Intradermal delivery of TLR agonists in a human explant skin model: preferential activation of migratory dendritic cells by polyribosinic-polyribocytidylic acid and peptidoglycans.

TLR agonists are attractive candidate adjuvants for therapeutic cancer vaccines as they can induce a balanced humoral and T cell-mediated immune response. With a dense network of dendritic cells (DCs) and draining lymphatics, the skin provides an ideal portal for vaccine delivery. Beside direct DC activation, TLR agonists may also induce DC activation through triggering the release of inflammatory mediators by accessory cells in the skin microenvironment. Therefore, a human skin explant model was used to explore the in vivo potential of intradermally delivered TLR agonists to stimulate Langerhans cells and dermal DCs in their natural complex tissue environment. The skin-emigrated DCs were phenotyped and analyzed for T cell stimulatory capacity. We report that, of six tested TLR-agonists, the TLR2 and -3 agonists peptidoglycan (PGN) and polyribosinic-polyribocytidylic acid (Poly I:C) were uniquely able to enhance the T cell-priming ability of skin-emigrated DCs, which, in the case of PGN, was accompanied by Th1 polarization. The enhanced priming capacity of Poly I:C-stimulated DCs was associated with a strong upregulation of appropriate costimulatory molecules, including CD70, whereas that of PGN-stimulated DCs was associated with the release of a broad array of proinflammatory cytokines. Transcriptional profiling further supported the notion that the PGN- and Poly I:C-induced effects were mediated through binding to TLR2/nucleotide-binding oligomerization domain 2 and TLR3/MDA5, respectively. These data warrant further exploration of PGN and Poly I:C, alone or in combination, as DC-targeted adjuvants for intradermal cancer vaccines.

CD40-targeted adenoviral cancer vaccines: the long and winding road to the clinic.

The ability of dendritic cells (DCs) to orchestrate innate and adaptive immune responses has been exploited to develop potent anti-cancer immunotherapies. Recent clinical trials exploring the efficacy of ex vivo modified autologous DC-based vaccines have reported some promising results. However, in vitro generation of autologous DCs for clinical administration, their loading with tumor associated antigens (TAAs) and their activation, is laborious and expensive, and, as a result of inter-individual variability in the personalized vaccines, remains poorly standardized. An attractive alternative approach is to load resident DCs in vivo by targeted delivery of TAAs, using viral vectors and activating them simultaneously. To this end, we have constructed genetically-modified adenoviral (Ad) vectors and bispecific adaptor molecules to retarget Ad vectors encoding TAAs to the CD40 receptor on DCs. Pre-clinical human and murine studies conducted so far have clearly demonstrated the suitability of a 'two-component' (i.e. Ad and adaptor molecule) configuration for targeted modification of DCs in vivo for cancer immunotherapy. This review summarizes recent progress in the development of CD40-targeted Ad-based cancer vaccines and highlights pre-clinical issues in the clinical translation of this approach.

Construction of a Vero Cell Line Expressing Human ICAM1 for the Development of Rhinovirus Vaccines.

Human rhinoviruses (HRVs) are small non-enveloped RNA viruses that belong to the Enterovirus genus within the Picornaviridae family and are known for causing the common cold. Though symptoms are generally mild in healthy individuals, the economic burden associated with HRV infection is significant. A vaccine could prevent disease. The Vero-cell-based viral vaccine platform technology was considered for such vaccine development. Unfortunately, most HRV strains are unable to propagate on Vero cells due to a lack of the major receptor of HRV group A and B, intercellular adhesion molecule (ICAM1, also known as CD54). Therefore, stable human ICAM1 expressing Vero cell clones were generated by transfecting the ICAM1 gene in Vero cells and selecting clones that overexpressed ICAM1 on the cell surface. Cell banks were made and expression of ICAM1 was stable for at least 30 passages. The Vero_ICAM1 cells and parental Vero cells were infected with four HRV prototypes, B14, A16, B37 and A57. Replication of all four viruses was detected in Vero_ICAM1, but not in the parental Vero cells. Altogether, Vero cells expressing ICAM1 could efficiently propagate the tested HRV strains. Therefore, ICAM1-expressing cells could be a useful tool for the development and future production of polyvalent HRV vaccines or other viruses that use ICAM1 as a receptor.

