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One in every three cancers diagnosed is a skin cancer. Europe has the global lead in the number of UV-attributable cancer cases with the highest number of melanoma cases worldwide and the second highest number of keratinocyte cancers (KC). Further increases are expected in Europe for the coming decades. Projected increases are highest for KC with increases in incidence around 40% and increases in mortality around 50%, with KC mortality in males approximating melanoma mortality in females. The two main drivers for this skin cancer epidemic are ageing of the population but especially UV exposure. In conclusion, skin cancer represents a major challenge in the cancer field in Europe today and will continue to do so in the coming decades. This calls for a European skin cancer action plan intended to reduce avoidable UV exposure and to prepare the healthcare system to safeguard early diagnosis and treatment of skin cancer., (© 2024 European Academy of Dermatology and Venereology.)

Background: Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are potentially life-threatening autoimmune blistering diseases. Treatment is based on long-term immunosuppression with high doses of glucocorticosteroids in combination with potentially corticosteroid-sparing agents and/or rituximab. Immunoadsorption (IA) has emerged as a fast-acting adjuvant treatment option., Objectives: To assess the clinical efficacy of IA in addition to best medical treatment (BMT)., Methods: We conducted a multicentre (26 centres from Germany and Austria) randomized controlled trial in 72 patients with newly diagnosed, relapsed or chronic active PV or PF (34 female patients and 38 male patients, aged 42-72 years) comparing BMT (prednisolone 1.0 mg kg-1 per day plus azathioprine or mycophenolate) with adjuvant IA (BMT + IA). Central 1 : 1 randomization was done at the coordinating centre for clinical trials (KKS Marburg). The primary endpoint was analysed using Kaplan-Meier and Cox regression methods., Results: The study was ended prematurely owing to safety concerns after random allocation of 72 patients to BMT + IA (n = 34) or BMT (n = 38). The primary endpoint, time to complete remission on therapy, was not significantly different for the two groups [hazard ratio (HR) 1.35, 95% confidence interval (CI) 0.68-2.69; P = 0.39]. The cumulative dose of prednisolone was significantly lower in the BMT + IA group compared with BMT alone (difference -1214, 95% CI -2225 to -70; P = 0.03). In a post hoc analysis, patients with more extensive PV/PF showed a tendency towards a shorter time to remission in the BMT + IA group compared with the BMT group (HR 1.87, P = 0.17 in patients with baseline Pemphigus Disease Area Index ≥ 15). While more adverse events were observed in patients in the BMT group (29 vs. 25), severe adverse events were more frequent in patients in the BMT + IA group (17 events in 10 patients vs. 11 events in 8 patients)., Conclusions: In this study, adjuvant IA did not demonstrate a shorter time to clinical remission, but a corticosteroid-sparing effect was observed. In patients with extensive PV/PF, post hoc analysis suggests that adjuvant IA may lead to earlier remission, but potential adverse events must be carefully weighed against the expected benefits., Competing Interests: Conflicts of interest N.v.B. has a joint project and a patent pending with Euroimmun and received honoraria from Fresenius Medical Care. E.S. has a scientific cooperation arrangement with Fresenius Medical Care and received honoraria from this company. M.H. and R.E. have received honoraria from Fresenius Medical Care., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists.)

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ -/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients., (© 2023. The Author(s).)

