Purpose: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma. Experimental Design: Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line). Results: Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (p < 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells' cytotoxicity. Conclusions: A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.

中文翻译：

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抗PD-1治疗的转移性黑素瘤患者中NK细胞的综合分子和免疫表型分析。

目的：抗PD-1治疗彻底改变了治疗方法，提高了IV期黑色素瘤患者的生存率。但是，由于免疫逃避机制，几乎一半的患者无法做出反应。已知的机制是下调主要组织相容性复合体（MHC）I类表达，这阻止了T细胞识别肿瘤。这项研究确定了转移性黑色素瘤中自然杀伤（NK）细胞数与抗PD-1治疗的临床反应之间的关系。实验设计：将25例抗PD-1治疗的转移性黑色素瘤患者分为缓解者（完全缓解（CR）/部分缓解（PR）/稳定疾病（SD）≥6 mo，n = 13）和无缓解者（根据RECIST反应，SD <6天/进行性疾病（PD），n = 12）。整个转录组测序和多重免疫荧光染色在治疗前和治疗过程中早期的肿瘤子集上进行。在多重免疫荧光图像上进行空间分布分析，以确定NK细胞与肿瘤细胞的接近程度。流式细胞术用于确定未治疗过的黑色素瘤患者（n = 5）的淋巴结转移中的NK表型。使用经抗PD-1或同种型对照处理并与3种不同的黑色素瘤细胞系和K562细胞（白血病细胞系）共培养的NK细胞进行细胞毒性测定。结果：差异表达分析确定了在有反应的患者（n = 11）和无反应的患者（n = 10）中有9个上调的NK细胞特异性基因（调整后p <0.05）。活组织检查的免疫荧光染色证实，在有反应的患者中，肿瘤内和肿瘤周围的CD16 +和颗粒酶B + NK细胞的密度显着更高（p <0.05）。有趣的是，与未应答的患者相比，在应答PD-1治疗的患者中NK细胞更接近肿瘤细胞。尽管I类MHC丢失，但对抗PD-1治疗有反应的患者的NK细胞密度要高于MHC I类表达低的患者。最后，功能测定表明PD-1阻断可诱导NK细胞的细胞毒性增加。结论：较高的肿瘤NK细胞密度与抗PD-1治疗的反应有关。NK细胞可能在介导抗PD-1治疗的反应中起重要作用，包括在下调MHC I类表达的部分肿瘤中。