Tumor-infiltrating CCR2+ inflammatory monocytes counteract specific immunotherapy.

Tumor development and progression is shaped by the tumor microenvironment (TME), a heterogeneous assembly of infiltrating and resident host cells, their secreted mediators and intercellular matrix. In this context, tumors are infiltrated by various immune cells with either pro-tumoral or anti-tumoral functions. Recently, we published our non-invasive immunization platform DIVA suitable as a therapeutic vaccination method, further optimized by repeated application (DIVA2). In our present work, we revealed the therapeutic effect of DIVA2 in an MC38 tumor model and specifically focused on the mechanisms induced in the TME after immunization. DIVA2 resulted in transient tumor control followed by an immune evasion phase within three weeks after the initial tumor inoculation. High-dimensional flow cytometry analysis and single-cell mRNA-sequencing of tumor-infiltrating leukocytes revealed cytotoxic CD8+ T cells as key players in the immune control phase. In the immune evasion phase, inflammatory CCR2+ PDL-1+ monocytes with immunosuppressive properties were recruited into the tumor leading to suppression of DIVA2-induced tumor-reactive T cells. Depletion of CCR2+ cells with specific antibodies resulted in prolonged survival revealing CCR2+ monocytes as important for tumor immune escape in the TME. In summary, the present work provides a platform for generating a strong antigen-specific primary and memory T cell immune response using the optimized transcutaneous immunization method DIVA2. This enables protection against tumors by therapeutic immune control of solid tumors and highlights the immunosuppressive influence of tumor infiltrating CCR2+ monocytes that need to be inactivated in addition for successful cancer immunotherapy.

Optimized dithranol-imiquimod-based transcutaneous immunization enables tumor rejection.

Introduction: Transcutaneous immunization (TCI) is a non-invasive vaccination method promoting strong cellular immune responses, crucial for the immunological rejection of cancer. Previously, we reported on the combined application of the TLR7 agonist imiquimod (IMQ) together with the anti-psoriatic drug dithranol as novel TCI platform DIVA (dithranol/IMQ based vaccination). In extension of this work, we further optimized DIVA in terms of drug dose, application pattern and established a new IMQ formulation. Methods: C57BL/6 mice were treated on the ear skin with dithranol and IMQ-containing ointments together with ovalbumin-derived peptides. T cell responses were determined by flow cytometry and IFN-ɤ ELISpot assay, local skin inflammation was characterized by ear swelling. Results: Applying the adjuvants on separate skin sites, a reduced number of specific CD8+ T cells with effector function was detectable, indicating that the local concurrence of adjuvants and peptide antigens is required for optimal vaccination. Likewise, changing the order of dithranol and IMQ resulted in an increased skin inflammatory reaction, but lower frequencies of antigen-specific CD8+ T cells indicating that dithranol is essential for superior T cell priming upon DIVA. Dispersing nanocrystalline IMQ in a spreadable formulation (IMI-Sol+) facilitated storage and application rendering comparable immune responses. DIVA applied one or two weeks after the first immunization resulted in a massive increase in antigen-specific T cells and up to a ten-fold increased memory response. Finally, in a prophylactic tumor setting, double but no single DIVA treatment enabled complete control of tumor growth, resulting in full tumor protection. Discussion: Taken together, the described optimized transcutaneous vaccination method leads to the generation of a strong cellular immune response enabling the effective control of tumor growth and has the potential for clinical development as a novel non-invasive vaccination method for peptide-based cancer vaccines in humans.

In Activated Murine Mast Cells, NFATc2 Is Critical for the Production of Autocrine IL-3, Thereby Promoting the Expression of IL-9.

IL-9 has lent its numerical designation to the Th9 subset of CD4+ Th cells, although it is also produced by additional cell types, including mast cells. It is a pleiotropic cytokine involved in allergic reactions, parasitic infections, autoimmune inflammation, and cancer immunity. In this article, we provide evidence that NFATc2 has contradictory functions in the expression of IL-9 in murine Th9 cells and bone marrow-derived mast cells (BMMC). The basis for this is our observation that the production of IL-9 in NFATc2-deficient Th9 cells is increased, whereas it is decreased in BMMC devoid of NFATc2. In addition, NFATc2 deficiency almost completely abrogates the expression of IL-3 in both cell types. However, selectively in BMMC, the production of IL-9 critically depends on autocrine IL-3 acting via the sustained activation of STAT5 on the expression of IL-9. Furthermore, we demonstrate that IL-3 acts independently and synergistically with IL-1ß on the production of IL-9. Taken together, we highlight NFATc2-driven production of autocrine IL-3 as a critical and cell type-specific component for IL-9 expression in BMMC.

