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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 IFN-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 intermetastatic 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.
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BACKGROUND: Immune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor T cell responses. Switching of melanoma toward a dedifferentiated cell state has recently been linked to T cell and therapy resistance. It remains to be determined whether RIG-I agonists affect melanoma differentiation, potentially leading to T cell resistance. METHODS: Patient metastases-derived melanoma cell lines were treated with the synthetic RIG-I agonist 3pRNA, and effects on tumor cell survival, phenotype and differentiation were determined. Transcriptomic data sets from cell lines and metastases were analyzed for associations between RIG-I (DDX58) and melanoma differentiation markers and used to define signaling pathways involved in RIG-I-driven dedifferentiation. The impact of 3pRNA-induced melanoma dedifferentiation on CD8 T cell activation was studied in autologous tumor T cell models. RESULTS: RIG-I activation by 3pRNA induced apoptosis in a subpopulation of melanoma cells, while the majority of tumor cells switched into a non-proliferative cell state. Those persisters displayed a dedifferentiated cell phenotype, marked by downregulation of the melanocytic lineage transcription factor MITF and its target genes, including melanoma differentiation antigens (MDA). Transition into the MITFlow/MDAlow cell state was JAK-dependent, with some cells acquiring nerve growth factor receptor expression. MITFlow/MDAlow persisters switched back to the proliferative differentiated cell state when RIG-I signaling declined. Consistent with our in vitro findings, an association between melanoma dedifferentiation and high RIG-I (DDX58) levels was detected in transcriptomic data from patient metastases. Notably, despite their dedifferentiated cell phenotype, 3pRNA-induced MITFlow/MDAlow persisters were still efficiently targeted by autologous CD8 tumor-infiltrating T lymphocytes (TILs). CONCLUSIONS: Our results demonstrate that RIG-I signaling in melanoma cells drives a transient phenotypic switch toward a non-proliferative dedifferentiated persister cell state. Despite their dedifferentiation, those persisters are highly immunogenic and sensitive toward autologous TILs, challenging the concept of melanoma dedifferentiation as a general indicator of T cell resistance. In sum, our findings support the application of RIG-I agonists as a therapeutic tool for the generation of long-term clinical benefit in non-resectable melanoma.
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Melanoma , Linfocitos T CD8-positivos , Línea Celular Tumoral , Humanos , Inmunidad Innata , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Transducción de SeñalRESUMEN
The highly plastic nature of melanoma enables its transition among diverse cell states to survive hostile conditions. However, the interplay between specific tumor cell states and intratumoral T cells remains poorly defined. With MAPK inhibitorâtreated BRAFV600-mutant tumors as models, we linked human melanoma state transition to CD8+ T cell responses. Repeatedly, we observed that isogenic melanoma cells could evolve along distinct differentiation trajectories on single BRAF inhibitor (BRAFi) treatment or dual BRAFi/MEKi treatment, resulting in BRAFiâinduced hyperdifferentiated and BRAFi/MEKiâinduced dedifferentiated resistant subtypes. Taking advantage of patient-derived autologous CD8+ tumor-infiltrating lymphocytes (TILs), we demonstrate that progressive melanoma cell state transition profoundly affects TIL function. Tumor cells along the hyperdifferentiation trajectory continuously gained sensitivity toward tumor-reactive CD8+ TILs, whereas those in the dedifferentiation trajectory acquired T cell resistance in part owing to the loss of differentiation antigens. Overall, our data reveal the tight connection of MAPKiâinduced temporary (drug-tolerant transition state) and stable (resistant state) phenotype alterations with T cell function and further broaden the current knowledge on melanoma plasticity in terms of sculpting local antitumor immune responses, with implications for guiding the optimal combination of targeted therapy and immunotherapy.
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Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Diferenciación Celular , Humanos , Activación de Linfocitos , Melanoma/tratamiento farmacológico , Melanoma/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Escape del TumorRESUMEN
Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 µg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics.
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Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Vesículas Extracelulares/inmunología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/inmunología , Animales , Membrana Externa Bacteriana/inmunología , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Lipopolisacáridos/inmunología , Ratones , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiologíaRESUMEN
Human FAT1 is overexpressed on the surface of most colorectal cancers (CRCs) and in particular a 25 amino acid sequence (D8) present in one of the 34 cadherin extracellular repeats carries the epitope recognized by mAb198.3, a monoclonal antibody which partially protects mice from the challenge with human CRC cell lines in xenograft mouse models. Here we present data in immune competent mice demonstrating the potential of the D8-FAT1 epitope as CRC cancer vaccine. We first demonstrated that the mouse homolog of D8-FAT1 (mD8-FAT1) is also expressed on the surface of CT26 and B16F10 murine cell lines. We then engineered bacterial outer membranes vesicles (OMVs) with mD8-FAT1 and we showed that immunization of BALB/c and C57bl6 mice with engineered OMVs elicited anti-mD8-FAT1 antibodies and partially protected mice from the challenge against CT26 and EGFRvIII-B16F10 cell lines, respectively. We also show that when combined with OMVs decorated with the EGFRvIII B cell epitope or with OMVs carrying five tumor-specific CD4+ T cells neoepitopes, mD8-FAT1 OMVs conferred robust protection against tumor challenge in C57bl6 and BALB/c mice, respectively. Considering that FAT1 is overexpressed in both KRAS+ and KRAS- CRCs, these data support the development of anti-CRC cancer vaccines in which the D8-FAT1 epitope is used in combination with other CRC-specific antigens, including mutation-derived neoepitopes.