In order to update recommendations on treatment, supportive care, education, and follow-up of patients with invasive cutaneous squamous cell carcinoma (cSCC), a multidisciplinary panel of experts from the European Association of Dermato-Oncology (EADO), the European Dermatology Forum (EDF), the European Society for Radiotherapy and Oncology (ESTRO), the European Union of Medical Specialists (UEMS), the European Academy of Dermatology and Venereology (EADV), and the European Organisation of Research and Treatment of Cancer (EORTC) was formed. Recommendations were based on an evidence-based literature review, guidelines, and expert consensus. Treatment recommendations are presented for common primary cSCC (low risk, high risk), locally advanced cSCC, regional metastatic cSCC (operable or inoperable), and distant metastatic cSCC. For common primary cSCC, the first-line treatment is surgical excision with postoperative margin assessment or micrographically controlled surgery. Achieving clear surgical margins is the most important treatment consideration for patients with cSCCs amenable to surgery. Regarding adjuvant radiotherapy for patients with high-risk localised cSCC with clear surgical margins, current evidence has not shown significant benefit for those with at least one high-risk factor. Radiotherapy should be considered as the primary treatment for non-surgical candidates/tumours. For cSCC with cytologically or histologically confirmed regional nodal metastasis, lymph node dissection is recommended. For patients with metastatic or locally advanced cSCC who are not candidates for curative surgery or radiotherapy, anti-PD-1 agents are the first-line systemic treatment, with cemiplimab being the first approved systemic agent for advanced cSCC by the Food and Drugs Administration/European Medicines Agency. Second-line systemic treatments for advanced cSCC, include epidermal growth factor receptor inhibitors (cetuximab) combined with chemotherapy or radiotherapy. Multidisciplinary board decisions are mandatory for all patients with advanced cSCC, considering the risks of toxicity, the age and frailty of patients, and co-morbidities, including immunosuppression. Patients should be engaged in informed, shared decision-making on management and be provided with the best supportive care to improve symptom management and quality of life. The frequency of follow-up visits and investigations for subsequent new cSCC depends on underlying risk characteristics., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Invasive cutaneous squamous cell carcinoma (cSCC) is one of the most common cancers in white populations, accounting for 20% of all cutaneous malignancies. Overall, cSCC mostly has very good prognosis after treatment, with 5-year cure rates greater than 90%. Despite the overall favourable prognosis and the proportionally rare deaths, cSCC is associated with a high total number of deaths due to its high incidence. A collaboration of multidisciplinary experts from the European Association of Dermato-Oncology (EADO), the European Dermatology Forum (EDF), the European Society for Radiotherapy and Oncology (ESTRO), the European Union of Medical Specialists (UEMS), the European Academy of Dermatology and Venereology (EADV) and the European Organization of Research and Treatment of Cancer (EORTC), was formed to update recommendations on cSCC, based on current literature and expert consensus. Part 1 of the guidelines addresses the updates on classification, epidemiology, diagnosis, risk stratification, staging and prevention in immunocompetent as well as immunosuppressed patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Stratigos reports personal fees and/or research support from Novartis, Roche, BMS, Abbvie, Sanofi, Regeneron, Genesis Pharma, outside the submitted work. Dr. Garbe reports personal fees from Amgen, personal fees from MSD, grants and personal fees from Novartis, grants and personal fees from NeraCare, grants and personal fees from BMS, personal fees from Pierre Fabre, personal fees from Philogen, grants and personal fees from Roche, grants and personal fees from Sanofi, outside the submitted work. Dr. Dessinioti has nothing to disclose. Dr. Lebbe reports grants and personal fees from Bristol-Myers Squibb, personal fees from MSD, personal fees from Novartis, personal fees from Amgen, grants and personal fees from Roche, personal fees from Avantis Medical Systems, personal fees from Pierre Fabre, personal fees from Pfizer, personal fees from Incyte, outside the submitted work. Dr. Bataille reports personal fees from Novartis, personal fees from Merck MSD, outside the submitted work. Dr. Bastholt reports personal fees for advisory board activity: BMS, Roche, Novartis, Pierre-Fabre, Astra Zeneca, InCyte, MSD/Merck, Bayer, outside the submitted work. Dr. Dréno reports grants and personal fees from BMS, personal fees from MSD, grants and personal fees from Roche, grants and personal fees from Fabre, grants and personal fees from Sanofi, outside the submitted work. Dr Dummer reports intermittent, project focused consulting and/or advisory relationships with Novartis, Merck Sharp & Dhome (MSD), Bristol-Myers Squibb (BMS), Roche, Amgen, Takeda, Pierre Fabre, Sun Pharma, Sanofi, Catalym, Second Genome, Regeneron, Alligator, T3 Pharma, MaxiVAX SA, Pfizer, Simcere and touchIME outside the submitted work. Dr. Fargnoli reports personal consulting fees from Almirall, honoraria from Sanofi-Regeneron and participation in Advisory Board for Sanofi-Regeneron, outside the submitted work. Dr. Forsea reports scientific consultant/speaker fee from Novartis, Merck, outside the submitted work. Dr. Harwood reports institutional research grants and honoraria from Sanofi, Novartis, Merck, Pfizer, Galderma, MEDA, Almirall, Pellepharm, Leo Pharma, CERIES, outside the submitted work. Dr. Hauschild reports grants and personal fees from Amgen, grants and personal fees from BMS, grants and personal fees from MerckPfizer, grants and personal fees from MSD/Merck, grants and personal fees from Philogen, grants and personal fees from Pierre Fabre, grants and personal fees from Regeneron, grants and personal fees from Roche, grants and personal fees from Sanofi-Genzyme, grants and personal fees from Novartis Pharma, grants and personal fees from Eisai, personal fees from Immunocore, grants and personal fees from Replimune, personal fees from Seagen, personal fees from IO Biotech, personal fees from Dermagnostix, personal fees from Incyte, grants and personal fees from NeraCare, personal fees from Highlight Therapeutics, grants from Huya Biosciences, personal fees from Kyowa Kirin, outside the submitted work. Dr. Hoeller reports personal fees from BMS, personal fees from Novartis, grants and personal fees from Amgen, personal fees from Almirall, personal fees from Pierre Fabre, personal fees from Sanofi, personal fees from MSD, outside the submitted work. Dr. Kandolf-Sekulovic reports speakers’ honoraria for Roche, Novartis, MSD, BMS, Janssen outside the submitted work. Dr. Kaufmann reports institutional research grants (clinical trials) from: AbbVie, Amgen, Biontech, BMS, Celgene, Galderma, Janssen, Leo, Lilly, Merck, MSD, Novartis, Pierre Fabre, Pfizer, Regeneron, Roche, Wyeth. Advisory Board and Honoraria from Merz, Roche, Novartis, outside the submitted work. Dr. Kelleners-Smeets reports grants from Netherlands Organization for Health Research and Development, other from Janssen-Cilag, other from AbbVie, other from Galderma, outside the submitted work. Dr. Malvehy reports research grants from Almirall, ISDIN, Leo Pharma, Galderma, GSK, Cantabria; participation in advisory board meetings for Almirall, Sunpharma, BMS, Roche, Novartis, Pierre-Fabre, outside the submitted work. Dr. del Marmol reports personal fees from MSD, from BMS, personal fees from Sanofi, grants and personal fees from ABVIE, grants from Jansen, outside the submitted work. Dr. Moreno-Ramírez has nothing to disclose. Dr. Pellecani reports grants from university of Modena and Reggio Emilia, during the conduct of the study; grants from Novartis, grants and personal fees from Almirall, grants from Leo Pharma, from null, outside the submitted work. Dr. Peris reports honoraria for advisory board and grants from AbbVie, Almirall, Biogen Celgene, Lilly, Galderma, Leo Pharma, Novartis, Roche, Sanofi, Sun Pharma, Sandoz outside the submitted work. Dr. Saiag reports honoraria for advisory board and grants from Amgen, Bristol-Myers Squibb, MSD, Merck-Serono, Novartis, Pfizer, Roche-Genentech, Pierre Fabre, and Sanofi, outside the submitted work. Dr. Vieira has nothing to disclose. Dr. Zalaudek reports honoraria and advisory board and grants from Sanofi, Sun Pharma, Novartis, Galderma, Roche, Celgene, Almirall, Leofarma, Mylan, Difa Cooper, Cieffe Labs, La Roche Posay, Pierre Fabre. Dr. Eggermont reports over the last 5 years personal fees as a consultant advisor for Biocad, BioInvent, Bristol Myers Squibb (BMS), CatalYm, Ellipses, Glaxo Smith Kline (GSK), HalioDX, Incyte, IO Biotech, ISA Pharmaceuticals, Merck Sharpe & Dohme (MSD), Novartis, Pfizer, Polynoma, Regeneron, Sanofi, Sellas, SkylineDx. Alexander M.M. EGGERMONT: Honoraria, consultancy, SAB, IDMC, lectures for the last 2 years: Agenus, BioInvent, BioNTech, BMS, Brenus, CatalYm, Clover Pharmaceuticals, Ellipses, Galecto, GenOway, GSK, IO Biotech, IQVIA, ISA Pharmaceuticals, Merck&Co/MSD, Pierre Fabre, Pfizer, Sairopa, Sellas, SkylineDx, TigaTx, Trained Therapeutics,. Equity : IO Biotech, Sairopa, SkylineDX.Dr. Grob reports personal fees for advisory board and as speaker from Amgen, Roche, GSK, Novartis, BMS, Pierre Fabre, Merck, Sanofi, Merck, Pfizer outside the submitted work. Alexander van Akkooi: Advisory Board / Consultancy Honoraria: Amgen, Bristol-Myers Squibb, Novartis, MSD-Merck, Merck-Pfizer, Pierre Fabre, Provectus, Sanofi, Sirius Medical, 4SC. Dr Lallas has nothing to disclose. Dr Ioannides reports support from Sanofi in a phase III clinical trial, outside the submitted work.Dr Lorigan reports honoraria (consulting/advisory/speakers' bureau) from BMS, Merck/MSD, Nektar, NeraCare, Amgen, Novartis, Oncology Education Canada, Pierre Fabre, Roche, outside the submitted work. Dr Rocken reports study grants from AB Science, Abbott, Abbvie, Alcedis, Almirall Hermal, Amgen, AnaptysBio, argenx, AstraZeneca, Bayer, Biogen idec, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, CureVac, DelArrivo, Dynavax Technologies, Eli Lilly, Galderma, Genentech, GSK, Hoffman-La Roche, Hokusai, Idera Pharmaceuticals, Ilkos Therapeutic, Immatics Biotechnologies, Incyte, Iovance Biotherapeutics, Janssen-Cilag, Johnson & Johnson, LEO Pharma, Merck, MSD Sharp & Dome, Novartis Pharmaceuticals, PellePharma, Pfizer, Philogen, Regeneron Pharmaceuticals, Sanofi-Aventis, Schering-Plough, Sun Pharma, Technische Universitat Dresden, Topaz Therapeutics, UCB, Universitatsklinikum Essen, Universitatsklinik Koln, 4SC, grants for research projects from Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, Wilhelm Sander-Stiftung, consulting fees from Galderma, support for attending meetings/travel from EADV, International League of Dermatological Societies, patent PCT/EP2020/072203, stock or stock options from Bristol-Myers Squibb, CureVac, Merck, Pfizer, outside the submitted work. Dr Arenberger has nothing to disclose. Dr Tagliaferri reports honoraria from Elekta, Igea Medical, SunPharma, Sanofi, Roche, Molipharma, Nanobiotix, Novartis, outside the submitted work. Dr Leiter reports personal fees from MSD, honoraria from MSD, Novartis, Sun Pharma, Sanofi, Almiral Hermal, support for attending meetings/travel from Pierre Fabre, Sun Pharma, participation in Advisory Board for MSD, Novartis, Sun Pharma, Sanofi, Almiral Hermal, outside the submitted work. Dr Trakatelli reports travel grants from Janssen, UCB, Abbvie, honoraria from UCB, Genesis Pharma, Pierre Fabre Greece, EADV courses, travel expenses for congresses from Abbvie, Pierre Fabre Greece, Genesis Pharma, outside the submitted work., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Recent advances in digital pathology have enabled accurate and standardised enumeration of tumour-infiltrating lymphocytes (TILs). Here, we aim to evaluate TILs as a percentage electronic TIL score (eTILs) and investigate its prognostic and predictive relevance in cutaneous melanoma., Methods: We included stage I to IV cutaneous melanoma patients and used hematoxylin-eosin-stained slides for TIL analysis. We assessed eTILs as a continuous and categorical variable using the published cut-off of 16.6% and applied Cox regression models to evaluate associations of eTILs with relapse-free, distant metastasis-free, and overall survival. We compared eTILs of the primaries with matched metastasis. Moreover, we assessed the predictive relevance of eTILs in therapy-naïve metastases according to the first-line therapy., Findings: We analysed 321 primary cutaneous melanomas and 191 metastatic samples. In simple Cox regression, tumour thickness (p < 0.0001), presence of ulceration (p = 0.0001) and eTILs ≤16.6% (p = 0.0012) were found to be significant unfavourable prognostic factors for RFS. In multiple Cox regression, eTILs ≤16.6% (p = 0.0161) remained significant and downgraded the current staging. Lower eTILs in the primary tissue was associated with unfavourable relapse-free (p = 0.0014) and distant metastasis-free survival (p = 0.0056). In multiple Cox regression adjusted for tumour thickness and ulceration, eTILs as continuous remained significant (p = 0.019). When comparing TILs in primary tissue and corresponding metastasis of the same patient, eTILs in metastases was lower than in primary melanomas (p < 0.0001). In therapy-naïve metastases, an eTILs >12.2% was associated with longer progression-free survival (p = 0.037) and melanoma-specific survival (p = 0.0038) in patients treated with anti-PD-1-based immunotherapy. In multiple Cox regression, lactate dehydrogenase (p < 0.0001) and eTILs ≤12.2% (p = 0.0130) were significantly associated with unfavourable melanoma-specific survival., Interpretation: Assessment of TILs is prognostic in primary melanoma samples, and the eTILs complements staging. In therapy-naïve metastases, eTILs ≤12.2% is predictive of unfavourable survival outcomes in patients receiving anti-PD-1-based therapy., Funding: See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript., Competing Interests: Declaration of interests SF received personal fees from Kyowa Kirin and Takeda Pharmaceuticals, institutional grants from NeraCare, SkylineDx, and BioNTech, all outside the submitted work. TA reports institutional grants and personal fees from Novartis, institutional grants from NeraCare, Sanofi, SkylineDx, personal fees from CeCaVa, Pierre Fabre, BMS all outside the submitted work, participate on a data safety monitoring board for Unicancer. DLR reports grants and personal fees from Amgen, Astra Zeneca, Cepheid, Konica—Minolta, Lilly, NextCure personal fees from Cell Signaling Technology, Danaher, Fluidigm, GSK, Merck, Monopteros, NanoString, Odonate, Paige. AI, Regeneron, Roche, Sanofi, Ventana and Verily, royalties from Rarecyte, all outside the submitted work. CG reports grants and personal fees from NeraCare, Novartis, Roche, Sanofi, personal fees from Amgen, BMS, MSD, and Philogen, all outside the submitted work. TE reports personal fees from Novartis, BMS, Almirall Hermal, CureVac, Sanofi, MSD, Pierre Fabre and institutional grants from MSD, Sanofi, BMS, Pfizer, GenenTech, Seagan, Regeneron all outside the submitted work. IB reports having received speaker fees from Bayer, Pfizer, Takeda and AstraZeneca. MR reports grants from AB Science, Abbott, AbbVie, Alcedis, Almirall Hermal, Amgen, Anaptys Bio, Argenx, AstraZeneca, Bayer, Biogen Idec, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, CureVac, DelArrivo, Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, Dynavax Tech, Eli Lilly, Galderma, Genentech, GSK, Hoffmann La Roche, Hokusai, Idera Pharmaceuticals, Ilkos Therapeutic, Immatics biotechnologies, Incyte, Iovance Biotherapeutics, Janssen Cilag, Johnson & Johnson, LEO Pharma, Merck, MSD Sharp &Dohme, Novartis Pharmaceuticals, PellePharm, Pfizer, Philogen, Regeneron Pharmaceuticals, Sanofi Aventis, Schering Plough, Sun Pharma, Technische Universitat Dresden, Topaz Therapeutics, UCB, Universitatsklinik Essen, Universitatklinik Koln, Wilhelm Sander-Stiftung, 4SC. The other authors report no potential conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Aim/Introduction: Despite the spectacular success of immune checkpoint inhibitor therapy (ICT) in patients with metastatic cancer, only a limited proportion of patients benefit from ICT. CD8 + cytotoxic T cells are important gatekeepers for the therapeutic response to ICT and are able to recognize MHC class I-dependent tumor antigens and destroy tumor cells. The radiolabeled minibody [ 89 Zr]Zr-Df-IAB22M2C has a high affinity for human CD8 + T cells and was successfully tested in a phase I study. Here, we aimed to gain the first clinical PET/MRI experience with the noninvasive assessment of the CD8 + T-cell distribution in cancer patients by in vivo [ 89 Zr]Zr-Df-IAB22M2C with a distinct focus of identifying potential signatures of successful ICT. Material and Methods: We investigated 8 patients with metastasized cancers undergoing ICT. Radiolabeling of Df-IAB22M2C with Zr-89 was performed according to Good Manufacturing Practice. Multiparametric PET/MRI was acquired 24 h after injection of 74.2±17.9 MBq [ 89 Zr]Zr-Df-IAB22M2C. We analyzed [ 89 Zr]Zr-Df-IAB22M2C uptake within the metastases and within primary and secondary lymphatic organs. Results: [ 89 Zr]Zr-Df-IAB22M2C injection was tolerated well without noticeable side effects. The CD8 PET/MRI data acquisitions 24 hours post-administration of [ 89 Zr]Zr-Df-IAB22M2C revealed good image quality with a relatively low background signal due to only low unspecific tissue uptake and marginal blood pool retention. Only two metastatic lesions showed markedly increased tracer uptake in our cohort of patients. Furthermore, we observed high interpatient variability in [ 89 Zr]Zr-Df-IAB22M2C uptake within the primary and secondary lymphoid organs. Four out of five ICT patients exhibited rather high [ 89 Zr]Zr-Df-IAB22M2C uptake in the bone marrow. Two of these four patients as well as two other patients yielded pronounced [ 89 Zr]Zr-Df-IAB22M2C uptake within nonmetastatic lymph nodes. Interestingly, cancer progression in ICT patients was associated with a relatively low [ 89 Zr]Zr-Df-IAB22M2C uptake in the spleen compared to the liver in 4 out of the 6 patients. Lymph nodes with enhanced [ 89 Zr]Zr-Df-IAB22M2C uptake revealed significantly reduced apparent diffusion coefficient (ADC) values in diffusion weighted MRI. Conclusion: Our first clinical experiences revealed the feasibility of [ 89 Zr]Zr-Df-IAB22M2C PET/MRI in assessing potential immune-related changes in metastases and primary and secondary lymphatic organs. According to our results, we hypothesize that alterations in [ 89 Zr]Zr-Df-IAB22M2C uptake in primary and secondary lymphoid organs might be associated with the response to ICT., Competing Interests: Competing Interests: R.K. and I.W. are employees of ImaginAB. ClF is an advisor of ImaginAB. The other authors report no conflicts of interest., (© The author(s).)