Transcriptomic data on the role of PEST-domain-enriched tyrosine phosphatase in the regulation of antigen-mediated activation and antiallergic action of glucocorticoids in mast cells.

Protein tyrosine phosphatases and glucocorticoids are known to regulate allergic and antiallergic action in activated mast cells. Here we provide RNA sequencing and quantitative real-time PCR data from bone marrow derived mast cells, for wild-type and PEST-domain-enriched tyrosine phosphatase (PEP) null mice, activated by immunoglobulin E sensitization and dinitrophenol treatment, and additionally treated with the glucocorticoid dexamethasone. The transcriptomics experiment was performed in duplicate with a total of 16 samples (GSE108972).

9-Phenanthrol enhances the generation of an CD8+ T cell response following transcutaneous immunization with imiquimod in mice.

BACKGROUND: Transcutaneous immunization (TCI) is a non-invasive vaccination strategy targeting the skin-associated lymphoid tissue. Topical application of the TLR7 agonist imiquimod as adjuvant in combination with peptide antigens activates the innate immune system and mounts cytotoxic T lymphocyte (CTL) responses. OBJECTIVE: Based on the commercial 5% imiquimod-containing drug Aldara we aimed to develop an improved formulation with superior vaccination efficiencies. The primary target was the enhancement of mast cell activation as important key for the function of the innate immune system. METHODS: We investigated the effects of 9-phenanthrol (9-phe) on the activation of mast cells in vitro and in vivo. For TCI, we applied 0.2% 9-phe in Aldara or Aldara alone as adjuvants in combination with the MHC class I - restricted peptide SIINFEKL. To monitor vaccination, mast cell degranulation, migration of DC and frequencies of epitope-specific CTL was assessed. In a transgenic tumor model, the efficiencies of prophylactic immunization against a tumor antigen were also monitored. RESULTS: 9-phe induced degranulation of mast cells in vitro and upon topical application in vivo. A mixture of 0.2% 9-phe in Aldara showed superior results regarding the migration of DC and the expansion of antigen-specific CTL. Consequently, prophylactic immunization with 0.2% 9-phe in Aldara caused enhanced protection against tumor inoculation. CONCLUSION: Our data demonstrate that a simple modification of an adjuvant formulation can yield superior results in experimental vaccination protocols by boosting critical steps leading to the generation of an efficient CTL response.

Transcutaneous immunization with a novel imiquimod nanoemulsion induces superior T cell responses and virus protection.

BACKGROUND: Transcutaneous immunization (TCI) is a novel vaccination strategy utilizing the skin associated lymphatic tissue to induce immune responses. TCI using a cytotoxic T lymphocyte (CTL) epitope and the Toll-like receptor 7 (TLR7) agonist imiquimod mounts strong CTL responses by activation and maturation of skin-derived dendritic cells (DCs) and their migration to lymph nodes. However, TCI based on the commercial formulation Aldara only induces transient CTL responses that needs further improvement for the induction of durable therapeutic immune responses. OBJECTIVE: Therefore we aimed to develop a novel imiquimod solid nanoemulsion (IMI-Sol) for TCI with superior vaccination properties suited to induce high quality T cell responses for enhanced protection against infections. METHODS: TCI was performed by applying a MHC class I or II restricted epitope along with IMI-Sol or Aldara (each containing 5% Imiquimod) on the shaved dorsum of C57BL/6, IL-1R, Myd88, Tlr7 or Ccr7 deficient mice. T cell responses as well as DC migration upon TCI were subsequently analyzed by flow cytometry. To determine in vivo efficacy of TCI induced immune responses, CTL responses and frequency of peptide specific T cells were evaluated on day 8 or 35 post vaccination and protection in a lymphocytic choriomeningitis virus (LCMV) infection model was assessed. RESULTS: TCI with the imiquimod formulation IMI-Sol displayed equal skin penetration of imiquimod compared to Aldara, but elicited superior CD8+ as well as CD4+ T cell responses. The induction of T-cell responses induced by IMI-Sol TCI was dependent on the TLR7/MyD88 pathway and independent of IL-1R. IMI-Sol TCI activated skin-derived DCs in skin-draining lymph nodes more efficiently compared to Aldara leading to enhanced protection in a LCMV infection model. CONCLUSION: Our data demonstrate that IMI-Sol TCI can overcome current limitations of previous imiquimod based TCI approaches opening new perspectives for transcutaneous vaccination strategies and allowing the use of this enhanced cutaneous drug-delivery system to be tailored for the improved prevention and treatment of infectious diseases and cancers.