An Intranasal OMV-Based Vaccine Induces High Mucosal and Systemic Protecting Immunity Against a SARS-CoV-2 Infection.

The development of more effective, accessible, and easy to administer COVID-19 vaccines next to the currently marketed mRNA, viral vector, and whole inactivated virus vaccines is essential to curtailing the SARS-CoV-2 pandemic. A major concern is reduced vaccine-induced immune protection to emerging variants, and therefore booster vaccinations to broaden and strengthen the immune response might be required. Currently, all registered COVID-19 vaccines and the majority of COVID-19 vaccines in development are intramuscularly administered, targeting the induction of systemic immunity. Intranasal vaccines have the capacity to induce local mucosal immunity as well, thereby targeting the primary route of viral entry of SARS-CoV-2 with the potential of blocking transmission. Furthermore, intranasal vaccines offer greater practicality in terms of cost and ease of administration. Currently, only eight out of 112 vaccines in clinical development are administered intranasally. We developed an intranasal COVID-19 subunit vaccine, based on a recombinant, six-proline-stabilized, D614G spike protein (mC-Spike) of SARS-CoV-2 linked via the LPS-binding peptide sequence mCramp (mC) to outer membrane vesicles (OMVs) from Neisseria meningitidis. The spike protein was produced in CHO cells, and after linking to the OMVs, the OMV-mC-Spike vaccine was administered to mice and Syrian hamsters via intranasal or intramuscular prime-boost vaccinations. In all animals that received OMV-mC-Spike, serum-neutralizing antibodies were induced upon vaccination. Importantly, high levels of spike-binding immunoglobulin G (IgG) and A (IgA) antibodies in the nose and lungs were only detected in intranasally vaccinated animals, whereas intramuscular vaccination only induced an IgG response in the serum. Two weeks after their second vaccination, hamsters challenged with SARS-CoV-2 were protected from weight loss and viral replication in the lungs compared to the control groups vaccinated with OMV or spike alone. Histopathology showed no lesions in lungs 7 days after challenge in OMV-mC-Spike-vaccinated hamsters, whereas the control groups did show pathological lesions in the lung. The OMV-mC-Spike candidate vaccine data are very promising and support further development of this novel non-replicating, needle-free, subunit vaccine concept for clinical testing.

Sunitinib-induced myeloid lineage redistribution in renal cell cancer patients: CD1c+ dendritic cell frequency predicts progression-free survival.

PURPOSE: A disturbed myeloid lineage development with abnormally abundant neutrophils and impaired dendritic cell (DC) differentiation may contribute to tumor immune escape. We investigated the effect of sunitinib, a tyrosine kinase inhibitor of fms-like tyrosine kinase-3, KIT, and vascular endothelial growth factor receptors, on myeloid differentiation in renal cell cancer (RCC) patients. EXPERIMENTAL DESIGN: Twenty-six advanced RCC patients were treated with sunitinib in a 4-week on/2-week off schedule. Enumeration and extensive phenotyping of myeloid subsets in the blood was done at baseline and at weeks 4 and 6 of the first treatment cycle. Baseline patient data were compared with sex- and age-matched healthy donor data. RESULTS: Baseline frequencies of DC subsets were lower in RCC patients than in healthy donors. After 4 weeks of sunitinib treatment, a generalized decrease in myeloid frequencies was observed. Whereas neutrophils and monocytes, which were both abnormally high at baseline, remained low during the 2-week off period, DC rates recovered, resulting in a normalized myeloid lineage distribution. Subsequent to sunitinib treatment, an increase to high levels of myeloid DC (MDC) subset frequencies relative to other myeloid subsets, was specifically observed in patients experiencing tumor regression. Moreover, high CD1c/BDCA-1(+) MDC frequencies were predictive for tumor regression and improved progression-free survival. CONCLUSION: The sunitinib-induced myeloid lineage redistribution observed in advanced RCC patients is consistent with an improved immune status. Immunologic recovery may contribute to clinical efficacy as suggested by the finding of highly increased MDC frequencies relative to other myeloid subsets in patients with tumor regression.

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.

