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-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.

摘要

已知外泌体，包括人类黑色素瘤衍生的外泌体 (HMEX) 会抑制免疫效应细胞的功能，对于 HMEX，这与免疫检查点配体 PD-L1 的表面存在有关。本研究调查了黑色素瘤细胞的 BRAF 突变状态与分泌的 HMEX 外泌体对抗原特异性人类 T 细胞的抑制之间的关系。从两种黑色素瘤细胞系 2183-Her4 和 888-mel 中分离出外泌体，它们是基因野生型 BRAF ^WT和 BRAF ^V600E， 分别。HMEX 使用改进的尺寸排阻色谱 (SEC) 方法进行分离，与超速离心分离相比，该方法显示可减少非外泌体相关细胞因子的共分离。外泌体的免疫抑制作用在体外对用 NY-ESO-1 抗原攻击的患者衍生的 NY-ESO-1 特异性 CD8 ⁺ T 细胞进行了测试。来自两种细胞系的 HMEX 可相当地抑制抗原特异性 T 细胞的免疫反应，这可以通过 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 已被证明是在 BRAF ^V600E突变细胞中维持增强的肿瘤发生的关键途径黑色素瘤细胞。因此，我们证明 HMEX 抑制抗原特异性 T 细胞反应，而与亲本细胞的 BRAF 突变状态无关。此外，PD-L1 和 IL-10 有助于 HMEX 介导的抗原特异性人类 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 细胞效力的疗法时，应考虑外泌体的抑制能力。