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Melanomas are malignant tumors that can partly and very rarely completely regress in response to immune responses. Analyzing the mechanisms underlying this immune-mediated rejection, melanomas became leading in developing general cancer immunotherapy. This resulted in the discovery of tumor-specific neoantigens and mutations autoantigens, now called tumor-associated antigens, and their specific recognition by cytotoxic T lymphocytes. Melanomas were of key importance for the development of adoptive T-cell therapy and active tumor vaccines, namely dendritic cell vaccines. Melanoma therapy with antibodies against CTLA-4 provided the proof of concept that solid cancers can be susceptible to cancer immunotherapy, and melanoma therapy with antibodies against PD-1 resulted in the clinical breakthrough of cancer immunotherapy. Still, about half of patients die from metastatic melanoma. Combining anti‒PD-1 with anti‒CTLA-4 antibodies to increase antitumor immune responses or with targeted therapy improves the overall survival only partially. Recent data revealed a close link between defects in the IFN-γ‒dependent induction of cell cycle control genes and resistance to immunotherapy, which may allow for identifying those patients that respond to immunotherapy and to develop novel therapies, combining cancer immunotherapy with cell cycle inhibitors., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The aim is to unravel the mechanism of mesalazine (5-ASA) on proinflammatory cytokines in PBMCs of patients with HLA-B27 +and HLA-B27 -acute anterior uveitis (AAU), and whether this may explain the different effects of 5-ASA in both disoders., Methods: PBMCs from 12 HLA-B27+ and 4 HLA-B27- AAU patients were preincubated with 5-ASA and stimulated with LPS. As mesalazine (5-ASA) could be involved in ER stress, proinflammatory and ER stress-associated cytokines and markers were measured., Results: Mesalazine (5-ASA) suppressed IL-6 mRNA in healthy donors and in HLA-B27+ and HLA-B27- patients but did not lead to induction and secretion of IL-1β. In HLA-B27 + or - patients the ER stress-associated markers CHOP (DDIT3) and ATF6 were suppressed., Conclusions: Here we show that mesalazine (5-ASA) inhibits the transcription of proinflammatory and (ER) stress associated cytokines and markers, independently of the HLA-B27 status. Results show the similarities of both AAU types but do not decipher the mechanism why the HLA-B27 status determines the therapeutic response to mesalazine in AAU.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX + CD8 + T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration., Competing Interests: Declaration of interests B.I. has received honoraria from consulting with Merck, Janssen Pharmaceuticals, Astra Zeneca, and Volastra Therapeutics. M.A.D. has been a consultant to Roche/Genentech, Array, Pfizer (New York, NY, United States of America), Novartis, BMS, GSK, Sanofi-Aventis (Bridgewater, NJ, United States of America), Vaccinex, Apexigen, EISAI, and ABM Therapeutics and he has been the PI of research grants to MD Anderson by Roche/Genentech (South San Francisco, CA, United States of America), GSK, Sanofi-Aventis, Merck, Myriad, and Oncothyreon. A.R. has received honoraria from consulting with CStone, Merck, and Vedanta, is or has been a member of the scientific advisory board and holds stock in Advaxis, Appia, Apricity, Arcus, Compugen, CytomX, Highlight, ImaginAb, ImmPact, ImmuneSensor, Inspirna, Isoplexis, Kite-Gilead, Lutris, MapKure, Merus, PACT, Pluto, RAPT, Synthekine, and Tango, has received research funding from Agilent (Santa Clara, CA, United States of America) and from Bristol-Myers Squibb through Stand Up to Cancer (SU2C), and patent royalties from Arsenal Bio. T.E. has acted as a consultant for Almiral Hermal, Bristol-Myers Squibb, MSD, Novartis, Pierre Fabre, and Sanofi. E.Z.M. is a consultant for Curio Bioscience. The other authors do not declare competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Immune checkpoint blockade (ICB) therapy is a central pillar of melanoma treatment leading to durable response rates. Important mechanisms of action of ICB therapy include disinhibition of CD4 + and CD8 + T cells. Stimulated CD4 + T helper 1 cells secrete the effector cytokines interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF), which induce senescence in tumor cells. Besides being growth-arrested, senescent cells are metabolically active and secrete a large spectrum of factors, which are summarized as senescence-associated secretory phenotype (SASP). This secretome affects the tumor growth. Here, we compared the SASP of cytokine-induced senescent (CIS) cells with the SASP of therapy-induced senescent (TIS) cells. Therefore, we established in vitro models for CIS and TIS in melanoma. The human melanoma cell lines SK-MEL-28 and WM115 were treated with the cytokines IFN-γ and TNF as CIS, the chemotherapeutic agent doxorubicin, and the cell cycle inhibitor palbociclib as TIS. Then, we determined several senescence markers, i.e., growth arrest, p21 expression, and senescence-associated β-galactosidase (SA-β-gal) activity. For SASP analyses, we measured the regulation and secretion of several common SASP factors using qPCR arrays, protein arrays, and ELISA. Each treatment initiated a stable growth arrest, enhanced SA-β-gal activity, and-except palbociclib-increased the expression of p21. mRNA and protein analyses revealed that gene expression and secretion of SASP factors were severalfold stronger in CIS than in TIS. Finally, we showed that treatment with the conditioned media (CM) derived from cytokine- and palbociclib-treated cells induced senescence characteristics in melanoma cells. Thus, we conclude that senescence induction via cytokines may lead to self-sustaining senescence surveillance of melanoma.

Chronic UV irradiation results in many changes in the skin, including hyperplasia, changes in dermal structures, and alteration of pigmentation. Exposure to UVB leads to cutaneous damage, which results in inflammation characterized by increased NF- κ B activation and the induction of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin- (IL-) 1, or IL-8. IL-1 secretion is the result of inflammasome activation which is besides apoptosis, a result of acute UVB treatment. Inflammasomes are cytosolic protein complexes whose formation results in the activation of proinflammatory caspase-1. Key substrates of caspase-1 are IL-1 β and IL-18, and the cytosolic protein gasdermin D (GSDMD), which is involved in inflammatory cell death. Here, we demonstrate that UVB-induced inflammasome activation leads to the formation of ASC specks. Our findings show that UVB provokes ASC speck formation in human primary keratinocytes prior to cell death, and that specks are, opposed to the perinuclear cytosolic localization in myeloid cells, formed in the nucleus. Additionally, we showed by RNAi that NLRP1 and not NLRP3 is the major inflammasome responsible for UVB sensing in primary human keratinocytes. Formation of ASC specks indicates inflammasome assembly and activation as their formation in hPKs depends on the presence of NLRP1 and partially on NLRP3. Nuclear ASC specks are not specific for NLRP1/NLRP3 inflammasome activation, as the activation of the AIM2 inflammasome by cytosolic DNA results in ASC specks too. These nuclear ASC specks putatively link cell death to inflammasome activation, possibly by binding of IFI16 (gamma-interferon-inducible protein) to ASC. ASC can interact upon UVB sensing via IFI16 with p53, linking cell death to ASC speck formation., Competing Interests: The authors report no conflict of interest., (Copyright © 2021 Nikola Smatlik et al.)

