Tumor-targeted therapy with BRAF-inhibitor recruits activated dendritic cells to promote tumor immunity in melanoma.

BACKGROUND: Tumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially dendritic cells (DC) during tumor-targeted therapy. METHODS: Here, we investigated therapy-mediated immunological alterations in the tumor microenvironment (TME) and tumor-draining lymph nodes (LN) in the D4M.3A preclinical melanoma mouse model (harboring the V-Raf murine sarcoma viral oncogene homolog B (BRAF)V600E mutation) by using high-dimensional multicolor flow cytometry in combination with multiplex immunohistochemistry. This was complemented with RNA sequencing and cytokine quantification to characterize the immune status of the tumors. The importance of T cells during tumor-targeted therapy was investigated by depleting CD4+ or CD8+ T cells in tumor-bearing mice. Tumor antigen-specific T-cell responses were characterized by performing in vivo T-cell proliferation assays and the contribution of conventional type 1 DC (cDC1) to T-cell immunity during tumor-targeted therapy was assessed using Batf3-/- mice lacking cDC1. RESULTS: Our findings reveal that BRAF-inhibitor therapy increased tumor immunogenicity, reflected by an upregulation of genes associated with immune activation. The T cell-inflamed TME contained higher numbers of activated cDC1 and cDC2 but also inflammatory CCR2-expressing monocytes. At the same time, tumor-targeted therapy enhanced the frequency of migratory, activated DC subsets in tumor-draining LN. Even more, we identified a cDC2 population expressing the Fc gamma receptor I (FcγRI)/CD64 in tumors and LN that displayed high levels of CD40 and CCR7 indicating involvement in T cell-mediated tumor immunity. The importance of cDC2 is underlined by just a partial loss of therapy response in a cDC1-deficient mouse model. Both CD4+ and CD8+ T cells were essential for therapy response as their respective depletion impaired therapy success. On resistance development, the tumors reverted to an immunologically inert state with a loss of DC and inflammatory monocytes together with the accumulation of regulatory T cells. Moreover, tumor antigen-specific CD8+ T cells were compromised in proliferation and interferon-γ-production. CONCLUSION: Our results give novel insights into the remodeling of the myeloid landscape by tumor-targeted therapy. We demonstrate that the transient immunogenic tumor milieu contains more activated DC. This knowledge has important implications for the development of future combinatorial therapies.

Dendritic cell-targeted therapy expands CD8 T cell responses to bona-fide neoantigens in lung tumors.

Cross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage.

Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization.

BACKGROUND: Chronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment. METHODS: We examined CD45-/CD31-/CD34+ preadipocytes and CD68+ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14+ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR. RESULTS: CD45-/CD31-/CD34+ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68+ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs. CONCLUSION: Preadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing.

Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Mouse dendritic cells and other myeloid subtypes in healthy lymph nodes and skin: 26-Color flow cytometry panel for immune phenotyping.

This novel 26-color flow cytometry panel allows the detailed immune phenotyping of the complex network of myeloid cells in murine lymph nodes and skin. With the optimized panel the different murine DC-subsets and other myeloid cell types can be identified and further characterized for co-stimulatory and inhibitory surface molecules.

Antigen targeting to dendritic cells: Still a place in future immunotherapy?

The hallmark of DCs is their potent and outstanding capacity to activate naive resting T cells. As such, DCs are the sentinels of the immune system and instrumental for the induction of immune responses. This is one of the reasons, why DCs became the focus of immunotherapeutical strategies to fight infections, cancer, and autoimmunity. Besides the exploration of adoptive DC-therapy for which DCs are generated from monocytes or purified in large numbers from the blood, alternative approaches were developed such as antigen targeting of DCs. The idea behind this strategy is that DCs resident in patients' lymphoid organs or peripheral tissues can be directly loaded with antigens in situ. The proof of principle came from mouse models; subsequent translational studies confirmed the potential of this therapy. The first clinical trials demonstrated feasibility and the induction of T-cell immunity in patients. This review will cover: (i) the historical aspects of antigen targeting, (ii) briefly summarize the biology of DCs and the immunological functions upon which this concept rests, (iii) give an overview on attempts to target DC receptors with antibodies or (glycosylated) ligands, and finally, (iv) discuss the translation of antigen targeting into clinical therapy.

Early antitumor activity of oral Langerhans cells is compromised by a carcinogen.

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αßT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin.

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses.

Specific Protein Antigen Delivery to Human Langerhans Cells in Intact Skin.

