CD4 Polarized T cells Reduce the Expansion of Tumor Infiltrating Lymphocytes in Melanoma Patients Treated with Adoptive Cell Therapy [RNA-seq]

Background: Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) is effective in treating PD-1 refractory melanoma, but requires adequate ex vivo expansion of TIL. Methods: CD4+ and CD8+ TIL from metastatic melanoma patients treated with TIL ACT were analyzed by RNA-seq (n=12) and ChIP-seq of acetylated histone 3 (n=19). Patients were grouped into “TIL high” and “TIL low” based on division at the median number of TIL infused. The number of TIL infused and CD4+ TIL frequency were correlated with overall survival (OS). Results: The number of TIL infused correlated with longer OS (R2=0.57, p=0.00076), and the percent of CD4+ infused was negatively correlated with the total number of TIL infused (R2=0.64, p=0.00047). RNA-seq analysis of CD4+ TIL showed increases in Th2/Th17/Treg transcripts and pathways in the TIL low group. ChIP-seq analysis of CD8+ TIL showed decreased acetylation in the TIL low group in genes upregulated during CD8+ activation. Conclusion: The numbers of TIL infused were associated with increased overall survival, while RNA-seq suggested that polarized CD4+ cells in the transferred TIL were associated with decreased overall expansion. These data suggest that improper CD4+ TIL polarization may reduce expansion and treatment efficacy. Overall design: Multi-omic analysis of tumor infiltrating lymphocytes expanded from melanoma patients enrolled in clinical trials NCT01005745, NCT01659151 and NCT01701674 for the treatment of melanoma. Anlyses include RNA-seq, panH3ac ChIP-seq and DNA methylation microarray. No healthy controls are included in the study design. Experimental design involved comparison of patients with high TIL expansion vs those with low TIL expansion.

背景：采用肿瘤浸润淋巴细胞（tumor infiltrating lymphocytes, TIL）的过继细胞疗法（adoptive cell therapy, ACT）在治疗PD-1耐药黑色素瘤方面疗效确切，但该疗法需对TIL进行充分的体外扩增。方法：对接受TIL ACT治疗的转移性黑色素瘤患者的CD4+与CD8+ TIL开展转录组测序（RNA-seq，n=12）以及乙酰化组蛋白3的染色质免疫共沉淀测序（ChIP-seq，n=19）。依据输注TIL的中位数将患者分为"TIL高"组与"TIL低"组，并分析输注TIL数量、CD4+ TIL占比与总生存期（overall survival, OS）的相关性。结果：输注TIL数量与更长的总生存期呈正相关（决定系数R²=0.57，p=0.00076）；输注的CD4+ TIL占比与输注TIL总数量呈负相关（R²=0.64，p=0.00047）。对CD4+ TIL的RNA-seq分析显示，TIL低组中Th2/Th17/Treg相关转录本及通路表达上调。对CD8+ TIL的ChIP-seq分析显示，TIL低组中CD8+激活过程中上调基因的乙酰化水平降低。结论：输注TIL数量与总生存期延长显著相关；RNA-seq结果提示，输注TIL中极化的CD4+细胞与整体扩增能力下降存在关联。上述数据表明，不恰当的CD4+ TIL极化可能会降低扩增效率与治疗效果。整体研究设计：本研究针对入组黑色素瘤治疗临床试验NCT01005745、NCT01659151及NCT01701674的患者，对其扩增获得的肿瘤浸润淋巴细胞开展多组学分析，分析内容涵盖RNA-seq、泛乙酰化组蛋白3（panH3ac）ChIP-seq以及DNA甲基化微阵列检测。本研究未设置健康对照样本。实验设计采用高TIL扩增患者与低TIL扩增患者的组间比较方案。