In malignant disease, CD4 + Foxp3 + regulatory T cells (Tregs) hamper antitumor immune responses and may provide a target for immunotherapy. Although immune checkpoint blockade (ICB) has become an established therapy for several cancer entities including lymphoma, its mechanisms have not been entirely uncovered. Using endogenously arising λ-MYC-transgenic mouse B-cell lymphomas, which can effectively be suppressed by either Treg ablation or ICB, we investigated which mechanisms are used by Tregs to suppress antitumor responses and how ICB affects these pathways. During tumor development, Tregs up-regulated Foxp3, CD25, CTLA-4 and IL-10, which correlated with enhanced immunosuppressive functions. Thus, in contrast to other tumors, Tregs did not become dysfunctional despite chronic stimulation in the tumor microenvironment and progressive up-regulation of PD-1. Immunosuppression was mediated by direct contacts between Tregs and effector T cells and by IL-10. When λ-MYC mice were treated with ICB antibodies, Tregs revealed a less profound up-regulation of Foxp3, CD25 and IL-10 and a decreased suppressive capacity. This may be due to the shift towards a pro-inflammatory milieu fostered by ICB. In summary, an ICB-induced interference with Treg-dependent immunosuppression may contribute to the success of ICB., (Copyright © 2021. Published by Elsevier Inc.)

CD4 + T helper cells are capable of mediating long-term antitumoral immune responses. We developed a combined immunotherapy (COMBO) using tumor antigen-specific T helper 1 cells (Tag-Th1), dual PD-L1/LAG-3 immune checkpoint blockade, and a low-dose total body irradiation (TBI) of 2 Gy, that was highly efficient in controlling the tumor burden of non-immunogenic RIP1-Tag2 mice with late-stage endogenous pancreatic islet carcinomas. In this study, we aimed to explore the impact of 2 Gy TBI on the treatment efficacy and the underlying mechanisms to boost CD4 + T cell-based immunotherapies. Methods: Heavily progressed RIP1-Tag2 mice underwent COMBO treatment and their survival was compared to a cohort without 2 Gy TBI. Positron emission tomography/computed tomography (PET/CT) with radiolabeled anti-CD3 monoclonal antibodies and flow cytometry were applied to investigate 2 Gy TBI-induced alterations in the biodistribution of endogenous T cells of healthy C3H mice. Migration and homing properties of Cy5-labeled adoptive Tag-Th1 cells were monitored by optical imaging and flow cytometric analyses in C3H and tumor-bearing RIP1-Tag2 mice. Splenectomy or sham-surgery of late-stage RIP1-Tag2 mice was performed before onset of COMBO treatment to elucidate the impact of the spleen on the therapy response. Results: First, we determined a significant longer survival of RIP1-Tag2 mice and an increased CD4 + T cell tumor infiltrate when 2 Gy TBI was applied in addition to Tag-Th1 cell PD-L1/LAG-3 treatment. In non-tumor-bearing C3H mice, TBI induced a moderate host lymphodepletion and a tumor antigen-independent accumulation of Tag-Th1 cells in lymphoid and non-lymphoid organs. In RIP1-Tag2, we found increased numbers of effector memory-like Tag-Th1 and endogenous CD4 + T cells in the pancreatic tumor tissue after TBI, accompanied by a tumor-specific Th1-driven immune response. Furthermore, the spleen negatively regulated T cell effector function by upregulation PD-1/LAG-3/TIM-3 immune checkpoints, providing a further rationale for this combined treatment approach. Conclusion: Low-dose TBI represents a powerful tool to foster CD4 + T cell-based cancer immunotherapies by favoring Th1-driven antitumoral immunity. As TBI is a clinically approved and well-established technique it might be an ideal addition for adoptive cell therapy with CD4 + T cells in the clinical setting., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Checkpoint inhibitors revolutionized the treatment of metastatic melanoma patients. Although tumor burden and lactate dehydrogenase (LDH) are associated with overall survival (OS), the impact of tumor growth kinetics remains elusive and in part contradictory. The aims of this study were to develop a novel simple and rapid method that estimates pretreatment metastatic growth rate (MGR) and to investigate its prognostic impact in melanoma patients treated with antiprogrammed death receptor-1 (PD-1) antibodies., Methods: MGR was assessed in three independent cohorts of a total of 337 unselected consecutive metastasized stage IIIB-IV melanoma patients (discovery cohort: n=53, confirmation cohort: n=126, independent multicenter validation cohort: n=158). MGR was computed during the pretreatment period before initiation of therapy with anti-PD-1 antibodies nivolumab or pembrolizumab by measuring the increase of the longest diameter of the largest target lesion. Tumor doubling time served as quality control. Kaplan-Meier analysis and univariable as well as multivariable Cox regression were used to examine the prognostic impact of MGR., Results: Pretreatment MGR >3.9 mm/month was associated with impaired OS in the discovery cohort (HR 6.19, 95% CI 2.92 to 13.10, p<0.0001), in the confirmation cohort (HR 3.62, 95% CI 2.19 to 5.98, p<0.0001) and in the independent validation cohort (HR 2.57, 95% CI 1.56 to 4.25, p=0.00023). Prior lines of systemic treatment did not influence the significance of MGR. Importantly, the prognostic impact of MGR was independent of total tumor burden, diameter of the largest metastasis, number of prior lines of systemic treatment, LDH, as well as liver and brain metastasis (discovery and confirmation cohorts: both p<0.0001). Superiority of MGR compared with these variables was confirmed in the independent multicenter validation cohort (HR 2.92, 95% CI 1.62 to 5.26, p=0.00036)., Conclusions: High pretreatment MGR is an independent strong prognostic biomarker associated with unfavorable survival of melanoma patients receiving anti-PD-1 antibodies. Further investigations are warranted to assess the predictive impact of MGR in distinct systemic therapeutic regimens., Competing Interests: Competing interests: NBW reports a consulting/advisory role for Sanofi and has received travel support from AbbVie and Amgen outside the submitted work. RL has received research funding from Bristol-Myers Squibb, Pierre Fabre and Roche, honoraria and travel support from Merck Sharp & Dohme and travel support from Amgen. AO is a consultant/advisory board member for MSD and Bristol-Myers Squibb. AF has received speaker’s honoraria from Bristol-Myers Squibb, MSD, Roche, and Novartis, is advisory board member for Roche and Novartis and received travel support from Roche, Novartis, and Bristol-Myers Squibb. UL is a consultant/advisory board member for and has received honoraria from MSD, Novartis, and Roche. MG is an advisory board member of Novartis and has received grants from Novartis outside the submitted work. CB has received speaker's honoraria and consultancy fees from Amgen, Bristol-Myers Squibb, Merck, MSD, Novartis, Pierre Fabre, Regeneron, Roche, and Sanofi-Aventis outside the submitted work, and has received travel support from Bristol-Myers Squibb. ER has received honoraria from and has a consultancy or advisory role for Amgen, Bayer, Bristol-Myers Squibb, MSD, Merck, Novartis, Pierre Fabre, Roche and Sanofi, reports speakers' bureau for Amgen, Bristol-Myers Squibb, MSD, Merck, Novartis, Pierre Fabre and Sanofi, received research funding (at the institution) from Amgen, Bristol-Myers Squibb, MSD, Novartis, Pierre Fabre and Roche, is a member of the research funding steering committee of Novartis, and received travel accommodations and/or expenses from Amgen, Bristol-Myers Squibb, MSD, Merck, Novartis, Pierre Fabre, Roche and Sanofi. JM has intermittent project focused consultant or advisory relationships with Merck/Pfizer, Merck Sharp & Dohme, Amgen, Novartis, Bristol-Myers Squibb and Pierre Fabre, and has received travel support from Ultrasun, L’Oreal, Merck Sharp & Dohme, Bristol-Myers Squibb and Pierre Fabre outside of the submitted work. PT has received honoraria and/or consultancy fees from Bristol-Myers Squibb, Curevac, Merck, MSD, Novartis, Pierre Fabre, Roche, and Sanofi outside the submitted work, and has received travel support from Bristol-Myers Squibb and Pierre-Fabre. CL is a consultant/advisory board member for and has received advisory board fees, speaker's fees and/or travel reimbursements from Roche, Pierre Fabre, Novartis, Bristol-Myers Squibb, Merck, MSD, Biontech, Almiral Hermal, Kyowa Kirin, Sun Pharma and Sanofi. VAN has received speaker’s honoraria and consultancy fees from Amgen, Bristol-Myers Squibb, GlaxoSmithKline, Merck Sharp & Dohme, Novartis, Roche, and Takeda outside of the submitted work. CGe has received personal speaker/advisory board fees from Amgen, Beiersdorf, BMS, MSD, Novartis, Pierre Fabre, Roche, Sun Pharma and Sanofi Genzyme. FM has received travel support or/and speaker’s fees or/and advisor’s honoraria by Novartis, Roche, BMS, MSD and Pierre Fabre, and research funding from Novartis and Roche. SD has received travel support from MSD. AC has received consultancy fees from AbbVie, BMS, Almirall, Amgen, Eli Lilly, Galderma, Janssen, Kyowa Kirin, Leo, Novartis, and Sanofi. LF reports grants from the Swiss National Science Foundation, Swiss Cancer League, Hookipa Pharma and Novartis Foundation as well as an advisory role for Novartis and Bristol-Myers Squibb. CGa has received grants and personal fees from Bristol-Myers Squibb, Roche, Novartis and personal fees from Amgen, MSD and Philogen outside the submitted work. TE is a consultant/advisory board member for Philogen, Bristol-Myers Squibb, Novartis, Roche and Sanofi. No other disclosures were reported., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Immune checkpoint blocking (ICB) is a promising new tool of cancer treatment. Yet, the underlying therapeutic mechanisms are not fully understood. Here we investigated the role of dendritic cells (DCs) for the therapeutic effect of ICB in a λ-MYC-transgenic mouse model of endogenously arising B-cell lymphoma. The growth of these tumors can be effectively delayed by antibodies against CTLA-4 and PD-1. Tumor-infiltrating DCs from mice having received therapy showed an upregulation of costimulatory molecules as well as an augmented IL-12/IL-10 ratio as compared to untreated controls. Both alterations seemed to be induced by interferon-γ (IFN-γ), which is upregulated in T cells and natural killer cells upon ICB. Furthermore, the enhanced IL-12/IL-10 ratio, which favors Th1-prone antitumor T-cell responses, was a consequence of direct interaction of ICB antibodies with DCs. Importantly, the capability of tumor-infiltrating DCs of stimulating peptide-specific or allogeneic T-cell responses in vitro was improved when DCs were derived from ICB-treated mice. The data indicate that ICB therapy is not only effective by directly activating T cells, but also by triggering a complex network, in which DCs play a pivotal role at the interface between innate and adaptive antitumor responses.

