Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8+ T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.

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.

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.

BACKGROUND Neoantigen-driven recognition and T cell–mediated killing contribute to tumor clearance following adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs). Yet how diversity, frequency, and persistence of expanded neoepitope-specific CD8 + T cells derived from TIL infusion products affect patient outcome is not fully determined. METHODS Using barcoded pMHC multimers, we provide a comprehensive mapping of CD8 + T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic melanoma patients who received ACT. RESULTS We identified 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observed neoepitope-specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we found that the frequency of neoepitope-specific CD8 + T cells in TIL infusion products correlated with increased survival and that neoepitope-specific CD8 + T cells shared with the infusion product in posttreatment blood samples were unique to responders of TIL-ACT. Finally, we found that a transcriptional signature for lymphocyte activity within the tumor microenvironment was associated with a higher frequency of neoepitope-specific CD8 + T cells in the infusion product. CONCLUSIONS These data support previous case studies of neoepitope-specific CD8 + T cells in melanoma and indicate that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8 + T cells. FUNDING NEYE Foundation; European Research Council; Lundbeck Foundation Fellowship; Carlsberg Foundation.

Adoptive T-cell therapy (ACT) relies on expansion of tumor-infiltrating lymphocytes and infusion into patients following lymphodepletion, which have yielded complete responders in up to 25% of treated patients suffering from metastatic melanoma. However, a large proportion of patients have no clinical benefit (50-60%). Previous studies have found that clinical outcome correlates with tumor mutational and putative neoantigen load. Furthermore, expression of core antigen-presentation pathways including MHC class I genes in tumors correlates with clinical benefit. Based on these findings, we hypothesize that the presence of neoepitope-specific CD8+ T cells within the infusion product of ACT is a key determinant of clinical benefit. We apply in silico prediction of neoepitopes along with DNA-barcoded dextran multimer libraries to screen for personal neoepitope-specific CD8+ T cells. We show persistence of neoepitope-specific CD8+ T cells within patient peripheral blood for up to 24 months after therapy. Furthermore, we show an almost complete lack of neoepitope-specific CD8+ T cells within the infusion products of patients with progressive disease. However, patients with stable disease and long-term responses only have a modest increase in the number of targeted neoepitopes, as wells as the total frequency of neoepitope-specific CD8+ T cells. For future experimentation, we therefore aim to include more patients as well as study the phenotypical differences within the pool neoepitope-specific CD8+ T cells, the ultimate goal being a better understanding of what parameters from neoepitope-specific CD8+ T cells correlate with clinical efficacy of ACT. Citation Format: Nikolaj Pagh Kristensen, Christina Heeke, Siri A. Tvingsholm, Anne-Mette Bjerregaard, Arianna Draghi, Amalie Kai Bentzen, Rikke Andersen, Marco Donia, Inge Marie Svane, Sine Reker Hadrup. Neoepitope-specific CD8+ T cells in adoptive T-cell transfer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A14.

Purpose: Recent studies have demonstrated HLA class II (HLA-II)-dependent killing of melanoma cells by cytotoxic CD4 T cells. We investigated evolution of HLA-II-loss tumors that escape cytotoxic CD4 T cell activity and contribute to immunotherapy resistance. Experimental Design: Melanoma cells from longitudinal metastases were studied for constitutive and interferon-inducible HLA-II expression, sensitivity towards autologous CD4 T cells, and immune evasion by HLA-II loss. Clinical significance of HLA-II-low tumors was determined by analysis of transcriptomic data sets from patients with immune checkpoint blockade (ICB). Results: Analysis of longitudinal samples revealed strong inter-metastatic heterogeneity in melanoma cell-intrinsic HLA-II expression and subclonal HLA-II loss. Tumor cells from early lesions either constitutively expressed HLA-II, sensitizing to cytotoxic CD4 T cells, or induced HLA-II and gained CD4 T cell sensitivity in the presence of IFNγ. In contrast, late outgrowing subclones displayed a stable CD4 T cell-resistant HLA-II-loss phenotype. These cells lacked not only constitutive but also IFNγ-inducible HLA-II due to JAK1/2-STAT1 pathway inactivation. Coevolution of JAK1/2 deficiency and HLA-II loss established melanoma cross-resistance to IFNγ and CD4 T cells, as detected in distinct stage IV metastases. In line with their immune-evasive phenotype, HLA-II-low melanomas showed reduced CD4 T cell infiltrates and correlated with disease progression under ICB. Conclusions: Our study links melanoma resistance to CD4 T cells, IFNγ, and ICB at the level of HLA-II, highlighting the significance of tumor cell-intrinsic HLA-II antigen presentation in disease control and calling for strategies to overcome its downregulation for improvement of patient outcome

