Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium.
Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium.
J Immunother Cancer. 2021 Feb;9(2). doi: 10.1136/jitc-2020-001749.
Modulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker.
We employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models.
We were able to discern two ti-Treg populations, one of which is characterized by the unique expression of in conjunction with Treg activation markers. is also expressed by dysfunctional CD4 and CD8 T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs.
Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.
调节性 T 细胞（Tregs）的调制和耗竭策略构成了抗肿瘤免疫治疗的有效方法，但由于缺乏对肿瘤浸润（ti）-Treg 群体的选择性，它们存在严重的副作用，这表明需要一种 ti-Treg 特异性生物标志物。
我们在非小细胞肺癌（NSCLC）的小鼠模型中使用单细胞 RNA 测序，全面了解肿瘤浸润 T 细胞区室，重点关注 ti-Treg 亚群。这些发现通过对小鼠（LLC-OVA、MC38 和 B16-OVA）和人类（NSCLC 和黑色素瘤）肿瘤样本的流式细胞分析进行了验证。我们生成了两种 CCR8 特异性纳米抗体（Nbs），它们识别 CCR8 细胞外结构域上的不同表位。这些 Nbs 被制成四价 Nb-Fc 融合蛋白，以实现最佳的 CCR8 结合和阻断，其中包含抗体依赖性细胞介导的细胞毒性（ADCC）缺陷或 ADCC 倾向的 Fc 区。我们评估了这些 Nb-Fc 融合蛋白的治疗用途，无论是作为单一疗法还是与抗程序性细胞死亡蛋白-1（抗 PD-1）联合治疗，在 LLC-OVA 和 MC38 小鼠模型中都进行了评估。
我们能够区分两种 ti-Treg 群体，其中一种群体的特征是与 Treg 激活标志物一起独特表达。还表达于功能失调的 CD4 和 CD8 T 细胞，但 CCR8 蛋白仅在小鼠和人类肿瘤的高度激活和强烈抑制性 ti-Treg 亚群上突出，在外周 Tregs 上未发现主要的 CCR8 阳性。CCR8 表达是 TCR 介导的 Treg 触发的结果，NF-κB 依赖性，但对这些细胞的募集、激活或抑制能力不是必需的。虽然用阻断 ADCC 缺陷型 Nb-Fc 治疗荷瘤小鼠不会影响肿瘤生长，但 ADCC 倾向型 Nb-Fc 与抗 PD-1 治疗协同引发抗肿瘤免疫并减少肿瘤生长。重要的是，ADCC 倾向型 Nb-Fc 以自然杀伤（NK）细胞依赖的方式特异性耗尽 ti-Tregs，而不影响外周 Tregs。
总的来说，我们的研究结果突出了针对 CCR8 以与抗 PD-1 治疗联合耗尽肿瘤促进的 ti-Tregs 的有效性和安全性。
