832 Unravelling human melanoma heterogeneity by 6-color multiplex immunofluorescence to overcome recurrence and resistance to therapy

Background Inter- and intralesional tumor heterogeneity is commonly seen in metastatic melanoma, likely playing a major role in resistance to therapy, immunotherapy included. This research project aims to identify by 6-color multiplex immunofluorescence melanoma cell subpopulations, to reveal those that are insufficiently targeted by current immunotherapies. Methods In silico analysis of single cell RNAseq data available from the literature for melanoma were performed and matched with a list of known cancer antigens. Genes discriminating between different subpopulations of melanoma cells were selected and included for multiplex immunofluorescence experiments. FFPE sections from pre- and post-immunotherapy (DC vaccination or ipilimumab) treated melanoma patients were stained by multiplex immunofluorescence for AXL, MITF, PRAME, melanoma lineage (comprising Melan-A, gp100 and tyrosinase), CD45 and CD8 expression. Results Single cell-based analysis of RNAseq data revealed two sets of genes discriminating between different subpopulations of melanoma cells and covering most melanoma cells. Set 1 was shown to be AXL high/MITF low and expressed PRAME, whereas set 2 was shown to be AXL low/MITF high and expressed melanoma lineage markers. The 6-color multiplex immunofluorescence panel could discriminate different melanoma subpopulations, thereby giving information on melanoma heterogeneity. Image analyses of melanoma phenotypes and immune infiltrates is still ongoing. These analyses also include the topographical location of different melanoma cell subpopulations with respect to immune cells, and their changes after immunotherapy. Conclusions Melanoma heterogeneity pre- and post-immunotherapy can be analyzed by 6-color multiplex immunofluorescence

Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to γ-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy

Effective Melanoma Immunotherapy in Mice by the Skin-Depigmenting Agent Monobenzone and the Adjuvants Imiquimod and CpG

Background: Presently melanoma still lacks adequate treatment options for metastatic disease. While melanoma is exceptionally challenging to standard regimens, it is suited for treatment with immunotherapy based on its immunogenicity. Since treatment-related skin depigmentation is considered a favourable prognostic sign during melanoma intervention, we here aimed at the reverse approach of directly inducing vitiligo as a shortcut to effective anti-melanoma immunity. Methodology and Principal Findings: We developed an effective and simple to use form of immunotherapy by combining the topical skin-bleaching agent monobenzone with immune-stimulatory imiquimod cream and cytosine-guanine oligodeoxynucleotides (CpG) injections (MIC therapy). This powerful new approach promptly induced a melanoma antigen-specific immune response, which abolished subcutaneous B16. F10 melanoma growth in up to 85% of C57BL/6 mice. Importantly, this regimen induced over 100 days of tumor-free survival in up to 60% of the mice, and forcefully suppressed tumor growth upon re-challenge either 65- or 165 days after MIC treatment cessation. Conclusions: MIC therapy is effective in eradicating melanoma, by vigilantly incorporating NK-, B-and T cells in its therapeutic effect. Based on these results, the MIC regimen presents a high-yield, low-cost and simple therapy, readily applicable in the clini

Genome-wide association studies of autoimmune vitiligo identify 23 new risk loci and highlight key pathways and regulatory variants

Vitiligo is an autoimmune disease in which depigmented skin results from the destruction of melanocytes1, with epidemiological association with other autoimmune diseases2. In previous linkage and genome-wide association studies (GWAS1 and GWAS2), we identified 27 vitiligo susceptibility loci in patients of European ancestry. We carried out a third GWAS (GWAS3) in European-ancestry subjects, with augmented GWAS1 and GWAS2 controls, genome-wide imputation, and meta-analysis of all three GWAS, followed by an independent replication. The combined analyses, with 4,680 cases and 39,586 controls, identified 23 new significantly associated loci and 7 suggestive loci. Most encode immune and apoptotic regulators, with some also associated with other autoimmune diseases, as well as several melanocyte regulators. Bioinformatic analyses indicate a predominance of causal regulatory variation, some of which corresponds to expression quantitative trait loci (eQTLs) at these loci. Together, the identified genes provide a framework for the genetic architecture and pathobiology of vitiligo, highlight relationships with other autoimmune diseases and melanoma, and offer potential targets for treatment