Immune modulating therapies and vaccines are in high demand, not least to the recent global spread of SARS-CoV2. To achieve efficient activation of the immune system, professional antigen presenting cells have proven to be key coordinators of such responses. Especially targeted approaches, actively directing antigens to specialized dendritic cells, promise to be more effective and accompanied by reduced payload due to less off-target effects. Although antibody and glycan-based targeting of receptors on dendritic cells have been employed, these are often expensive and time-consuming to manufacture or lack sufficient specificity. Thus, we applied a small-molecule ligand that specifically binds Langerin, a hallmark receptor on Langerhans cells, conjugated to a model protein antigen. Via microneedle injection, this construct was intradermally administered into intact human skin explants, selectively loading Langerhans cells in the epidermis. The ligand-mediated cellular uptake outpaces protein degradation resulting in intact antigen delivery. Due to the pivotal role of Langerhans cells in induction of immune responses, this approach of antigen-targeting of tissue-resident immune cells offers a novel way to deliver highly effective vaccines with minimally invasive administration.

Differential infection of murine and human dendritic cell subsets by oncolytic vesicular stomatitis virus variants.

Oncolytic viruses (OVs) can eradicate tumor cells and elicit antitumor immunity. VSV-GP, a chimeric vesicular stomatitis virus (VSV) with the glycoprotein (GP) of the lymphocytic choriomeningitis virus, is a promising new OV candidate. However, the interaction of VSV-GP with host immune cells is not fully understood. Dendritic cells (DCs) are essential for inducing efficient antitumor immunity. Thus, we aimed to investigate the interaction of VSV-GP with different murine and human DCs subsets in direct comparison to the less cytopathic variant VSV-dM51-GP and wild type VSV. Immature murine bone marrow-derived DCs (BMDCs) were equally infected and killed by VSV and VSV-GP. Human monocyte-derived DCs (moDCs) were more permissive to VSV. Interestingly, VSV-dM51-GP induced maturation instead of killing in both BMDCs and moDCs as well as a pronounced release of pro-inflammatory cytokines. Importantly, matured BMDCs and moDCs were no longer susceptible to VSV-GP infection. Mouse splenic conventional DC type 1 (cDC1) could be infected ex vivo by VSV and VSV-GP to a higher extent than cDC2. Systemic infection of mice with VSV-GP and VSV-dM51-GP resulted in strong activation of cDCs despite low infection rates in spleen and tumor tissue. Human blood cDC1 were equally infected by VSV and VSV-GP, whereas cDC2 showed preferential infection with VSV. Our study demonstrated differential DC infection, activation, and cytokine production after the treatment with VSV and VSV-GP variants among species and subsets, which should be taken into account when investigating immunological mechanisms of oncolytic virotherapy in mouse models and human clinical trials.

Laser-assisted epicutaneous immunization to target human skin dendritic cells.

Dendritic cells (DC) are promising targets for immunotherapy of cancer. Clinically, immunization against cancer antigens by means of the most potent antigen-presenting cells, that is DC, remains an important treatment option in combination with the modern immune checkpoint approaches. Instead of adoptively transferring in vitro monocyte-derived DC, they can also be loaded in situ by antibody-mediated targeting of antigen. Conventionally, these vaccines are delivered by classical intradermal injections. Here, we tested an alternative approach, namely laser-assisted epicutaneous immunization. With an infrared laser ("Precise Laser Epidermal System"/P.L.E.A.S.E.® Laser System), we created micropores in human skin and applied monoclonal antibodies (mAbs) against C-type lectins, for example DEC-205/CD205 and Langerin/CD207. Optimal parameters for formation of pores in epidermis and dermis were determined. We could induce pores of defined depths without enhanced apoptosis around them. Antibodies applied epicutaneously to the laser-porated skin could be detected both in Langerhans cells (LC) in situ in the epidermis and in migratory skin DC subsets from short term human skin explant culture, demonstrating uptake and transport of Langerin and DEC-205 mAbs. Efficacy of targeting was similar between the different laser treatments and pore depths. Thus, laser-assisted epicutaneous immunization may be a valuable alternative to intradermal injection, yet the loading efficacy of DC needs to be further improved.

Skin dendritic cells in melanoma are key for successful checkpoint blockade therapy.

