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An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8⁺ T Cells is Dependent on IL-10 and Independent of BRAF^V600E Mutation in Melanoma Cell Lines.

ABSTRACT: Exosomes, including human melanoma-derived exosomes (HMEX), are known to suppress the function of immune effector cells, which for HMEX has been associated with the surface presence of the immune checkpoint ligand PD-L1. This study investigated the relationship between the BRAF mutational status of melanoma cells and the inhibition of secreted HMEX exosomes on antigen-specific human T cells. Exosomes were isolated from two melanoma cell lines, 2183-Her4 and 888-mel, which are genetically wild-type BRAF^WT and BRAF^V600E, respectively. HMEX were isolated using a modified, size-exclusion chromatography (SEC) method shown to reduce co-isolation of non-exosome-associated cytokines compared to ultracentrifugation isolation. The immunoinhibitory effect of the exosomes was tested in vitro on patient-derived NY-ESO-1-specific CD8⁺ T cells challenged with NY-ESO-1 antigen. HMEX from both cell lines inhibited the immune response of antigen-specific T cells comparably, as evidenced by the reduction of IFN-? and TNF-? in NY-ESO-1 tetramer-positive cells. This inhibition could be partially reversed by the presence of anti-PD-L1 and anti-IL-10 antibodies. IL-10 has been demonstrated to be a critical pathway for sustaining enhanced tumorigenesis in BRAF^V600E mutant cells compared to BRAF^WT melanoma cells. Thus, we demonstrate that HMEX inhibit antigen-specific T cell responses independent of the BRAF mutational status of the parent cells. In addition, PD-L1 and IL-10 contribute to the HMEX-mediated immunosuppression of antigen-specific human T cells. The inhibitory capacity of exosomes should be taken into consideration when developing therapies that are reliant upon the potency of customized, antigen-specific effector T cells.

SUBMITTER: Shu S

PROVIDER: S-EPMC7816128 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Publications

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8<sup>+</sup> T Cells is Dependent on IL-10 and Independent of BRAF<sup>V600E</sup> Mutation in Melanoma Cell Lines.

Shu ShinLa S Matsuzaki Junko J Want Muzamil Y MY Conway Alexis A Benjamin-Davalos Shawna S Allen Cheryl L CL Koroleva Marina M Battaglia Sebastiano S Odunsi Adekunle A Minderman Hans H Ernstoff Marc S MS

Immunological investigations 20200817 7

Exosomes, including human melanoma-derived exosomes (HMEX), are known to suppress the function of immune effector cells, which for HMEX has been associated with the surface presence of the immune checkpoint ligand PD-L1. This study investigated the relationship between the BRAF mutational status of melanoma cells and the inhibition of secreted HMEX exosomes on antigen-specific human T cells. Exosomes were isolated from two melanoma cell lines, 2183-Her4 and 888-mel, which are genetically wild-ty ...[more]

PMID: 32799717

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Project description:BackgroundMost mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.MethodsDNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.ResultsThree cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines.ConclusionCell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

Dataset Information

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8⁺ T Cells is Dependent on IL-10 and Independent of BRAF^V600E Mutation in Melanoma Cell Lines.

Publications

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8<sup>+</sup> T Cells is Dependent on IL-10 and Independent of BRAF<sup>V600E</sup> Mutation in Melanoma Cell Lines.

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Dataset Information

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8+ T Cells is Dependent on IL-10 and Independent of BRAFV600E Mutation in Melanoma Cell Lines.

Publications

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8<sup>+</sup> T Cells is Dependent on IL-10 and Independent of BRAF<sup>V600E</sup> Mutation in Melanoma Cell Lines.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8⁺ T Cells is Dependent on IL-10 and Independent of BRAF^V600E Mutation in Melanoma Cell Lines.