Adoptive cell transfer therapies (ACTs) with cytotoxic T cells that target melanocytic antigens can achieve remissions in patients with metastatic melanomas, but tumours frequently relapse. Hypotheses explaining the acquired resistance to ACTs include the selection of antigen-deficient tumour cell variants and the induction of T-cell tolerance. However, the lack of appropriate experimental melanoma models has so far impeded clear insights into the underlying mechanisms. Here we establish an effective ACT protocol in a genetically engineered mouse melanoma model that recapitulates tumour regression, remission and relapse as seen in patients. We report the unexpected observation that melanomas acquire ACT resistance through an inflammation-induced reversible loss of melanocytic antigens. In serial transplantation experiments, melanoma cells switch between a differentiated and a dedifferentiated phenotype in response to T-cell-driven inflammatory stimuli. We identified the proinflammatory cytokine tumour necrosis factor (TNF)-α as a crucial factor that directly caused reversible dedifferentiation of mouse and human melanoma cells. Tumour cells exposed to TNF-α were poorly recognized by T cells specific for melanocytic antigens, whereas recognition by T cells specific for non-melanocytic antigens was unaffected or even increased. Our results demonstrate that the phenotypic plasticity of melanoma cells in an inflammatory microenvironment contributes to tumour relapse after initially successful T-cell immunotherapy. On the basis of our work, we propose that future ACT protocols should simultaneously target melanocytic and non-melanocytic antigens to ensure broad recognition of both differentiated and dedifferentiated melanoma cells, and include strategies to sustain T-cell effector functions by blocking immune-inhibitory mechanisms in the tumour microenvironment.

Our understanding of pathways leading to antitumor immunity may depend on an undistorted knowledge of the primary antigenic targets of patients' autologous T cell responses. In the melanoma model derived from patient DT, we applied cryopreserved shortterm autologous mixed lymphocyte-tumor cell cultures (MLTCs) in combination with an IFN-␥ enzyme-linked immunospot (ELISPOT) assay to cDNA expression screening. We identified three previously unknown peptides processed from melanosomal proteins tyrosinase (presented by HLA-A*2601 and -B*3801) and gp100 (presented by HLA-B*07021) and five neoantigens generated by somatic point mutations in the patient's melanoma. The mutations were found in the genes SIRT2, GPNMB, SNRP116, SNRPD1, and RBAF600. Peptides containing the mutated residues were presented by HLA-A*03011, -B*07021, and -B*3801. Mutation-induced functional impairment was so far demonstrated for SIRT2. Within MLTC responder populations that were independently expanded from the patient's peripheral blood lymphocytes of different years, T cells against mutated epitopes clearly predominated. These results document a high degree of individuality for the cellular antitumor response and support the need for individualizing the monitoring and therapeutic approaches to the primary targets of the autologous T cell response, which may finally lead to a more effective cancer immunotherapy.expression cloning ͉ peptide ͉ tumor antigen ͉ mixed lymphocyte-tumor cell culture ͉ cytotoxic T lymphocytes

Purpose: CD8 þ T lymphocytes can kill autologous melanoma cells, but their activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here, we analyzed three consecutive melanoma lesions obtained within one year of developing stage IV disease for their recognition by autologous T cells. Experimental Design: One skin (Ma-Mel-48a) and two lymph node (Ma-Mel-48b, Ma-Mel-48c) metastases were analyzed for T-cell infiltration. Melanoma cell lines established from the respective lesions were characterized, determining the T-cell-stimulatory capacity, expression of surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor-T-cell interaction.Results: Metastases Ma-Mel-48a and Ma-Mel-48b, in contrast with Ma-Mel-48c, were infiltrated by T cells. The T-cell-stimulatory capacity was found to be strong for Ma-Mel-48a, lower for Ma-Mel-48b, and completely abrogated for Ma-Mel-48c cells. The latter proved to be HLA class I-negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. The same deletion was already present in Ma-Mel-48a and Ma-Mel-48b cells, pointing to an early acquired genetic event predisposing to development of b2m deficiency. Notably, the same chronology of genetic alterations was also observed in a second b2m-deficient melanoma model. Conclusion: Our study reveals a progressive loss in melanoma immunogenicity during the course of metastatic disease. The genetic evolvement of T-cell resistance suggests screening tumors for genetic alterations affecting immunogenicity could be clinically relevant in terms of predicting patient responses to T-cell-based immunotherapy. Clin Cancer Res; 20(24); 6593-604. Ó2014 AACR.