To establish strategies for immunotherapy of B-cell lymphoma, it is mandatory to gain deeper insights into the mechanisms of tumor immune escape. In a mouse model of endogenously arising lymphoma, we investigated the impact of IL-10 on the regulation of antitumor responses. Despite progressive functional impairment of NK cells and lack of IFN-γ in the tumor milieu, we found an augmented fraction of T helper type 1 (Th1) cells, which continued to express IFN-γ but also upregulated IL-10 during disease development. Using a lymphoma microenvironment in vitro, we showed that Th1 cells were converted to Foxp3-negative T regulatory type 1 (Tr1) cells, which coexpressed IFN-γ and IL-10 and upregulated PD-1. This differentiation required pre-existing IL-10, which was primarily provided by malignant B cells and dendritic cells. IFN-γ only declined in cells with the uppermost PD-1 levels. Importantly, antibody-mediated IL-10 ablation in vivo improved effector cell functions and significantly suppressed tumor development. While the contribution of IL-10 to cancer immune escape has been controversially discussed in the past, we show that IL-10 suppresses ongoing, potentially protective immune responses in lymphoma and might be a target for immunotherapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Hepatic immune-related adverse events (irAE) including elevated liver function tests (transaminases) occur in 1.4-22.3% of melanoma patients receiving immune checkpoint inhibitors (ICPI) and constitute a potentially serious toxicity that is challenging to treat. In contrast to the liver transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST), only little is known about the frequency and impact of gamma-glutamyl transferase (GGT) elevations., Methods: GGT determined prior to and during therapy of metastatic melanoma patients treated with ICPI were retrospectively assessed in two independent cohorts (PD-1: n = 218, Ipi + Nivo: n = 148). Overall survival (OS) and best objective response were analyzed according to baseline and immune-related GGT (irGGT) elevations during treatment., Results: In multivariate analysis, OS was reduced in patients with elevated baseline GGT (PD-1 group: hazard ratio [HR] 1.76, p = .0073; Ipi + Nivo group: HR 1.77, p = .032). Immune-related GGT elevation was recorded in 17% (PD-1 group) and 38.5% (Ipi + Nivo group). Of these patients, the majority (81 and 68%, respectively) had normal ALT and AST and showed no clinical signs of hepatotoxicity. Patients who experienced irGGT elevation had superior response (PD-1 group: odds ratio [OR] 3.57, p = .00072; Ipi + Nivo group: OR 1.74, p = .12) and OS (PD-1 group: HR 0.37, p = .0016; Ipi + Nivo group: HR 0.33, p = .00050)., Conclusions: The frequency of hepatic irAE is currently underestimated. The addition of the sensitive enzyme GGT to the laboratory panel before and during therapy with ICPI allows to detect two to three times more patients developing hepatic or hepatobiliary toxicity than known so far. Immune-related GGT elevations correlate with response and favorable survival. Precis for use in the Table of Contents The frequency of hepatotoxicity under immune checkpoint blockade is currently underestimated. We suggest the addition of gamma-glutamyl transferase to the laboratory panel in checkpoint inhibitor patients for the detection of hepatobiliary toxicity.

Rationale: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important regulators of inflammation. The exact impact of ROS/RNS on cutaneous delayed-type hypersensitivity reaction (DTHR) is controversial. The aim of our study was to identify the dominant sources of ROS/RNS during acute and chronic trinitrochlorobenzene (TNCB)-induced cutaneous DTHR in mice with differently impaired ROS/RNS production. Methods: TNCB-sensitized wild-type, NADPH oxidase 2 (NOX2)- deficient (gp91 phox-/- ), myeloperoxidase-deficient (MPO -/- ), and inducible nitric oxide synthase-deficient (iNOS -/- ) mice were challenged with TNCB on the right ear once to elicit acute DTHR and repetitively up to five times to induce chronic DTHR. We measured ear swelling responses and noninvasively assessed ROS/RNS production in vivo by employing the chemiluminescence optical imaging (OI) probe L-012. Additionally, we conducted extensive ex vivo analyses of inflamed ears focusing on ROS/RNS production and the biochemical and morphological consequences. Results: The in vivo L-012 OI of acute and chronic DTHR revealed completely abrogated ROS/RNS production in the ears of gp91 phox-/- mice, up to 90 % decreased ROS/RNS production in the ears of MPO -/- mice and unaffected ROS/RNS production in the ears of iNOS -/- mice. The DHR flow cytometry analysis of leukocytes derived from the ears with acute DTHR confirmed our in vivo L-012 OI results. Nevertheless, we observed no significant differences in the ear swelling responses among all the experimental groups. The histopathological analysis of the ears of gp91 phox-/- mice with acute DTHRs revealed slightly enhanced inflammation. In contrast, we observed a moderately reduced inflammatory immune response in the ears of gp91 phox-/- mice with chronic DTHR, while the inflamed ears of MPO -/- mice exhibited the strongest inflammation. Analyses of lipid peroxidation, 8-hydroxy-2'deoxyguanosine levels, redox related metabolites and genomic expression of antioxidant proteins revealed similar oxidative stress in all experimental groups. Furthermore, inflamed ears of wild-type and gp91 phox-/- mice displayed neutrophil extracellular trap (NET) formation exclusively in acute but not chronic DTHR. Conclusions: MPO and NOX2 are the dominant sources of ROS/RNS in acute and chronic DTHR. Nevertheless, depletion of one primary source of ROS/RNS exhibited only marginal but conflicting impact on acute and chronic cutaneous DTHR. Thus, ROS/RNS are not a single entity, and each species has different properties at certain stages of the disease, resulting in different outcomes., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Although antibodies blocking immune checkpoints have already been approved for clinical cancer treatment, the mechanisms involved are not yet completely elucidated. Here we used a λ-MYC transgenic model of endogenously growing B-cell lymphoma to analyze the requirements for effective therapy with immune checkpoint inhibitors., Methods: Growth of spontaneous lymphoma was monitored in mice that received antibodies targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein-4, and the role of different immune cell compartments and cytokines was studied by in vivo depletion experiments. Activation of T and natural killer cells and the induction of tumor senescence were analyzed by flow cytometry., Results: On immune checkpoint blockade, visible lymphomas developed at later time points than in untreated controls, indicating an enhanced tumor control. Importantly, 20% to 30% of mice were even long-term protected and did never develop clinical signs of tumor growth. The therapeutic effect was dependent on cytokine-induced senescence in malignant B cells. The proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor (TNF) were necessary for the survival benefit as well as for senescence induction in the λ-MYC model. Antibody therapy improved T-cell functions such as cytokine production, and long-time survivors were only observed in the presence of T cells. Yet, NK cells also had a pronounced effect on therapy-induced delay of tumor growth. Antibody treatment enhanced numbers, proliferation and IFN-γ expression of NK cells in developing tumors. The therapeutic effect was fully abrogated only after depletion of both, T cells and NK cells, or after ablation of either IFN-γ or TNF., Conclusions: Tumor cell senescence may explain why patients responding to immune checkpoint blockade frequently show stable growth arrest of tumors rather than complete tumor regression. In the lymphoma model studied, successful therapy required both, tumor-directed T-cell responses and NK cells, which control, at least partly, tumor development through cytokine-induced tumor senescence., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Aberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P < 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P < 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor-sensitive and -resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The detection of somatic driver mutations by next-generation sequencing (NGS) is becoming increasingly important in the care of advanced melanoma patients. In our study, we evaluated the NGS results of 82 melanoma patients from clinical routine in 2017. Besides determining the tumor mutational burden (TMB) and annotation of all genetic driver alterations, we investigated their potential as a predictor for resistance to immune checkpoint inhibitors (ICI) and as a distinguishing feature between melanoma subtypes. Melanomas of unknown primary had a similar mutation pattern and TMB to cutaneous melanoma, which hints at its cutaneous origin. Besides the typical hotspot mutation in BRAF and NRAS , we frequently observed CDKN2A deletions. Acral and mucosal melanomas were dominated by CNV alterations affecting PDGFRA , KIT , CDK4 , RICTOR , CCND2 and CHEK2 . Uveal melanoma often had somatic SNVs in GNA11/Q and amplification of MYC in all cases. A significantly higher incidence of BRAF V600 mutations and EGFR amplifications, PTEN and TP53 deletions was found in patients with disease progression while on ICI. Thus, NGS might help to characterize melanoma subtypes more precisely and to identify possible resistance mechanisms to ICI therapy. Nevertheless, NGS based studies, including larger cohorts, are needed to support potential genetic ICI resistance mechanisms.