Cylindromatosis (Cyld) gene mutation in T cells promotes the development of an IL-9-dependent allergic phenotype in experimental asthma.

Cylindromatosis (CYLD) is a ubiquitously expressed deubiquitinating enzyme which removes activating ubiquitin residues from important signaling molecules of the NF-κB pathway. In CYLDex7/8 transgenic mice, a naturally occurring short isoform (sCYLD) is overexpressed in the absence of full length CYLD, leading to excessive NF-κB activity. Herein, we investigated the impact of the CYLDex7/8 mutation selectively in T cells on the development of experimental allergic airway disease induced by sensitization and challenge with ovalbumin. Compared with their wildtype littermates, mice bearing the T cell-specific mutation (CD4+CYLDex7/8) display stronger eosinophilia and mucus production in the lungs and higher IgE serum levels. The reason for these observations is excessive production of T cell-derived IL-9, a cytokine to whom allergy-promoting properties were ascribed. Consequently, blockade of IL-9 in CD4+CYLDex7/8 mice alleviates the development of disease symptoms. Thus, by polarization of the T cell cytokine response, sCYLD can favor the development of allergic airway disease.

Tick Salivary Sialostatin L Represses the Initiation of Immune Responses by Targeting IRF4-Dependent Transcription in Murine Mast Cells.

Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1ß and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.

Mast cells: innate attractors recruiting protective CD8 T cells to sites of cytomegalovirus infection.

Reactivation of latent cytomegalovirus (CMV) in the transient immunocompromised state after hematoablative treatment is a major concern in patients undergoing hematopoietic cell transplantation (HCT) as a therapy of hematopoietic malignancies. Timely reconstitution of antiviral CD8 T cells and their efficient recruitment to the lungs is crucial for preventing interstitial pneumonia, the most severe disease manifestation of CMV in HCT recipients. Here, we review recent work in a murine model, implicating mast cells (MC) in the control of pulmonary infection. Murine CMV (mCMV) productively infects MC in vivo and triggers their degranulation, resulting in the release of the CC chemokine ligand 5 (CCL5) that attracts CD8 T cells to infiltrate infected tissues. Comparing infection of MC-sufficient C57BL/6 mice and congenic MC-deficient Kit (W-sh/W-sh) "sash" mutants revealed an inverse relation between the number of lung-infiltrating CD8 T cells and viral burden in the lungs. Specifically, reduced lung infiltration by CD8 T cells in "sash" mutants was associated with an impaired infection control. The causal, though indirect, involvement of MC in antiviral control was confirmed by reversion of the deficiency phenotype in "sash" mutants reconstituted with MC. These recent findings predict that efficient MC reconstitution facilitates the control of CMV infection also in immunocompromised HCT recipients.

Mast cells as rapid innate sensors of cytomegalovirus by TLR3/TRIF signaling-dependent and -independent mechanisms.

The succinct metaphor, 'the immune system's loaded gun', has been used to describe the role of mast cells (MCs) due to their storage of a wide range of potent pro-inflammatory and antimicrobial mediators in secretory granules that can be released almost instantly on demand to fight invaders. Located at host-environment boundaries and equipped with an arsenal of pattern recognition receptors, MCs are destined to be rapid innate sensors of pathogens penetrating endothelial and epithelial surfaces. Although the importance of MCs in antimicrobial and antiparasitic defense has long been appreciated, their role in raising the alarm against viral infections has been noted only recently. Work on cytomegalovirus (CMV) infection in the murine model has revealed MCs as players in a novel cross-talk axis between innate and adaptive immune surveillance of CMV, in that infection of MCs, which is associated with MC degranulation and release of the chemokine CCL5, enhances the recruitment of protective CD8 T cells to extravascular sites of virus replication, specifically to lung interstitium and alveolar epithelium. Here, we have expanded on these studies by investigating the conditions for MC activation and the consequent degranulation in response to host infection. Surprisingly, the data revealed two temporally and mechanistically distinct waves of MC activation: an almost instant indirect activation that depended on TLR3/TRIF signaling and delayed activation by direct infection of MCs that did not involve TLR3/TRIF signaling. Cell type-specific Cre-recombination that yielded eGFP-expressing reporter virus selectively originating from MCs identified MC as a new in vivo, first-hit target cell of productive murine CMV infection.

The Wnt/ß-catenin pathway attenuates experimental allergic airway disease.