Tumor-infiltrating T-cells: important players in clinical outcome of advanced melanoma patients

Immune escape mechanisms are prevalent in tumors, while their influence on the potency of antitumor immunotherapy has yet to be distinguished. We recently showed that increased numbers of intratumoral T cells rather than immune-escape-mechanisms significantly correlated with clinical outcome of advanced melanoma patients to subsequent autologous tumor cell vaccination. Our data emphasize the therapeutic relevance of tumor-infiltrating T cells for the clinical outcom

Autoimmune etiology of generalized vitiligo

Vitiligo is characterized by progressive skin depigmentation resulting from an autoimmune response targeting epidermal melanocytes. Melanocytes are particularly immunogenic by virtue of the contents of their melanosomes, generating the complex radical scavenging molecule melanin in a process that involves melanogenic enzymes and structural components, including tyrosinase, MART-1, gp100, TRP-2 and TRP-1. These molecules are also prime targets of the immune response in both vitiligo and melanoma. The immunogenicity of melanosomal proteins can partly be explained by the dual role of melanosomes, involved both in melanin synthesis and processing of exogenous antigens. Melanocytes are capable of presenting antigens in the context of MHC class II, providing HLA-DR+ melanocytes in perilesional vitiligo skin the option of presenting melanosomal antigens in response to trauma and local inflammation. Type I cytokine-mediated immunity to melanocytes in vitiligo involves T cells reactive with melanosomal antigens, similar to T cells observed in melanoma. In vitiligo, however, T cell tuning allows T cells with higher affinity for melanocyte differentiation antigens to enter the circulation after escaping clonal deletion in primary lymphoid organs. The resulting efficacious and progressive autoimmune response to melanocytes provides a roadmap for melanoma therap

Ectopic hTERT expression extends the life span of human CD4(+) helper and regulatory T-cell clones and confers resistance to oxidative stress-induced apoptosis

Human somatic cells have a limited life span in vitro. Upon aging and with each cell division, shortening of telomeres occurs, which eventually will lead to cell cycle arrest. Ectopic hTERT expression has been shown to extend the life span of human T cells by preventing this telomere erosion. In the present study, we have shown that ectopic hTERT expression extends the life span of CD4(+) T helper type 1 or 2 and,regulatory T-cell clones and affected neither the in vitro cytokine production profile. nor their specificity for antigen. In mixed cell cultures, ectopic hTERT-expressing clones were found to expand in greater numbers than untransduced cells of the same replicative age. This ectopic hTERT-induced growth ad vantage was not due to an enhanced cell division rate or number of divisions following, T-cell receptor-mediated activation, as determined in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling experiments. Moreover, the susceptibility to activation-induced cell death of both cell types was similar. However, cultures of resting hTERT-transduced T cells contained higher frequencies of Bcl-2-expressing cells and lower active caspase-3-expressing cells, compared with wild-type cells. Furthermore, hTERT-transduced cells were more resistant to oxidative stress, which causes preferential DNA damage in telomeres. Taken together, these results show that ectopic hTERT expression not only protects proliferating T cells from replicative senescence but also confers resistance to apoptosis induced by oxidative stress. (C) 2003 by The American Society of Hematolog

Vitiligo induced by immune checkpoint inhibitors in melanoma patients: an expert opinion

Introduction: Treatment with immune checkpoint inhibitors in melanoma patients can cause immune-related adverse effects, such as vitiligo. In vitiligo, specific autoimmunity against melanocytes results in depigmentation of the skin. Melanoma-associated vitiligo occurring in melanoma patients treated with immune checkpoint inhibitors can be seen as a good prognostic sign as higher survival rates in melanoma-associated vitiligo cases have been reported. Areas covered: This review gives an insight into the pathophysiology, clinical presentation, and management of melanoma-associated vitiligo caused by immune checkpoint inhibitors. Expert opinion: Development of melanoma-associated vitiligo induced by immune checkpoint inhibitors could be a good clinical marker for response and overall survival. Induction of vitiligo in these patients could also potentially lead to better response and survival rates. Further research should focus on several aspects of melanoma-associated vitiligo, such as better screening and registration, more understanding of pathophysiology of the type of immune response and the predictive value of melanoma-associated in patients treated with immune checkpoint inhibitors