BACKGROUND: Immunotherapy with checkpoint inhibitors has shown impressive results in patients with melanoma, but still many do not benefit from this line of treatment. A lack of tumor-infiltrating T cells is a common reason for therapy failure but also a loss of intratumoral dendritic cells (DCs) has been described. METHODS: We used the transgenic tg(Grm1)EPv melanoma mouse strain that develops spontaneous, slow-growing tumors to perform immunological analysis during tumor progression. With flow cytometry, the frequencies of DCs and T cells at different tumor stages and the expression of the inhibitory molecules programmed cell death protein-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells were analyzed. This was complemented with RNA-sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR) analysis to investigate the immune status of the tumors. To boost DC numbers and function, we administered Fms-related tyrosine 3 ligand (Flt3L) plus an adjuvant mix of polyI:C and anti-CD40. To enhance T cell function, we tested several checkpoint blockade antibodies. Immunological alterations were characterized in tumor and tumor-draining lymph nodes (LNs) by flow cytometry, CyTOF, microarray and RT-qPCR to understand how immune cells can control tumor growth. The specific role of migratory skin DCs was investigated by coculture of sorted DC subsets with melanoma-specific CD8+ T cells. RESULTS: Our study revealed that tumor progression is characterized by upregulation of checkpoint molecules and a gradual loss of the dermal conventional DC (cDC) 2 subset. Monotherapy with checkpoint blockade could not restore antitumor immunity, whereas boosting DC numbers and activation increased tumor immunogenicity. This was reflected by higher numbers of activated cDC1 and cDC2 as well as CD4+ and CD8+ T cells in treated tumors. At the same time, the DC boost approach reinforced migratory dermal DC subsets to prime gp100-specific CD8+ T cells in tumor-draining LNs that expressed PD-1/TIM-3 and produced interferon γ (IFNγ)/tumor necrosis factor α (TNFα). As a consequence, the combination of the DC boost with antibodies against PD-1 and TIM-3 released the brake from T cells, leading to improved function within the tumors and delayed tumor growth. CONCLUSIONS: Our results set forth the importance of skin DC in cancer immunotherapy, and demonstrates that restoring DC function is key to enhancing tumor immunogenicity and subsequently responsiveness to checkpoint blockade therapy.

CD73 contributes to anti-inflammatory properties of afferent lymphatic endothelial cells in humans and mice.

CD73 is an important ectoenzyme responsible for the production of extracellular adenosine. It is involved in regulating inflammatory responses and cell migration and is overexpressed in various cancers. The functions of CD73 in blood endothelial cells are understood in detail, but its role on afferent lymphatics remains unknown. Moreover, anti-CD73 antibodies are now used in multiple clinical cancer trials, but their effects on different endothelial cell types have not been studied. This study reveals that a previously unknown role of CD73 on afferent lymphatics is to dampen immune responses. Knocking it out or suppressing it by siRNA leads to the upregulation of inflammation-associated genes on lymphatic endothelial cells and a more pro-inflammatory phenotype of interacting dendritic cells in vitro and in vivo. In striking contrast, anti-CD73 antibodies had only negligible effects on the gene expression of lymphatic- and blood-endothelial cells. Our data thus reveal new functions of lymphatic CD73 and indicate a low likelihood of endothelial cell-related adverse effects by CD73 targeting therapeutic antibodies.

Notch-Mediated Generation of Monocyte-Derived Langerhans Cells: Phenotype and Function.

Langerhans cells (LCs) in the skin are a first line of defense against pathogens but also play an essential role in skin homeostasis. Their exclusive expression of the C-type lectin receptor Langerin makes them prominent candidates for immunotherapy. For vaccine testing, an easily accessible cell platform would be desirable as an alternative to the time-consuming purification of LCs from human skin. Here, we present such a model and demonstrate that monocytes in the presence of GM-CSF, TGF-ß1, and the Notch ligand DLL4 differentiate within 3 days into CD1a+Langerin+cells containing Birbeck granules. RNA sequencing of these monocyte-derived LCs (moLCs) confirmed gene expression of LC-related molecules, pattern recognition receptors, and enhanced expression of genes involved in the antigen-presenting machinery. On the protein level, moLCs showed low expression of costimulatory molecules but prominent expression of C-type lectin receptors. MoLCs can be matured, secrete IL-12p70 and TNF-α, and stimulate proliferation and cytokine production in allogeneic CD4+ and CD8+ T cells. In regard to vaccine testing, a recently characterized glycomimetic Langerin ligand conjugated to liposomes demonstrated specific and fast internalization into moLCs. Hence, these short-term in vitro‒generated moLCs represent an interesting tool to screen LC-based vaccines in the future.