Signaling via the Wnt/ß-catenin pathway plays crucial roles in embryogenesis and homeostasis of adult tissues. In the lung, the canonical Wnt/ß-catenin pathway has been implicated in remodeling processes, development of emphysema, and fibrosis. However, its relevance for the modulation of allergic responses in the lung remains unclear. Using genetically modified mice with lung-specific inducible (doxycycline) Wnt-1 expression (CCSP-rtTA × tetO-Wnt1), the impact of Wnt on the development of allergic airway disease was analyzed. Overexpression of Wnt during the allergen challenge phase attenuated the development of airway inflammation in an acute model, as well as in a more therapeutic model of secondary challenge. These findings were further supported by treatment of allergen-sensitized mice with LiCl during challenge. Similar to Wnt, LiCl prevented the degradation of ß-catenin and, thus, attenuated allergic airway inflammation and hyperresponsiveness. Migration studies revealed that lung-specific expression of Wnt reduced the migration of Ag-loaded dendritic cells (DCs) into the draining lymph nodes following allergen challenge. Administration of in vitro allergen-loaded DCs overcame Wnt-mediated suppression of airway inflammation. Furthermore, in vitro studies confirmed that DC-dependent T cell activation is impaired by blocking ß-catenin degradation. These results demonstrate an important role for the canonical Wnt/ß-catenin pathway in the DC-mediated regulation of allergic responses in the lung.

Intronic promoters and their noncoding transcripts: a new source of cancer-associated genes.

Recent studies of mammalian genomes suggest that alternative promoters are associated with various disorders, including cancer. Here we present an intronic promoter of the murine proteinase 3 gene, which drives the expression of an alternative mRNA in intron 2 of the prtn3 gene. The proximal promoter sequences were identified and a series of promoter deletion constructs were used to identify the sequence elements that are required for basal promoter activity. Expression of the homeobox transcription factor CUX1 p75 isoform was found to suppress the activity of the alternative PR3 promoter. Data base analyses, multiple alignments and expression data showed that the intronic PR3 promoter is active in leukemia and other tumor cells as well as in mouse embryo, male mammary gland and bone marrow. In the spleen, the transcript is exclusively expressed by Gr-1(int) /CD11b(+) cells, which are also known as myeloid-derived suppressor cells (MDSCs). In humans, an alternative transcript of the PR3-gene could be detected in the bone marrow and in various cancer cell lines but not in primary leukemia cells, suggesting a species-overarching function of this kind of promoter. Therefore, the alternative PR3 promoter and its mRNA may be useful tools to investigate the fate of hematopoietic stem cells.

Oxidative burst and neutrophil elastase contribute to clearance of Aspergillus fumigatus pneumonia in mice.

Polymorphonuclear neutrophils (PMN) are important for the control of invasive aspergillosis (IA), a major threat to immunocompromised individuals. For clearance of Aspergillus fumigatus infections, PMN employ their potent oxidative and non-oxidative mechanisms. To clarify the relative contribution of these mechanisms, we analyzed p47(phox-/-), gp91(phox-/-) and elastase (ELA) deficient mice (ELANE) after intratracheal infection with A. fumigatus. Infected p47(phox-/-) and gp91(phox-/-) mice died within 4 days and had a significant higher fungal burden in the lungs compared to wild-type controls. Interestingly, the survival of ELANE mice after infection was unimpaired suggesting that ELA is not essential here. Nevertheless, A. fumigatus clearance was delayed in ELANE mice indicating a partial contribution of ELA to fungal immunity. Comparing p47(phox-/-), gp91(phox-/-) or ELANE mice for PMN activation and recruitment to the lungs, we were unable to detect significant differences in vitro or in vivo among mutant or wild-type strains suggesting intact PMN functionality of basic effector mechanisms. Fungal killing in vitro by ELA deficient PMN was comparably reduced as in p47(phox-/-) and gp91(phox-/-) deficient PMN corroborating the importance of oxidative and non-oxidative PMN mechanisms for the control of fungal outgrowth. Taken together, this suggests that intact oxidative as well as non-oxidative PMN effector functions are highly relevant for the control of A. fumigatus infections in vitro and in vivo. While ELA contributes to clearance of A. fumigatus, the oxidative functions are essential for survival.