Immune checkpoint blockade (ICB) is standard-of-care for patients with metastatic melanoma. It may re-invigorate T cells recognizing tumors, and several tumor antigens have been identified as potential targets. However, little is known about the dynamics of tumor antigen-specific T cells in the circulation, which might provide valuable information on ICB responses in a minimally invasive manner. Here, we investigated individual signatures composed of up to 167 different melanoma-associated epitope (MAE)-specific CD8+ T cells in the blood of stage IV melanoma patients before and during anti-PD-1 treatment, using a peptide-loaded multimer-based high-throughput approach. Additionally, checkpoint receptor expression patterns on T cell subsets and frequencies of myeloid-derived suppressor cells and regulatory T cells were quantified by flow cytometry. Regression analysis using the MAE-specific CD8+ T cell populations was applied to identify those that correlated with overall survival (OS). The abundance of MAE-specific CD8+ T cell populations, as well as their dynamics under therapy, varied between patients. Those with a dominant increase of these T cell populations during PD-1 ICB had a longer OS and progression-free survival than those with decreasing or balanced signatures. Patients with a dominantly increased MAE-specific CD8+ T cell signature also exhibited an increase in TIM-3+ and LAG-3+ T cells. From these results, we created a model predicting improved/reduced OS by combining data on dynamics of the three most informative MAE-specific CD8+ T cell populations. Our results provide insights into the dynamics of circulating MAE-specific CD8+ T cell populations during ICB, and should contribute to a better understanding of biomarkers of response and anti-cancer mechanisms.

BackgroundAdoptive cell transfer (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has proven to be one of the most successful immune therapies with overall response rates of around 50% in patients with metastatic melanoma including complete responses in up to 20% of the patients. Current protocols combine a first expansion of TILs from tumor fragments or tumor digest with high-dose IL-2, followed by further expansion with a rapid-expansion-protocol (REP) using allogeneic feeder cells, αCD3 and IL-2. Following this protocol, TIL production takes 4–7 weeks, and many patients deteriorate before they can receive therapy. With success rates of TIL expansion ranging from 70–90%, a TIL product cannot be generated for every patient. Furthermore, clinical response to TIL therapy is lower in other solid tumors such as ovarian cancer, likely due to a lower number of expanded TILs and lower frequencies of tumor reactive CD8+ T cells.Therefore, there is a clinical need for improvement of current TIL expansion protocols to make this therapy available to more cancer patients.MethodsIn this project, TILs from tumor fragments of patients with various solid tumors have been cultured under two different conditions using novel culture vessels. TIL yield, viability, composition, phenotype, reactivity and expansion time were compared to TILs expanded following the standard protocol.ResultsUsing the two novel culture conditions, success rates of expansion across all tumor types increased from 70% to >95%. Additionally, significantly higher frequency and total numbers of viable CD8+ T cells per fragment were obtained compared to standard expansion with IL-2 alone. The majority of these CD8+ T cells exhibit an effector memory phenotype with elevated levels of exhaustion markers. A third of CD8+ T cells can be activated unspecifically and express any combination of the cytotoxicity markers IFNg, TNFa or CD107a. Overall, the initial expansion time could be reduced from 2–5 weeks to 10–14 days and the rapid expansion time from 2 weeks to 8–12 days. Thus, the total culture time was shortened from 4–7 weeks to 18–26 days. As the REP TILs still comprise a majority of CD8+ T cells (~75%), the clinical dose would contain a total number of 3–10 billion functional CD8+ T cells.ConclusionsWith this study, we show that by improving the initial culture conditions, we can shorten expansion time while simultaneously improving the characteristics of the TIL product with a high dose of functional CD8+ T cells with potential anti-tumor activity.