Purpose: Reactive oxygen and nitrogen species (ROS/RNS) production and the NF-κB activation are critically involved in inflammatory responses, but knowledge about the temporal dynamics during acute and chronic inflammation is limited. Here, we present a comparative longitudinal in vivo study of both parameters in an experimental model of acute and chronic T cell-driven delayed-type hypersensitivity reaction (DTHR) using noninvasive optical imaging., Procedures: Trinitrochlorobenzene (TNCB)-sensitized NF-κB-luciferase-reporter and wild-type mice were TNCB challenged on the right ear to elicit acute DTHR and then repetitively challenged (up to five times) to induce chronic DTHR. Mice were treated with the ROS-scavenging and NF-κB inhibiting molecule N-acetylcysteine (NAC) or underwent sham treatment. ROS/RNS production was noninvasively analyzed in vivo using the ROS-/RNS-sensitive chemiluminescent probe L-012, and NF-κB activation was measured using NF-κB-luciferase-reporter mice. H&E staining, CD3 and myeloperoxidase (MPO) immunohistochemistry (IHC), and quantitative PCR (qPCR) analyses were employed to investigate immune cell infiltration and expression of NF-κB- and ROS-/RNS-driven genes., Results: In acute DTHR, we found strongly elevated ROS/RNS production and NF-κB activation 12 h after the 1st TNCB ear challenge, peaking at 24 h after the challenge. In chronic DTHR, ROS production peaked as early as 4 h after the 5th TNCB challenge, whereas NF-κB activity peaked after 12 h. The increase in ROS/RNS production in acute DTHR was higher than the increase in NF-κB activity but the relationship was inverse in chronic DTHR. Treatment with the ROS scavenger NAC had differential effects on ROS/RNS production and NF-κB activation during acute and chronic DTHR. Ex vivo cross-validation by histopathology and qPCR analysis correlated closely with the in vivo imaging results., Conclusions: Noninvasive in vivo imaging is capable of assessing the temporal dynamics of ROS/RNS production and NF-κB activation during progression from acute to chronic DTHR and enables monitoring of anti-inflammatory treatment responses.

Immune checkpoint blockade (ICB)-based or natural cancer immune responses largely eliminate tumours. Yet, they require additional mechanisms to arrest those cancer cells that are not rejected. Cytokine-induced senescence (CIS) can stably arrest cancer cells, suggesting that interferon-dependent induction of senescence-inducing cell cycle regulators is needed to control those cancer cells that escape from killing. Here we report in two different cancers sensitive to T cell-mediated rejection, that deletion of the senescence-inducing cell cycle regulators p16 Ink4a /p19 Arf (Cdkn2a) or p21 Cip1 (Cdkn1a) in the tumour cells abrogates both the natural and the ICB-induced cancer immune control. Also in humans, melanoma metastases that progressed rapidly during ICB have losses of senescence-inducing genes and amplifications of senescence inhibitors. Metastatic cells also resist CIS. Such genetic and functional alterations are infrequent in metastatic melanomas regressing during ICB. Thus, activation of tumour-intrinsic, senescence-inducing cell cycle regulators is required to stably arrest cancer cells that escape from eradication.

Purpose: Cancer immunotherapy depends on a systemic immune response, but the basic underlying mechanisms are still largely unknown. Despite the very successful and widespread use of checkpoint inhibitors in the clinic, the majority of cancer patients do not benefit from this type of treatment. In this translational study, we investigated whether noninvasive in vivo positron emission tomography (PET) imaging using 2-[ 18 F]fluoro-2-deoxy-D-glucose ( 18 F-FDG) is capable of detecting immunotherapy-associated metabolic changes in the primary and secondary lymphoid organs and whether this detection enables the prediction of a successful anti-cancer immune response. Methods: RIP1-Tag2 mice with progressed endogenous insular cell carcinomas underwent a combined cancer immunotherapy consisting of CD4 + T cells plus monoclonal antibodies (mAbs) against programmed death ligand-1 (PD-L1) and lymphocyte activation gene-3 (LAG-3) or a sham treatment after radiation-mediated immune cell depletion. A second cohort of RIP1-Tag2 mice underwent exclusive checkpoint inhibitor therapy (CIT) using anti-PD-L1/LAG-3 mAbs or sham treatment without initial immune cell depletion to mimic the clinical situation. All mice were monitored by 18 F-FDG-PET combined with anatomical magnetic resonance imaging (MRI). In addition, we retrospectively analyzed PET / computed tomography (CT) scans (PET/CT) regarding 18 F-FDG uptake of CIT-treated metastatic melanoma patients in the spleen (n=23) and bone marrow (BM; n=20) as well as blood parameters (n=17-21). Results: RIP1-Tag2 mice with advanced insular cell carcinomas treated with combination immunotherapy exhibited significantly increased 18 F-FDG uptake in the spleen compared to sham-treated mice. Histopathology of the spleens from treated mice revealed atrophy of the white pulp with fewer germinal centers and an expanded red pulp with hyperplasia of neutrophils than those of sham-treated mice. Immunohistochemistry and flow cytometry analyses of the spleens revealed a lower number of T cells and a higher number of neutrophils compared to those in the spleens of sham-treated mice. Flow cytometry of the BM showed enhanced activation of T cells following the treatment schemes that included checkpoint inhibitors. The ratio of 18 F-FDG uptake at baseline to the uptake at follow-up in the spleens of exclusively CIT-treated RIP1-Tag2 mice was significantly enhanced, but the ratio was not enhanced in the spleens of the sham-treated littermates. Flow cytometry analysis confirmed a reduced number of T cells in the spleens of exclusively CIT-treated mice compared to that of sham-treated mice. A retrospective analysis of clinical 18 F-FDG-PET/CT scans revealed enhanced 18 F-FDG uptake in the spleens of some successfully CIT-treated patients with metastatic melanoma, but there were no significant differences between responders and non-responders. The analysis of the BM in clinical 18 F-FDG-PET/CT scans with a computational segmentation tool revealed significantly higher baseline 18 F-FDG uptake in patients who responded to CIT than in non-responders, and this relationship was independent of bone metastasis, even in the baseline scan. Conclusions: Thus, we are presenting the first translational study of solid tumors focusing on the metabolic patterns of primary and secondary lymphoid organs induced by the systemic immune response after CIT. We demonstrate that the widely available 18 F-FDG-PET modality is an applicable translational tool that has high potential to stratify patients at an early time point., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell-cell adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3 -/- mouse skin also indicated an increase of p53/p21 WAF1/CIP1 and cleaved caspase-3 relative to Dsg3 +/- controls. Finally, we evaluated whether this pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell-cell adhesion. Collectively, our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is disrupted in PV.

Immune checkpoint blockade has revolutionized cancer treatment. Patients developing immune mediated adverse events, such as colitis, appear to particularly benefit from immune checkpoint inhibition. Yet, the contributing mechanisms are largely unknown. We identified a systemic LPS signature in melanoma patients with colitis following anti-cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) checkpoint inhibitor treatment and hypothesized that intestinal microbiota-derived LPS contributes to therapeutic efficacy. Because activation of immune cells within the tumor microenvironment is considered most promising to effectively control cancer, we analyzed human and murine melanoma for known sentinels of LPS. We identified mast cells (MCs) accumulating in and around melanomas and showed that effective melanoma immune control was dependent on LPS-activated MCs recruiting tumor-infiltrating effector T cells by secretion of CXCL10. Importantly, CXCL10 was also upregulated in human melanomas with immune regression and in patients with colitis induced by anti-CTLA-4 antibody. Furthermore, we demonstrate that CXCL10 upregulation and an MC signature at the site of melanomas are biomarkers for better patient survival. These findings provide conclusive evidence for a "Trojan horse treatment strategy" in which the plasticity of cancer-resident immune cells, such as MCs, is used as a target to boost tumor immune defense.

