloid plasticity to re-direct dendritic cell (DC) differentiation from T cell stimulatory subsets to immune-suppressive subsets that can interfere with anti-tumor immunity. Lined by a dense network of easily accessible DC the skin is a preferred site for the delivery of DC-targeted vaccines. Various groups have recently been focusing on functional aspects of DC subsets in the skin and how these may be affected by tumor-derived suppressive factors. IL-6, Prostaglandin-E2, and IL-10 were identified as factors in cultures of primary human tumors responsible for the inhibited development and activation of skin DC as well as monocyte-derived DC. IL-10 was found to be uniquely able to convert fully developed DC to immature macrophage-like cells with functional M2 characteristics in a physiologically highly relevant skin explant model in which the phenotypic and functional traits of "crawl-out" DC were studied. Mostly from mouse studies, the JAK2/STAT3 signaling pathway has emerged as a "master switch" of tumor-induced immune suppression. Our lab has additionally identified p38-MAPK as an important signaling element in human DC suppression, and recently validated it as such in ex vivo cultures of single-cell suspensions from melanoma metastases. Through the identification of molecular mechanisms and signaling events that drive myeloid immune suppression in human tumors, more effective DC-targeted cancer vaccines may be designed.

IL-10 conditioning of human skin affects the distribution of migratory dendritic cell subsets and functional T cell differentiation.

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.

Functional characterization of a STAT3-dependent dendritic cell-derived CD14+ cell population arising upon IL-10-driven maturation.

Interleukin (IL)-10 is a major cancer-related immunosuppressive factor, exhibiting a unique ability to hamper the maturation of dendritic cells (DCs). We have previously reported that IL-10 induces the conversion of activated, migratory CD1a+ DCs found in the human skin to CD14+CD141+ macrophage-like cells. Here, as a model of tumor-conditioned DC maturation, we functionally assessed CD14- and CD14+ DCs that matured in vitro upon exposure to IL-10. IL-10-induced CD14+ DCs were phenotypically characterized by a low maturation state as well as by high levels of BDCA3 and DC-SIGN, and as such they closely resembled CD14+ cells infiltrating melanoma metastases. Compared with DC matured under standard conditions, CD14+ DCs were found to express high levels of B7-H1 on the cell surface, to secrete low levels of IL-12p70, to preferentially induce TH2 cells, to have a lower allogeneic TH cell and tumor antigen-specific CD8+ T-cell priming capacity and to induce proliferative T-cell anergy. In contrast to their CD14+ counterparts, CD14- monocyte-derived DCs retained allogeneic TH priming capacity but induced a functionally anergic state as they completely abolished the release of effector cytokines. Transcriptional and cytokine release profiling studies indicated a more profound angiogenic and pro-invasive signature of CD14+ DCs as compared with DCs matured in standard conditions or CD14- DCs matured in the presence of IL-10. Importantly, signal transducer and activator of transcription 3 (STAT3) depletion by RNA interference prevented the development of the IL-10-associated CD14+ phenotype, allowing for normal DC maturation and providing a potential means of therapeutic intervention.

Preferential Langerhans cell differentiation from CD34(+) precursors upon introduction of ABCG2 (BCRP).

Epidermal Langerhans cells (LC) and dermal interstitial dendritic cells (IDC) were found to express the ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP; ABCG2). Also, low BCRP expression was present on CD34(+) blood DC precursors and expression was increased upon their differentiation to LC. The CD34(+) acute myeloid leukemia-derived DC cell line MUTZ3 can be cultured into LC or IDC, depending on the cytokine cocktail used. Introduction of functional BCRP in MUTZ3 progenitor cells through retroviral transduction resulted in the emergence of typical LC-characteristics in IDC cultures; the majority of cells remained negative for the IDC-specific C-type lectin DC-SIGN, but rather displayed enhanced expression of the LC-specific C-type lectin Langerin and characteristic high expression levels of CD1a. BCRP-induced skewing toward LC-like differentiation coincided with early RelB expression in 'IDC', derived from MUTZ3-BCRP, and depended on endogenous transforming growth factor beta (TGF-ß) production. Intriguingly, cellular BCRP localization differed between skin LC and IDC, and a more cytoplasmic BCRP localization, as observed in primary skin LC, seemed to relate to LC-like differentiation in IDC cultures upon BCRP introduction in MUTZ3 progenitors. Together these data support a role for BCRP in preferential LC differentiation from CD34(+) myeloid DC progenitors.

Glioblastoma-induced inhibition of Langerhans cell differentiation from CD34(+) precursors is mediated by IL-6 but unaffected by JAK2/STAT3 inhibition.

AIMS: Langerhans cell (LC) infiltration has been observed in glioblastoma, but the glioblastoma microenvironment may be conditioned to resist antitumor immune responses. As little is known about how glioblastoma may affect dendritic cell differentiation, here we set out to delineate the effects of glioblastoma-derived soluble factors on LC differentiation. METHODS: CD34(+) precursor cells of the human myeloid cell line MUTZ-3 were differentiated into LC in the presence of conditioned media of the human glioblastoma cell lines U251 or U373 and phenotypically and functionally characterized. RESULTS: Glioblastoma-conditioned media inhibited LC differentiation, resulting in functional impairment, as determined by allogeneic mixed leukocyte reactivity, and induction of STAT3 activation. IL-6 blockade completely abrogated these glioblastoma-induced immunosuppressive effects and reduced STAT3 phosphorylation. However, neither addition of JSI-124 (cucurbitacin-I; a JAK2/STAT3 inhibitor), nor of GW5074 (a Raf-1 inhibitor), both of which interfere with signaling pathways reported to act downstream of the IL-6 receptor, prevented the observed inhibitory effects on LC differentiation. CONCLUSION: Glioblastoma-derived IL-6 is responsible for the observed suppression of LC differentiation from CD34(+) precursors but appears to exert this effect in a STAT3 and Raf-1 independent fashion.