Gene-directed enzyme prodrug therapy for osteosarcoma: sensitization to CPT-11 in vitro and in vivo by adenoviral delivery of a gene encoding secreted carboxylesterase-2.

Despite improvement in the treatment of osteosarcoma (OS), there are still many patients who cannot benefit from current treatment modalities. This warrants exploration of new treatment options. To that end, we investigated gene-directed enzyme prodrug therapy (GDEPT) with the use of human liver carboxylesterase-2 (CE2) and the anticancer agent CPT-11. CPT-11 is a clinically approved prodrug that needs to be metabolized into the active drug SN-38 by CEs, which occurs rather inefficiently in humans. GDEPT aims at high production of CE2 at the tumor site, resulting in efficient local conversion of CPT-11 into SN-38. Here, we show that OS cells transduced with an adenoviral vector containing the cDNA encoding a secreted form of CE2 (Ad-sCE2) expressed and efficiently secreted CE2. In vitro, transduction of a panel of OS cell lines with Ad-sCE2 resulted in sensitization up to 2800-fold to CPT-11 treatment. Primary OS short-term cultures, derived from patients suffering from a classic high-grade OS, demonstrated increased CPT-11 sensitivity up to 70-fold after transduction with Ad-sCE2 in vitro. When mice bearing s.c. MG-63 OS xenografts were intratumorally injected with Ad-sCE2 and CPT-11, this resulted in a significant difference in time to reach 2000 mm(3) in tumor volume as compared with animals receiving Ad-sCE2 or CPT-11 treatment (P < 0.05). Taken together, these data suggest that OS cells are sensitive for the combination of Ad-sCE2 and CPT-11.

Hematopoietic cancer cell lines can support replication of Sabin poliovirus type 1.

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus.

Animal models for cutaneous vaccine delivery.

Main challenges in skin vaccination are overcoming the stratum corneum (SC) barrier and targeting the antigen presenting cells (APC) in the epidermis and the dermis. For this purpose many delivery techniques are being developed. In vivo immunogenicity and safety studies in animals are mandatory before moving to clinical trials. However, the results obtained in animals may or may not be predictive for humans. Knowledge about differences and similarities in skin architecture and immunology within a species and between species is crucial. In this review, we discuss variables, including skin morphology, skin barrier function, mechanical properties, site of application and immunology, which should be taken into account when designing animal studies for vaccination via the skin in order to support the translation to clinical trial outcomes.

Pronounced antitumor efficacy by extracellular activation of a doxorubicin-glucuronide prodrug after adenoviral vector-mediated expression of a human antibody-enzyme fusion protein.

Tumor-specific activation of the glucuronide prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate (DOX-GA3), by beta-glucuronidase present in necrotic tumor areas might be improved after transduction of tumor cells to secrete a targeted form of beta-glucuronidase. To that end, we constructed an adenovirus vector, designated Ad/C28-GUSh, encoding human beta-glucuronidase fused to a human single-chain Fv (scFv) against the epithelial cell adhesion molecule (EpCAM), C28, and preceded by a signal sequence for secretion. Antibody specificity and enzyme activity were retained in the fusion protein secreted by tumor cells infected with Ad/C28-GUSh. Diffusion of fusion protein from transduced tumor cells within MCF-7 multicellular spheroids was visualized by immunohistochemistry. Treatment of spheroids with Ad/C28-GUSh and DOX-GA3 resulted in growth inhibition comparable to treatment with doxorubicin alone. Treatment of well-established FMa human ovarian cancer xenografts with intravenous injection of DOX-GA3 (500 mg/kg) resulted in a tumor volume-doubling time of 23.8 days compared to 8.0 days for phosphate-buffered saline (PBS)-treated mice. Intratumoral administration of Ad/C28-GUSh before DOX-GA3 enhanced the growth inhibition and increased the tumor volume-doubling time to 43.1 days (p < 0.01), while virus alone had no effect. Thus, we have successfully shown that an adenovirus vector encoding a secreted, targeted form of human beta-glucuronidase can further improve DOX-GA3 monotherapy.

Conditionally replicating adenoviruses as anticancer agents and ways to improve their efficacy.