Heterogeneity of cancer-associated fibroblasts (CAFs) can result from activation of distinct signaling pathways. We show that in primary human dermal fibroblasts (HDFs), fibroblast growth factor (FGF) and transforming growth factor β (TGF-β) signaling oppositely modulate multiple CAF effector genes. Genetic abrogation or pharmacological inhibition of either pathway results in induction of genes responsive to the other, with the ETV1 transcription factor mediating the FGF effects. Duality of FGF/TGF-β signaling and differential ETV1 expression occur in multiple CAF strains and fibroblasts of desmoplastic versus non-desmoplastic skin squamous cell carcinomas (SCCs). Functionally, HDFs with opposite TGF-β versus FGF modulation converge on promoting cancer cell proliferation. However, HDFs with increased TGF-β signaling enhance invasive properties and epithelial-mesenchymal transition (EMT) of SCC cells, whereas HDFs with increased FGF signaling promote macrophage infiltration. The findings point to a duality of FGF versus TGF-β signaling in distinct CAF populations that promote cancer development through modulation of different processes., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cysteine-type cathepsins such as cathepsin B are involved in various steps of inflammatory processes such as antigen processing and angiogenesis. Here, we uncovered the role of cysteine-type cathepsins in the effector phase of T cell-driven cutaneous delayed-type hypersensitivity reactions (DTHR) and the implication of this role on therapeutic cathepsin B-specific inhibition. Methods : Wild-type, cathepsin B-deficient (Ctsb -/- ) and cathepsin Z-deficient (Ctsz -/- ) mice were sensitized with 2,4,6-trinitrochlorobenzene (TNCB) on the abdomen and challenged with TNCB on the right ear to induce acute and chronic cutaneous DTHR. The severity of cutaneous DTHR was assessed by evaluating ear swelling responses and histopathology. We performed fluorescence microscopy on tissue from inflamed ears and lymph nodes of wild-type mice, as well as on biopsies from psoriasis patients, focusing on cathepsin B expression by T cells, B cells, macrophages, dendritic cells and NK cells. Cathepsin activity was determined noninvasively by optical imaging employing protease-activated substrate-like probes. Cathepsin expression and activity were validated ex vivo by covalent active site labeling of proteases and Western blotting. Results : Noninvasive in vivo optical imaging revealed strong cysteine-type cathepsin activity in inflamed ears and draining lymph nodes in acute and chronic cutaneous DTHR. In inflamed ears and draining lymph nodes, cathepsin B was expressed by neutrophils, dendritic cells, macrophages, B, T and natural killer (NK) cells. Similar expression patterns were found in psoriatic plaques of patients. The biochemical methods confirmed active cathepsin B in tissues of mice with cutaneous DTHR. Topically applied cathepsin B inhibitors significantly reduced ear swelling in acute but not chronic DTHR. Compared with wild-type mice, Ctsb -/- mice exhibited an enhanced ear swelling response during acute DTHR despite a lack of cathepsin B expression. Cathepsin Z, a protease closely related to cathepsin B, revealed compensatory expression in inflamed ears of Ctsb -/- mice, while cathepsin B expression was reciprocally elevated in Ctsz -/- mice. Conclusion : Cathepsin B is actively involved in the effector phase of acute cutaneous DTHR. Thus, topically applied cathepsin B inhibitors might effectively limit DTHR such as contact dermatitis or psoriasis. However, the cathepsin B and Z knockout mouse experiments suggested a complementary role for these two cysteine-type proteases., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Melanoma incidence rates rise as people age, but the impact of aging on distant metastasis is unclear., Objective: To investigate how timing, pattern, and extent of distant metastasis is influenced by age., Methods: Analysis of a single-center cohort of 1457 patients of the German Central Malignant Melanoma Registry with prospectively documented follow-up. Findings were compared with those for 1682 patients from 5 different institutions. All patients presented initially with stage IA to IIC and developed distant metastasis in their further disease course., Results: The number of metastatic sites decreased with increasing age at melanoma diagnosis (P < .001). The rate of stage M1d disease decreased from 50.2% in patients aged 50 years or younger to 30.1% in patients older than 70 years, and the rate of stage M1b disease increased from 5.8% to 21.5%. The rate of lung metastases remained stable in all investigated age groups (P = .54). Distant metastases occurred earlier and were more synchronized in patients older than 70 years than in patients aged 50 years or younger. An age-dependent decrease in metastatic sites and stable rate of lung metastasis were found and confirmed by data on the multi-institutional cohort., Limitations: The study was not population based., Conclusion: Pattern, timing, and extent of distant metastasis change as people age. These findings may be considered when treating patients with melanoma of different ages., (Copyright © 2019 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Cancer treatment with adoptively transferred tumor-associated antigen-specific CD4+ T-helper cells is a promising immunotherapeutic approach. In the pancreatic cancer model RIP-Tag2, the intraperitoneal (i.p.) application of Tag-specific TH1 cells exhibited a profound antitumoral efficiency. We investigated, whether an intravenous (i.v.) application of Tag-TH1 cells induces an equivalent therapeutic effect. Adoptively transferred fluorescent Tag-TH1 cells revealed a pronounced homing to the tumors after either i.p. or i.v. transfer, and both routes induced an almost equivalent therapeutic effect as demonstrated by magnetic resonance imaging, blood glucose level course and histology. The i.v. administration of Tag-TH1 cells induced p16INK4-positive/Ki67-negative tumor senescence more efficiently than i.p. administration. Both routes replenish host CD4+ T cells by transferred T cells and recruitment of B and dendritic cells to the tumors while reducing CD8+ T cells and depleting macrophages. Both administration routes efficiently induced a similar antitumoral efficiency despite the pronounced senescence induction after i.v. administration. Thus, a combinatory i.v./i.p. injection of therapeutic cells might overcome limitations of the individual routes and improve therapeutic efficacy in solid tumors., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Foxp3 + regulatory T cells (Tregs) sustain immune homeostasis and may contribute to immune escape in malignant disease. As a prerequisite for developing immunologic approaches in cancer therapy, it is necessary to understand the ontogeny and the antigenic specificities of tumor-infiltrating Tregs. We addressed this question by using a λ-MYC transgenic mouse model of endogenously arising B-cell lymphoma, which mirrors key features of human Burkitt lymphoma. We show that Foxp3 + Tregs suppress antitumor responses in endogenous lymphoma. Ablation of Foxp3 + Tregs significantly delayed tumor development. The ratio of Treg to effector T cells was elevated in growing tumors, which could be ascribed to differential proliferation. The Tregs detected were mainly natural Tregs that apparently recognized self-antigens. We identified MHC class II-restricted nonmutated self-epitopes, which were more prevalent in lymphoma than in normal B cells and could be recognized by Tregs. These epitopes were derived from proteins that are associated with cellular processes related to malignancy and may be overexpressed in the tumor., (©2019 American Association for Cancer Research.)

Enlargement of the lymphatic vascular network in tumor-draining lymph nodes (LNs) often precedes LN metastasis, likely providing a lymphovascular niche for tumor cells. We investigated morphological and molecular changes associated with the lymphatic remodeling process, using the 4T1 breast cancer and B16F10 melanoma models. Lymphatic expansion in tumor-draining LNs is mediated by sprouting and proliferation of lymphatic endothelial cells (LECs) as early as 4 days after tumor implantation. RNA sequencing revealed an altered transcriptional profile of LECs from tumor-draining compared to naive LNs with similar changes in both tumor models. Integrin αIIb is upregulated in LECs of tumor-draining LNs and mediates LEC adhesion to fibrinogen in vitro. LEC-associated fibrinogen was also detected in LNs in vivo, suggesting a role of integrin αIIb in lymphatic remodeling. Together, our results identify specific responses of LN LECs to tumor stimuli and provide insights into the mechanisms of lymphovascular niche formation in tumor-draining LNs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immune checkpoints are accessory molecules that either promote or inhibit T-cell activation. Two inhibitory molecules, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), got high attention, as inhibition of CTLA-4 or PD-1 signaling provides the first immune therapy that significantly improves the survival of patients with metastatic solid cancers. Inhibition of CTLA-4 or PD-1 was first studied in and approved for patients with metastatic melanoma. Blocking immune checkpoints is also efficient in non-small-cell lung cancer, renal cell cancers, hypermutated gastrointestinal cancers, and others. Immune responses, whether directed against infections or against tumors, are divided into 2 phases: an initiation phase and an activation phase, where the immune system recognizes a danger signal and becomes activated by innate signals to fight the danger. This reaction is fundamental for the control of infections and cancer, but needs to be turned off once the danger is controlled, because persistence of this activation ultimately causes severe tissue damage. Therefore, each activation of the immune system is followed by a termination phase, where endogenous immune suppressor molecules arrest immune responses to prevent harmful damage. In the case of cancer immune therapies, therapeutic approaches classically enhanced the initiation and activation of immune responses to increase the emergence and the efficacy of cytotoxic T lymphocytes (CTL) against cancers. In sharp contrast, immune checkpoint blockade focuses on the termination of immune responses by inhibiting immune suppressor molecules. It thus prevents the termination of immune responses or even awakes those CTLs that became exhausted during an immune response. Therefore, blocking negatively regulating immune checkpoints restores the capacity of exhausted CTL to kill the cancer they infiltrate. In addition, they drive surviving cancer cells into a still poorly defined state of dormancy. As the therapy also awakes self-reactive CTL, one downside of the therapy is the induction of organ-specific autoimmune diseases. The second downside is the exorbitant drug price that withdraws patients in need from a therapy that was developed by academic research, which impairs further academic treatment development and financially charges the public health system., (Copyright © 2018. Published by Elsevier Inc.)

