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-γ and TNF-α 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.

已知包括人类黑素瘤来源的外来体（HMEX）在内的外来体会抑制免疫效应细胞的功能，对于HMEX，这种效应与免疫检查点配体PD-L1的表面存在有关。这项研究调查了黑色素瘤细胞的BRAF突变状态与抗原特异性人T细胞对分泌型HMEX外泌体的抑制之间的关系。从两个黑色素瘤细胞系2183-Her4和888-mel中分离出外泌体，它们是野生型的BRAF ^WT和BRAF ^V600E， 分别。使用改良的尺寸排阻色谱法（SEC）分离HMEX，结果表明与超速离心分离相比，该混合物可减少非外泌体相关细胞因子的共分离。在体外测试了外泌体对受NY-ESO-1抗原攻击的患者来源的NY-ESO-1特异性CD8 ⁺ T细胞的免疫抑制作用。来自两种细胞系的HMEX都可同等地抑制抗原特异性T细胞的免疫反应，这可通过NY-ESO-1四聚体阳性细胞中IFN-γ和TNF-α的减少来证明。抗PD-L1和抗IL-10抗体的存在可以部分逆转这种抑制作用。与BRAF ^WT相比，IL-10已被证明是维持BRAF ^V600E突变细胞中增强的肿瘤发生的关键途径。黑色素瘤细胞。因此，我们证明了HMEX抑制了抗原特异性T细胞反应，而与亲本细胞的BRAF突变状态无关。另外，PD-L1和IL-10有助于HMEX介导的抗原特异性人T细胞的免疫抑制。在开发依赖于定制的抗原特异性效应T细胞的功效的疗法时，应考虑外泌体的抑制能力。