Malignant melanomas are highly resistant to chemotherapy. First-line chemotherapeutics used in melanoma therapy are the methylating agents dacarbazine (DTIC) and temozolomide (TMZ) and the chloroethylating agents BCNU and fotemustine. Here, we determined the mode of cell death in 11 melanoma cell lines upon exposure to TMZ and fotemustine. We show for the first time that TMZ induces apoptosis in melanoma cells, using therapeutic doses. For both TMZ and fotemustine apoptosis is the dominant mode of cell death. The contribution of necrosis to total cell death varied between 10 and 40%. The O 6 -methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg À1 protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and fotemustine. MGMT inactivation by O 6 -benzylguanine sensitized all melanoma cell lines expressing MGMT to TMZ and fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O 6 -alkylguanines are critical lesions involved in the initiation of programmed melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to fotemustine. Although these data indicate that methylating drug resistance of melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in melanoma cells induced by TMZ and fotemustine was accompanied by double-strand break (DSB) formation (as determined by H2AX phosphorylation) and caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for fotemustine a correlation was not found. Melanoma lines expressing p53 wild-type were more resistant to TMZ and fotemustine than p53 mutant melanoma lines, which is in marked contrast to previous data reported for glioma cells treated with TMZ. Overall, the findings are in line with the model that in melanoma cells TMZ-induced O 6 -methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner.

Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.

In melanoma patients, one of the main reasons for tumor immune escape and therapy failure is the immunosuppressive tumor microenvironment. Herein, suppressive immune cells and inhibitory factors secreted by the tumor itself play a central role.In the present study we show that the Treg activation marker GARP (glycoprotein A repetitions predominant), known to induce peripheral tolerance in a TGF-β dependent way, is also expressed on human primary melanoma. Interestingly, membrane bound GARP is shed from the surface of both, activated Treg and melanoma cells, and, in its soluble form (sGARP), not only induces peripheral Treg but also a tumor associated (M2) macrophage phenotype. Notably, proliferation of cytotoxic T cells and their effector function is inhibited in the presence of sGARP. GARP expression on Treg and melanoma cells is significantly decreased in the presence of agents such as IFN-α, thus explaining at least in part a novel mechanism of action of this adjuvant therapy.In conclusion, GARP in its soluble and membrane bound form contributes to peripheral tolerance in a multipronged way, potentiates the immunosuppressive tumor microenvironment and thus acts as a negative regulator in melanoma patients. Therefore, it may qualify as a promising target and a new checkpoint for cancer immunotherapy.

Melanoma often recurs after a latency period of several years, presenting a T cell-edited phenotype that reflects a role for CD8(+) T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen-specific CD8(+) T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8(+) T cell-resistant, HLA class I-negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8(+) T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Jun(high)/MITF(low) phenotype, possibly associated with immunosuppression, which contrasted with a c-Jun(low)/MITF(high) phenotype of T cell-edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347-58. ©2016 AACR.

The ability to use circulating peripheral blood cells and matched tumor sequencing data as a basis for neoantigen prediction has exciting possibilities for application in the personalized treatment of cancer patients. We have used a high-throughput screening approach, combining whole-exome sequence data, mRNA microarrays, and publicly available epitope prediction algorithm output to identify mutated proteins processed and displayed by patient tumors and recognized by circulating immune cells. Matched autologous melanoma cell lines and peripheral blood mononuclear cells were used to create mixed lymphocyte tumor cell cultures, resulting in an expansion of tumor-reactive T cells to use for mutated peptide screening. Five patients were investigated, three of whom had a durable complete response (CR; 15þ years) in an autologous melanoma-pulsed dendritic cell clinical trial. We identified seven mutated antigens in total that stimulated T-effector memory cells in two of the five patients. While the procedure did not result in clinically applicable neoantigens for all patients, those identified were likely important in tumor clearance, leading to durable CR. The nature of the screening process allows results to be obtained rapidly and is easily applicable to a wide variety of different tumor types.