Inherited forms of epidermolysis bullosa are blistering diseases of the skin and mucosa resulting from various gene mutations. Transplantation of bone marrow-derived stem cells might be a promising systemic treatment for severe dystrophic or junctional epidermolysis bullosa, but many key questions remain unresolved. Two open questions of clinical interest are whether systemically transplanted bone marrow-derived stem cells of mesodermal origin might be able to transdifferentiate into keratinocytes with an ectodermal phenotype and whether these cells are also capable of repairing a specific intraepidermal gene defect. To address these questions, we transplanted bone marrow-derived stem cells into mice with a blistering disease exclusively localized to the epidermis resulting from a functional knockout of desmoglein-3 (Dsg3). We found that Dsg3 + donor-derived cells migrate into the recipient epidermis. However, these cells failed to restore the missing Dsg3 mRNA and DSG3 protein expression in the transplanted Dsg3 -/- mice. The donor-derived cells found in the epidermis preserved their CD45 + hematopoietic origin, and no transdifferentiation into integrin α6 + keratinocytes or integrin α6 + /CD34 + epidermal stem cells occurred. Our results indicate that bone marrow-derived stem cells preserve their mesodermal fate after systemic transplantation and are not capable of treating patients with epidermolysis bullosa with an intraepidermal skin defect., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Staphylococcus epidermidis colonises human skin without apparent inflammation, but a dominance of S. epidermidis and S. aureus is characteristic of cutaneous microbial dysbiosis in atopic dermatitis (AD). While S. aureus can trigger AD, the role of S. epidermidis is less understood. We characterised consequences of innate immune sensing of lipoteichoic acid (LTA) preparations derived from S. epidermidis (epi-LTA) or S. aureus (aureus-LTA). Therefore, dendritic cell (DC) activation and consecutive priming of antigen-specific T cells following exposure of DC to epi-LTA or aureus-LTA were investigated. Mimicking acute AD, exposure of DC to IL-4 and LTAs was analysed. Exposure to epi-LTA or aureus-LTA activated human immune cells and murine dendritic cells (DCs) via TLR2/MyD88, however, resulting in divergent immune profiles. Differences between LTAs were significant for IL-6, IL-12p40 and IL-12p70 but not for IL-10, which was best reflected by the IL-12p70-to-IL-10 ratio being IL-10-balanced for epi-LTA but pro-inflammatory for aureus-LTA. LTA-exposed DCs activated CD4+ T cells; however, while T-cell-derived IL-10 was equivalent between LTAs, IFN-γ and IL-17 were significantly higher for aureus-LTA. Mimicking acute AD by exposing DCs to IL-4 and LTAs revealed that IL-4 significantly and uniformly suppressed epi-LTA-induced cytokine production, keeping the IL-12p70-to-IL-10 ratio balanced. In contrast, exposure of DCs to aureus-LTA and IL-4 enhanced IL-12p70 but suppressed IL-10 levels, further unbalancing the IL-12p70-to-IL-10 ratio. These data demonstrate opposing immune consequences following exposure to staphylococcal LTAs. Epi-LTA induced IL-10-balanced, aureus-LTA pro-inflammatory immune profiles., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Complete lymph node dissection (CLND) after a positive sentinel node (SN) biopsy provides important prognostic information in melanoma patients but has been questioned for therapeutic use recently. We explored whether quantification of the tumour spread to SNs may replace histopathology of non-sentinel nodes (NSNs) for staging purposes., Patients and Methods: We quantified melanoma spread in SNs and NSNs in 128 patients undergoing CLND for a positive SN. In addition to routine histopathology, one-half of each of all 1496 SNs and NSNs was disaggregated into a single cell suspension and stained immunocytochemically to determine the number of melanoma cells per 10 6 lymph node cells, i.e. the disseminated cancer cell density (DCCD)., Results: We uncovered melanoma spread to NSNs in the majority of patients; however, the tumour load and the proportion of positive nodes were significantly lower in NSNs than in SNs. The relation between SN and NSN spread could be described by a mathematical function with DCCD NSN  = DCCD SN c /10 1 - c (c = 0.69; 95% confidence interval [CI]: 0.62-0.76). At a median follow-up of 67 months, multivariable Cox regression analyses revealed that DCCD SN (p = 0.02; HR 1.34, 95% CI: 1.05-1.71) and the total number of pathologically positive nodes (p = 0.02; HR 1.53, 95% CI: 1.07-2.22) were significant risk factors after controlling for age, gender, thickness of melanoma and ulceration status. A prognostic model based on DCCD SN and melanoma thickness predicted outcome as accurately as a model including pathological information of both SNs and NSNs., Conclusion: The assessment of DCCD SN renders CLND for staging purposes unnecessary., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background/aims: Cellular senescence, or permanent growth arrest, is known as an effective tumor suppressor mechanism that can be induced by different stressors, such as oncogenes, chemotherapeutics or cytokine cocktails. Previous studies demonstrated that the growth-repressing state of oncogene-induced senescent cells depends on argonaute protein 2 (Ago2)-mediated transcriptional gene silencing and Ago2/Rb corepression of E2F-dependent cell cycle genes. Cytokine-induced senescence (CIS) likewise depends on activation of the p16Ink4a/Rb pathway, and consecutive inactivation of the E2F family of transcription factors. In the present study, we therefore analyzed the role of Ago2 in CIS., Methods: Human cancer cell lines were treated with interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) to induce senescence. Senescence was determined by growth assays and measurement of senescence-associated β-galactosidase (SA-β-gal) activity, Ago2 translocation by Ago2/ Ki67 immunofluorescence staining and western blot analysis, and gene transcription by quantitative polymerase chain reaction (qPCR)., Results: IFN-γ and TNF permanently stopped cell proliferation and time-dependently increased SA-β-gal activity. After 24 - 48 h of cytokine treatment, Ago2 translocated from the cytoplasm into the nucleus of Ki67-negative cells, an effect which was shown to be reversible. Importantly, the proinflammatory cytokine cocktail suppressed Ago2-regulated cell cycle control genes, and siRNA-mediated depletion of Ago2 interfered with cytokine-induced growth inhibition., Conclusion: IFN-γ and TNF induce a stable cell cycle arrest of cancer cells that is accompanied by a fast nuclear Ago2 translocation and repression of Ago2-regulated cell cycle control genes. As Ago2 downregulation impairs cytokine-induced growth regulation, Ago2 may contribute to tissue homeostasis in human cancers., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Autoimmune diseases are characterized by dendritic cell (DC)-driven activation of pro-inflammatory T cell responses. Therapeutic options for these severe diseases comprise small molecules such as dimethyl fumarate, or "gasotransmitters" such as CO. Herein we describe the synthesis of bifunctional enzyme-triggered CO-releasing molecules (ET-CORMs) that allow the simultaneous intracellular release of both CO and methyl fumarate. Using bone-marrow-derived DCs the impressive therapeutic potential of these methyl fumarate-derived compounds (FumET-CORMs) is demonstrated by strong inhibition of lipopolysaccharide-induced pro-inflammatory signaling pathways and blockade of downstream interleukin-12 or -23 production. The data also show that FumET-CORMs are able to transform DCs into an anti-inflammatory phenotype. Thus, these novel compounds have great clinical potential, for example, for the treatment of psoriasis or other inflammatory conditions of the skin., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cutaneous squamous cell carcinoma (cSCC) is an increasing health burden in white populations. We prospectively assessed risk factors for tumor-specific and overall survival in 1,434 patients who underwent surgery for cSCC between January 24, 2005, and May 29, 2015. A total of 2,149 invasive cSCCs were analyzed. Univariate and multivariate survival analyses included tumor thickness, horizontal size, body site, histological differentiation, desmoplastic growth, history of multiple cSCCs, and immunosuppression. The primary endpoint was time to tumor-specific death. During a median follow-up period of 36.5 months (range = 0-137 months), 515 patients died; 40 because of cSCC (2.8%). Of those, 12 died because of visceral metastases and 28 because of tumor growth by local infiltration. On multivariate analyses, prognostic factors for tumor-specific survival were increased vertical tumor thickness (hazard ratio = 6.73; 95% confidence interval = 3.47-13.08; P < 0.0001), desmoplastic growth (hazard ratio = 4.14; 95% confidence interval = 2.68-9.83; P < 0.0001), and immunosuppression (hazard ratio = 2.07; 95% confidence interval = 1.04-4.12; P = 0.039). Defining a point list out of those factors and grouping them into four cohorts resulted in comprehensively separating survival curves (P < 0.001). Using a cut-off for tumor thickness of 6 mm or greater, the presence of desmoplastic growth and immunosuppression identifies patients at high risk for tumor-specific death., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Chronic inflammatory skin diseases are characterized by controlled proliferation of keratinocytes. Here, activating transcription factor 3 (ATF3) might play a fundamental role. In these inflammatory diseases, proliferation is controlled and only rarely leads to cancer development which can be supported by an inflammatory microenvironment. ATF3 is a dual function protein as it suppresses pro-inflammatory IL-6 and IL-8, but also acts as a pro-oncogenic factor by the suppression of p53. We therefore analyzed ATF3 expression comparing myeloid cells with keratinocytes., Objective: To dissect the bi-modal role of ATF3 we pharmacologically induced ATF3 and analyzed its influence on cytokine expression and secretion in a cell type specific manner., Methods: Since inflammatory skin diseases can be treated systemically with Cyclosporin A or Dimethylfumarate we stimulated myeloid cells and primary human keratinocytes with these drugs and analyzed gene expression by quantitative real-time PCR. Cytokine secretion was measured by ELISA., Results: In the present study, we could show that ATF3 is induced in PBMCs by DMF and weakly by Ebselen, while CsA is the most prominent inducer of ATF3 in keratinocytes without enhancing HO-1 transcription. Further we could show that induction of stress by LPS treatment elevates IL-1β and IL-6 and weakly ATF3 transcription in PBMCs. While transcription of both cytokines is elevated, LPS treatment mediates IL-6 secretion with only little IL-1β secretion. Treatment with DMF dampens LPS-induced transcription., Conclusions: Taken together, our results shed light into the different carcinogenic potential of CsA and DMF, which both target ATF3. Collectively our data demonstrate that CsA strongly induces pro-carcinogenic ATF3 in keratinocytes, whereas ATF3 induction by DMF in myeloid cells acts anti-inflammatory., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Atopic dermatitis (AD) is a common inflammatory skin disease with dysfunction of the skin barrier, an abnormal immune response and frequent allergies to environmental antigens like food antigens. Clinical observations suggest that certain diets can influence the course of AD., Objective: Here we compared the phenotype of food allergen-specific T cells activated through skin or gut allergen exposure to transfer skin inflammation into naïve recipients upon epicutaenous allergen challenge., Methods: Ovalbumin (OVA) TCR-transgenic mice were treated epicutaneously with OVA or were fed OVA. CD4 + T cells from skin lymph nodes or mesenteric lymph nodes were transferred into naïve BALB/c mice, which were challenged with OVA epicutaneously. Skin inflammation was determined by histological parameters. In addition, we analyzed the phenotype of the immune response in lymphoid tissues and in skin tissue., Results: TCR-transgenic T cells activated through epicutaneous or oral OVA exposure both migrate to skin lymph nodes after adoptive transfer and epicutaenous OVA exposure. AD-like skin inflammation could only be induced by the transfer of epicutaneously primed OVA T cells. Analysis of the immune phenotype demonstrated an IL-22/IL-17A-dominated immune phenotype of skin-pathogenic T cells., Conclusion: IL-22 seems to be the critical cytokine for the development of AD and is induced in this model by epicutaneous sensitization with OVA., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

The immune response is a first-line systemic defense to curb tumorigenesis and metastasis. Much effort has been invested to design antitumor interventions that would boost the immune system in its fight to defeat or contain cancerous growth. Tumor vaccination protocols, transfer of tumor-associated-antigen-specific T cells, T cell activity-regulating antibodies, and recombinant cytokines are counted among a toolbox filled with immunotherapeutic options. Although the mechanistic underpinnings of tumor immune control remain to be deciphered, these are studied with the goal of cancer cell destruction. In contrast, tumor dormancy is considered as a dangerous equilibrium between cell proliferation and cell death. There is, however, emerging evidence that tumor immune control can be achieved in the absence of overt cancer cell death. Here, we propose cytokine-induced senescence (CIS) by transfer of T helper-1 cells (T H 1) or by recombinant cytokines as a novel therapeutic intervention for cancer treatment. Immunity-induced senescence triggers a stable cell cycle arrest of cancer cells. It engages the immune system to construct defensive, isolating barriers around tumors, and prevents tumor growth through the delivery or induction of T H 1-cytokines in the tumor microenvironment. Keeping cancer cells in a non-proliferating state is a strategy, which directly copes with the lost homeostasis of aggressive tumors. As most studies show that even after efficient cancer therapies minimal residual disease persists, we suggest that therapies should include immune-mediated senescence for cancer surveillance. CIS has the goal to control the residual tumor and to transform a deadly disease into a state of silent tumor persistence.