BRAF and MEK inhibition in melanoma patients enables reprogramming of tumor infiltrating lymphocytes.

BACKGROUND: Combined inhibition of BRAF/MEK is an established therapy for melanoma. In addition to its canonical mode of action, effects of BRAF/MEK inhibitors on antitumor immune responses are emerging. Thus, we investigated the effect of these on adaptive immune responses. PATIENTS, METHODS AND RESULTS: Sequential tumor biopsies obtained before and during BRAF/MEK inhibitor treatment of four (n = 4) melanoma patients were analyzed. Multiplexed immunofluorescence staining of tumor tissue revealed an increased infiltration of CD4+ and CD8+ T cells upon therapy. Determination of the T-cell receptor repertoire usage demonstrated a therapy induced increase in T-cell clonotype richness and diversity. Application of the Grouping of Lymphocyte Interactions by Paratope Hotspots algorithm revealed a pre-existing immune response against melanoma differentiation and cancer testis antigens that expanded preferentially upon therapy. Indeed, most of the T-cell clonotypes found under BRAF/MEK inhibition were already present in lower numbers before therapy. This expansion appears to be facilitated by induction of T-bet and TCF7 in T cells, two transcription factors required for self-renewal and persistence of CD8+ memory T cells. CONCLUSIONS: Our results suggest that BRAF/MEK inhibition in melanoma patients allows an increased expansion of pre-existing melanoma-specific T cells by induction of T-bet and TCF7 in these.

Combining chemotherapy and autologous peptide-pulsed dendritic cells provides survival benefit in stage IV melanoma patients.

BACKGROUND AND OBJECTIVES: We examined retrospectively whether the combination of standard dacarbazine (DTIC) and/or fotemustine chemotherapy and autologous peptide-loaded dendritic cell (DC) vaccination may improve survival of stage IV melanoma patients. Furthermore, a small cohort of long-term survivors was studied in more detail. PATIENTS AND METHODS: Between 1998 and 2008, 41 patients were vaccinated at least three times with DCs while receiving chemotherapy and compared to all other 168 patients in our database who only received chemotherapy (1993-2008). RESULTS: Median life expectancy of patients receiving additional DC-vaccination was 18 months, compared to eleven months for patients under standard chemotherapy alone. In contrast to patients with other haplotypes, the HLA-A1/A1 subset of DC-treated patients showed significantly lower median survival (12 vs. 25 months). Autoantibodies were frequently detected in serum of both vaccinated and non-vaccinated patients, and there was no correlation between titers, loss or appearance of autoantibodies and survival. Additionally, phenotyping of DCs and PBMCs also did not reveal any conspicuous correlation with survival. CONCLUSIONS: Combining standard chemotherapy and DC vaccination appears superior to chemotherapy alone. The impact of HLA haplotypes on survival emphasizes the importance of a careful selection of patients with specific, well-defined HLA haplotypes for future vaccination trials using peptide-pulsed DCs, possibly combined with checkpoint inhibitors.

Epidermal activation of Hedgehog signaling establishes an immunosuppressive microenvironment in basal cell carcinoma by modulating skin immunity.

Genetic activation of hedgehog/glioma-associated oncogene homolog (HH/GLI) signaling causes basal cell carcinoma (BCC), a very frequent nonmelanoma skin cancer. Small molecule targeting of the essential HH effector Smoothened (SMO) has proven an effective therapy of BCC, though the frequent development of drug resistance poses major challenges to anti-HH treatments. In light of recent breakthroughs in cancer immunotherapy, we analyzed the possible immunosuppressive mechanisms in HH/GLI-induced BCC in detail. Using a genetic mouse model of BCC, we identified profound differences in the infiltration of BCC lesions with cells of the adaptive and innate immune system. Epidermal activation of Hh/Gli signaling led to an accumulation of immunosuppressive regulatory T cells, and to an increased expression of immune checkpoint molecules including programmed death (PD)-1/PD-ligand 1. Anti-PD-1 monotherapy, however, did not reduce tumor growth, presumably due to the lack of immunogenic mutations in common BCC mouse models, as shown by whole-exome sequencing. BCC lesions also displayed a marked infiltration with neutrophils, the depletion of which unexpectedly promoted BCC growth. The study provides a comprehensive survey of and novel insights into the immune status of murine BCC and serves as a basis for the design of efficacious rational combination treatments. This study also underlines the need for predictive immunogenic mouse models of BCC to evaluate the efficacy of immunotherapeutic strategies in vivo.