Characterization of four conventional dendritic cell subsets in human skin-draining lymph nodes in relation to T-cell activation.

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(-) LN-residing 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 IFNγ 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.

Cross-talk between tumor and myeloid cells: how to tip the balance in favor of antitumor immunity.

Myeloid differentiation is often disturbed in cancer, leading to reduced frequencies of immunostimulatory dendritic cells and an over-representation of immunosuppressive immature myeloid cells, granulocytes and macrophages. As a result of this skewed myeloid differentiation, a highly immunosuppressive myeloid subset becomes prevalent during cancer development; these myeloid-derived suppressor cells are also recruited as a collateral to certain protumorigenic inflammatory processes, resulting in an effective downregulation of T-cell-mediated immune surveillance and antitumor immunity. In this article, some of the important myeloid cell subsets and mediators involved in cancer-related immune suppression are reviewed. Furthermore, cross-talk between tumors and the myeloid compartment, and ways in which it can suppress effective cell-mediated immunity, are discussed, as well as possible therapeutic approaches to tip the balance in favor of antitumor immunity.

Attenuation of invariant natural killer T-cell anergy induction through intradermal delivery of alpha-galactosylceramide.

CD1d restricted, alpha-galactosylceramide (alphaGC) responsive invariant (i)NKT cells positively regulate immune responses. Both intravenous and intradermal administered alphaGC are known to activate iNKT cells. iNKT cells become unresponsive to a second intravenous alphaGC injection, whereas no data are available regarding potential anergy upon intradermal administration. Here, comparative analysis of two intradermal versus two intravenous injections in mice demonstrated that iNKT cell anergy was prevented by intradermal injection and when combined with a vaccine, superior tumor protection afforded by intradermally administered alphaGC. Moreover, human skin dendritic cells (DC) took up intradermally injected alphaGC and activated iNKT cells upon migration, while iNKT cells in human skin-draining lymph nodes expanded in response to alphaGC presented either by exogenously added DC or by CD1d positive antigen presenting cells in the lymph nodes. In conclusion, glycolipids such as alphaGC may greatly improve the efficacy of skin immunization strategies, targeting cutaneous and lymph node DC.

Selective transduction of mature DC in human skin and lymph nodes by CD80/CD86-targeted fiber-modified adenovirus-5/3.

In vivo targeting of dendritic cells (DC) represents an attractive alternative to currently apply ex vivo DC-based genetic tumor vaccination protocols. Finding the optimal vector for in vivo targeting of DC is important for such strategies. We, therefore, tested a panel of subgroup C/B chimeric and fiber-modified adenoviruses (Ads) for their relative capacity to transduce human DC. We made use of in vitro generated Langerhans cells, and of ex vivo human skin and melanoma-draining lymph node derived DC. Of the tested viruses the C/B-chimeric adenovirus serotype 5 (Ad5)/3 virus most efficiently transduced in vitro generated Langerhans cells. In addition, Ad5/3 preferentially targeted mature myeloid DC from human skin and draining lymph node and transduced them at significantly higher frequencies than Ad5. In addition, Ad5/3 was more specific for mature human skin-derived CD1a+ CD83+ DC than the previously reported DC-transducing C/B-chimeric vector Ad5/35, infecting less bystander cells. It was previously reported that Ad5/3 transduced human monocyte-derived DC by binding to the B7 molecules CD80 and CD86. High-efficiency transduction of mature skin-derived DC was similarly shown to be mediated through binding to CD80/CD86 and not to interfere with subsequent T-cell priming. We conclude that Ad5/3, in combination with DC-activating adjuvants, represents a promising therapeutic tool for the in vivo transduction of mature DC, and may be less likely to induce unwanted side effects such as immune tolerance through the infection of nonprofessional antigen-presenting cells.

A role for multidrug resistance protein 4 (MRP4; ABCC4) in human dendritic cell migration.

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 down-regulation 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.

Identification and characterization of ErbB-3-binding protein-1 as a target for immunotherapy.

Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients' sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1). Immunohistochemical staining of colorectal tumors and neighboring normal colon tissue indicated the observed cancer-related immunogenicity of Ebp1 to be related to overexpression. Via reverse immunology, five potential HLA-A2-restricted T cell epitopes were identified, of which two (Ebp1(45-54) and Ebp1(59-67)) bound HLA-A2 with intermediate and high affinity, respectively. Analysis of their immunogenicity in vitro indicated that only the high-affinity Ebp1(59) epitope gave rise to CD8(+) T cells capable of recognizing both exogenously loaded Ebp1 peptide and endogenously expressed Ebp1 on target cells. In addition, in vivo CD8(+) T cell responsiveness against the Ebp1(59) epitope could be detected in two of nine and three of six cancer patients PBMC and tumor draining lymph nodes, respectively, but not in nine of nine healthy donors tested. These data confirm that Ebp1 is an immunogenic protein, capable of eliciting CD8-mediated responses in vivo and in vitro, providing a rationale for further exploration of Ebp1 as a possible target for anticancer immunotherapy.