Conditionally replicating adenoviruses (CRAds) were developed as new tools for cancer therapy. CRAds specifically replicate in and kill cancer cells. Within a solid tumor mass, release of newly formed infectious particles from infected cancer cells allows additional cell layers to be infected and destroyed. When used as monotherapeutic agents, CRAds showed only limited effects in clinical trials. Combination studies consisting of CRAd virotherapy with chemo-, radio-or enzyme prodrug therapy, however, showed additive or even synergistic effects and encouraging results in clinical studies. Furthermore, increased CRAd efficacy could be achieved in preclinical models by targeting CRAd infection specifically to tumor cells or via insertion of therapeutic genes in the CRAd genome. In this review, different mechanisms to achieve CRAd specificity, the efficacy of CRAds in clinical trials and ways to enhance their oncolytic potency are discussed.

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.

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.

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.

Epstein-Barr virus-encoded BARF1 protein is a decoy receptor for macrophage colony stimulating factor and interferes with macrophage differentiation and activation.

Epstein-Barr virus (EBV), like many other persistent herpes viruses, has acquired numerous mechanisms for subverting or evading immune surveillance. This study investigates the role of secreted EBV-encoded BARF1 protein (sBARF1) in creating an immune evasive microenvironment. Wild-type consensus BARF1 was expressed in the human 293 cell line and purified. This native hexameric sBARF1 had inhibitory capacity on macrophage colony stimulating factor (M-CSF)-stimulated, and not on granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated growth and differentiation of myeloid cells. Antibodies specific to hexameric sBARF1 were able to block this effect. M-CSF was shown to interact with sBARF1 via the protruding N-terminal loops involving Val38 and Ala84. Each BARF1 hexamer was capable of binding three M-CSF dimers. Mutations in the BARF1 loops greatly affected M-CSF interaction, and showed loss of growth inhibition. Analysis of the activation state of the M-CSF receptor c-fms and its downstream kinase pathways showed that sBARF1 prevented M-CSF-induced downstream phosphorylation. Since M-CSF is an important factor in macrophage differentiation, the effect of sBARF1 on the function of monocyte-derived macrophages was evaluated. sBARF1 affected overall survival and morphology and significantly reduced expression of macrophage differentiation surface markers such as CD14, CD11b, CD16, and CD169. Macrophages differentiating in the presence of sBARF1 showed impaired responses to lipopolysaccharide and decreased oxygen radical formation as well as reduced phagocytosis of apoptotic cells. In conclusion, EBV sBARF1 protein is a potent decoy receptor for M-CSF, hampering the function and differentiation of macrophages. These results suggest that sBARF1 contributes to the modulation of immune responses in the microenvironment of EBV-positive carcinomas.

Tumor-mediated inhibition of human dendritic cell differentiation and function is consistently counteracted by combined p38 MAPK and STAT3 inhibition.

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.

Irritant-induced migration of Langerhans cells coincides with an IL-10-dependent switch to a macrophage-like phenotype.

Langerhans cells (LCs) migrate after topical exposure of the skin to irritants, despite the supposed independence of irritant contact dermatitis from adaptive immunity. Whereas allergen-activated LCs are known to migrate to the draining lymph nodes (LNs), the fate of migrated LCs upon topical irritant exposure is unknown. Here, we identified a phenotypic switch of LCs after their migration into the dermis upon irritant exposure. With the aid of ex vivo intact human skin and epidermal sheets, we show that dermal fibroblasts are necessary for an IL-10-dependent postmigrational phenotypic switch of LCs into macrophage-like cells. Exposure of ex vivo skin to a panel of seven irritants resulted in a decrease in the number of CD1a(+) cells and an increase in CD14(+)/CD68(+) cells in the dermis. Neutralizing antibodies against IL-10 totally inhibited the phenotypic LC-to-macrophage transition, but did not influence the migration of CD1a(+) cells. Exposure of epidermal sheets to irritants resulted in a fibroblast-dependent LC-to-CD14(+)/CD68(+) switch coinciding with migration, which could be totally inhibited by neutralizing antibodies against either IL-10 or CCL2/CCL5 (two chemokines responsible for epidermal-to-dermal migration). We have thus identified an IL-10-dependent phenotypic switch of LCs into macrophage-like cells upon irritant exposure and emigration from the epidermis.

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.

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.