Distinct signaling cascades of TREM-1, TLR and NLR in neutrophils and monocytic cells.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is an important mediator of innate inflammatory responses in microbial infections and sepsis. TREM-1 ligation on neutrophils (PMN) or monocytes results in the production of proinflammatory cytokines. Engagement of TREM-1 induces the activation of MAP kinases as well as rapid Ca(2+) mobilization. However, a detailed understanding of TREM-1 signaling pathways is currently lacking. We evaluated the TREM-1 signaling hierarchy in monocytic cells and found that the acute myeloid leukemia cell line MUTZ-3 expresses TREM-1 in a natural and functional manner. We compared essential signaling molecules of the TREM-1, TLR and NLR cascade in MUTZ-3 cells as well as primary monocytes or PMN by Western blot analysis. These studies confirmed the essential role of phosphatidyl inositide 3-kinase (PI3K) and p38MAPK in the TREM-1 as well as the TLR or NLR cascade of monocytic cells. Importantly, PI3K and p38MAPK signals in monocytic cells both control Ca(2+) mobilization and are directly connected in the TREM-1 signaling hierarchy, which contrasts previous results obtained in PMN. Taken together, our results indicate cell type-specific differences in the TREM-1 signaling cascade and contribute to an enhanced understanding of the regulation of innate inflammatory responses.

Mast cell-derived mediators promote murine neutrophil effector functions.

Mast cells are able to trigger life-saving immune responses in murine models for acute inflammation. In such settings, several lines of evidence indicate that the rapid and protective recruitment of neutrophils initiated by the release of mast cell-derived pro-inflammatory mediators is a key element of innate immunity. Herein, we investigate the impact of mast cells on critical parameters of neutrophil effector function. In the presence of activated murine bone marrow-derived mast cells, neutrophils freshly isolated from bone marrow rapidly lose expression of CD62L and up-regulate CD11b, the latter being partly driven by mast cell-derived TNF and GM-CSF. Mast cells also strongly enhance neutrophil phagocytosis and generation of reactive oxygen species. All these phenomena partly depend on mast cell-derived TNF and to a greater extend on GM-CSF. Furthermore, spontaneous apoptosis of neutrophils is greatly diminished due to the ability of mast cells to deliver antiapoptotic GM-CSF. Finally, we show in a murine model for acute lung inflammation that neutrophil phagocytosis is impaired in mast cell-deficient Kit (W-sh) /Kit (W-sh) mice but can be restored upon mast cell engraftment. Thus, a previously underrated feature of mast cells is their ability to boost neutrophil effector functions in immune responses.

Mast cell-deficient Kit(W-sh) "Sash" mutant mice display aberrant myelopoiesis leading to the accumulation of splenocytes that act as myeloid-derived suppressor cells.

Mast cell-deficient Kit(W-sh) "sash" mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that Kit(W-sh) causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell-independent phenomenon. Thus, the Kit(W-sh) mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research.

The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine IL-9 and is effective in the prevention of experimental asthma.

Ticks developed a multitude of different immune evasion strategies to obtain a blood meal. Sialostatin L is an immunosuppressive cysteine protease inhibitor present in the saliva of the hard tick Ixodes scapularis. In this study, we demonstrate that sialostatin L strongly inhibits the production of IL-9 by Th9 cells. Because we could show recently that Th9-derived IL-9 is essentially involved in the induction of asthma symptoms, sialostatin L was used for the treatment of experimental asthma. Application of sialostatin L in a model of experimental asthma almost completely abrogated airway hyperresponsiveness and eosinophilia. Our data suggest that sialostatin L can prevent experimental asthma, most likely by inhibiting the IL-9 production of Th9 cells. Thus, alternative to IL-9 neutralization sialostatin L provides the basis for the development of innovative therapeutic strategies to treat asthma.

Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy.

UV exposure boosts transcutaneous immunization and improves tumor immunity: cytotoxic T-cell priming through the skin.

Immunologic approaches to combat cancer aim at the induction of tumor-reactive immune responses to achieve long-term protection. In this context, we recently developed a transcutaneous immunization (TCI) method using the Toll-like receptor (TLR) 7 agonist imiquimod and a peptide epitope. Application onto intact skin induces potent cytotoxic T lymphocyte (CTL) responses and protection against transplanted tumors. The purpose of this study was to explore the effects of UV irradiation on imiquimod-based TCI. Here we show that skin exposure to low-dose UV light before TCI with imiquimod strongly boosts specific CTL responses leading to memory formation and enhanced tumor protection. Toward the mechanisms, we show that the activation of bone-marrow-derived dermal dendritic cells (DCs), but not Langerin-expressing DCs, is responsible for enhanced CTL activation. We describe an optimized TCI method that mediates enhanced CTL and antitumor responses by a DC- and TLR-dependent mechanism. These data may provide the basis for the future development of advanced vaccination protocols against tumors and persistent virus infections.