Christoph H. Tripp

Langerhans cells – dendritic cells of the epidermis

Langerhans cells (LC) are dendritic cells of the epidermis. They are highly specialized leukocytes that serve immunogenic and tolerogenic purposes. Here, we review some aspects of LC biology, emphasizing those areas where LC are or may turn out to be special.

Langerhans cells cross-present antigen derived from skin

Dendritic cells (DC) efficiently cross-present exogenous antigen on MHC class I molecules to CD8+ T cells. However, little is known about cross-presentation by Langerhans cells (LC), the DCs of the epidermis. Therefore, we investigated this issue in detail. Isolated murine LCs were able to cross-present soluble ovalbumin protein on MHC-class I molecules to antigen-specific CD8+ T cells, albeit less potently than the CD8+ DC subsets from spleen. Furthermore, LCs cross-presented cell-associated ovalbumin peptide and protein expressed by neighboring keratinocytes. Use of transporter associated with antigen processing (TAP-1)-deficient mice suggested a TAP-dependent pathway. Similar observations were made with migratory LC. Antigen expressed in the epidermis was ingested by LCs during migration from the epidermis and presented to antigen-specific T cells in vitro. Cross-presentation of ovalbumin protein by LCs induced IFN-γ production and cytotoxicity in antigen-specific CD8+ T cells. Additionally, epicutaneous application of ovalbumin protein induced in vivo proliferation of OT-I T cells in the draining lymph nodes; this was markedly enhanced when antigen was applied to inflamed, barrier-disrupted skin. Thus, LCs cross-present exogenous antigen to CD8+ T cells and induce effector functions, like cytokine production and cytotoxicity, and may thereby critically contribute in epicutaneous vaccination approaches.

Tumor immunotherapy by epicutaneous immunization requires langerhans cells.

A role for Langerhans cells (LC) in the induction of immune responses in the skin has yet to be conclusively demonstrated. We used skin immunization with OVA protein to induce immune responses against OVA-expressing melanoma cells. Mice injected with OVA-specific CD8+ T cells and immunized with OVA onto barrier-disrupted skin had increased numbers of CD8+ T cells in the blood that produced IFN-γ and killed target cells. These mice generated accelerated cytotoxic responses after secondary immunization with OVA. Prophylactic or therapeutic immunization with OVA onto barrier-disrupted skin inhibited the growth of B16.OVA tumors. LC played a critical role in the immunization process because depletion of LC at the time of skin immunization dramatically reduced the tumor-protective effect. The topically applied Ag was presented by skin-derived LC in draining lymph nodes to CD8+ T cells. Thus, targeting of tumor Ags to LC in vivo is an effective strategy for tumor immunotherapy.

Epidermal Langerhans Cells Rapidly Capture and Present Antigens from C-Type Lectin-Targeting Antibodies Deposited in the Dermis

Antigen-presenting cells can capture antigens that are deposited in the skin, including vaccines given subcutaneously. These include different dendritic cells (DCs) such as epidermal Langerhans cells (LCs), dermal DCs, and dermal langerin+ DCs. To evaluate access of dermal antigens to skin DCs, we used mAb to two C-type lectin endocytic receptors, DEC-205/CD205 and langerin/CD207. When applied to murine and human skin explant cultures, these mAbs were efficiently taken up by epidermal LCs. In addition, anti-DEC-205 targeted langerin+ CD103+ and langerin- CD103- mouse dermal DCs. Unexpectedly, intradermal injection of either mAb, but not isotype control, resulted in strong and rapid labeling of LCs in situ, implying that large molecules can diffuse through the basement membrane into the epidermis. Epidermal LCs targeted in vivo by ovalbumin-coupled anti-DEC-205 potently presented antigen to CD4+ and CD8+ T cells in vitro. However, to our surprise, LCs targeted through langerin were unable to trigger T-cell proliferation. Thus, epidermal LCs have a major function in uptake of lectin-binding antibodies under standard vaccination conditions.

Targeting Skin Dendritic Cells to Improve Intradermal Vaccination

Vaccinations in medicine are typically administered into the muscle beneath the skin or into the subcutaneous fat. As a consequence, the vaccine is immunologically processed by antigen-presenting cells of the skin or the muscle. Recent evidence suggests that the clinically seldom used intradermal route is effective and possibly even superior to the conventional subcutaneous or intramuscular route. Several types of professional antigen-presenting cells inhabit the healthy skin. Epidermal Langerhans cells (CD207/langerin(+)), dermal langerin(neg), and dermal langerin(+) dendritic cells (DC) have been described, the latter subset so far only in mouse skin. In human skin langerin(neg) dermal DC can be further classified based on their reciprocal expression of CD1a and CD14. The relative contributions of these subsets to the generation of immunity or tolerance are still unclear. Yet, specializations of these different populations have become apparent. Langerhans cells in human skin appear to be specialized for induction of cytotoxic T lymphocytes; human CD14(+) dermal DC can promote antibody production by B cells. It is currently attempted to rationally devise and improve vaccines by harnessing such specific properties of skin DC. This could be achieved by specifically targeting functionally diverse skin DC subsets. We discuss here advances in our knowledge on the immunological properties of skin DC and strategies to significantly improve the outcome of vaccinations by applying this knowledge.

Lapatinib and doxorubicin enhance the Stat1‐dependent antitumor immune response

The dual erbB1/2 tyrosine kinase inhibitor lapatinib as well as the anthracycline doxorubicin are both used in the therapy of HER2‐positive breast cancer. Using MMTV‐neu mice as an animal model for HER2‐positive breast cancer, we observed enhanced tumor infiltration by IFN‐γ‐secreting T cells after treatment with doxorubicin and/or lapatinib. Antibody depletion experiments revealed a contribution of CD8+ but not CD4+ T cells to the antitumor effect of these drugs. Doxorubicin treatment additionally decreased the content of immunosuppressive tumor‐associated macrophages (TAMs) in the tumor bed. In contrast, Stat1‐deficient mice were resistant to tumor growth inhibition by lapatinib and/or doxorubicin and exhibited impaired T‐cell activation and reduced T‐cell infiltration of the tumor in response to drug treatment. Furthermore, Stat1‐deficiency resulted in reduced expression of the T‐cell chemotactic factors CXCL9, CXCL10, and CXCL11 in the tumor epithelium. The inhibition of TAM infiltration of the tumor by doxorubicin and the immunosuppressive function of TAMs were found to be Stat1 independent. Taken together, the results point to an important contribution toward enhancing T‐cell and IFN‐γ‐based immunity by lapatinib as well as doxorubicin and emphasize the role of Stat1 in building an effective antitumor immune response.

Sphingosine-1-phosphate receptor type-1 agonism impairs blood dendritic cell chemotaxis and skin dendritic cell migration to lymph nodes under inflammatory conditions

SEW2871 is a potent sphingosine-1-phosphate receptor type-1 (S1P(1))-selective agonist that induces peripheral lymphopenia through sequestration of lymphocytes into secondary lymphoid organs, similar to the non-selective sphingosine-1-phosphate (S1P) receptor agonist FTY720. FTY720 has been reported to interfere with human dendritic cell (DC) effector functions and both FTY720 and SEW2871 have been shown to modulate murine DC trafficking in vivo. Little is known about the possible effects of SEW2871 on human and murine DC functions. Here, we demonstrate that in contrast to FTY720, SEW2871 does not induce down-regulation of S1P(1) in human DCs and thus does not exert a functional antagonism at S1P(1). Notably, the compound was found to impair chemotaxis of immature and mature human DCs in vitro, possibly by interfering with the activation of p44/p42 and p38 mitogen-activated protein kinase signaling pathways. Comparative FACS analyses show that SEW2871 mediates CD18 down-regulation on mature human DCs. The influence on DC migration could be confirmed with in vivo assays using BALB/c mice in which SEW2871 impairs the migration of CD11c+ DC and CD207+ Langerhans cells (LC) to the draining lymph nodes (LNs) under inflammatory conditions. These results suggest that the S1P-S1P(1) axis might not only control lymphocyte trafficking but also play a pivotal role in DC migration from the skin to LN.

Murine Langerin+ dermal dendritic cells prime CD8+ T cells while Langerhans cells induce cross‐tolerance

Skin dendritic cells (DCs) control the immunogenicity of cutaneously administered vaccines. Antigens targeted to DCs via the C‐type lectin Langerin/CD207 are cross‐presented to CD8+ T cells in vivo. We investigated the relative roles of Langerhans cells (LCs) and Langerin+ dermal DCs (dDCs) in different vaccination settings. Poly(I:C) and anti‐CD40 agonist antibody promoted cytotoxic responses upon intradermal immunization with ovalbumin (OVA)‐coupled anti‐Langerin antibodies (Langerin/OVA). This correlated with CD70 upregulation in Langerin+ dDCs, but not LCs. In chimeric mice where Langerin targeting was restricted to dDCs, CD8+ T‐cell memory was enhanced. Conversely, providing Langerin/OVA exclusively to LCs failed to prime cytotoxicity, despite initial antigen cross‐presentation to CD8+ T cells. Langerin/OVA combined with imiquimod could not prime CD8+ T cells and resulted in poor cytotoxicity in subsequent responses. This tolerance induction required targeting and maturation of LCs. Altogether, Langerin+ dDCs prime long‐lasting cytotoxic responses, while cross‐presentation by LCs negatively influences CD8+ T‐cell priming. Moreover, this highlights that DCs exposed to TLR agonists can still induce tolerance and supports the existence of qualitatively different DC maturation programs.

Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4+ T cells, but also on MHC-class I molecules to CD8+ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4+ and CD8+ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin+ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin+ dermal dendritic cell population located in the upper dermis. The preferential capture of topically applied antigen by Langerhans cells and their ability to induce potent CD4+ and CD8+ T cell responses emphasizes their potential for epicutaneous immunization strategies.

Langerhans cells as targets for immunotherapy against skin cancer

Cancer is the second most common cause of death in the world. Treatment of cancer is very challenging and immunotherapy has been developed as a potential way to fight cancer. The main obstacle with immunotherapy is that cancer cells evolve from healthy body cells in response to an accumulation of genetic mutations. As a consequence, the immune system struggles to detect the abnormal cells as they are mainly recognized as self. This implies that equipping the immune system to eliminate cancer cells is tricky, yet represents a very efficient way to constrain the growth of tumors. We became interested in developing immunotherapeutical strategies against skin cancer in the context of our observations that Langerhans cells (LC) are very potent antigen presenting cells and are able to incorporate protein antigens and present them to CD4+ and CD8+ T cells in the skin‐draining lymph nodes. As a consequence, we developed an immunization strategy through the skin, termed epicutaneous immunization. Protein antigen applied onto barrier‐disrupted skin induces long‐lasting cytotoxic T‐cell responses, potent enough to control and inhibit tumor growth. In this review, we suggest that immunization strategies through the skin could be a promising new approach for the treatment of skin cancer.