Understanding how IL-2 cytokine synergizes with PD-1 directed immunotherapy during chronic viral infection [scRNA-seq]. Understanding how IL-2 cytokine synergizes with PD-1 directed immunotherapy during chronic viral infection [scRNA-seq]

In this study we have examined how the cytokine interleukin-2 (IL-2) synergizes with programmed cell death-1 (PD-1) directed immunotherapy during chronic lymphocytic choriomeningitis virus (LCMV) infection. PD-1 blockade in combination with IL-2 is one of the most effective combination therapies in this very stringent LCMV mouse model of life-long chronic infection with irreversible T-cell exhaustion. Our paper makes the following points: First, we show that the more effective viral control seen after PD-1/IL-2 combination therapy compared to PD-1 monotherapy is mediated by the CD8+T-cell response. Then we identify the virus-specific CD8+T cells that proliferate and respond to the combination therapy and show that these are the same lymphoid resident PD-1+TCF-1+stem-like CD8+T cells that act as resource cells to maintain the CD8+T-cell response during chronic infection and also respond to PD-1 blockade. However, the combination therapy dramatically changes the differentiation program of these chronic resource CD8+T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+T cells that resemble highly functional effector CD8+T cells seen after an acute viral infection. In contrast, PD-1 monotherapy doesnot modify the differentiation program and one gets more virus-specific CD8+T cells but they are transcriptionally and epigenetically similar to what is seen in untreated chronically infected mice. This epigenetic inflexibility of exhausted CD8+T cells is a potential barrier to PD-1 therapy and the ability of this combination therapy to modify the epigenetic signature of virus-specific CD8+T cells during chronic infection could be an important determinant of the striking synergy seen between IL-2 therapy and PD-1 blockade. We also highlight the importance of blocking the PD-1/PD-L1 inhibitory pathway at the target site for effective viral control. Expanding the CD8+ Tcell population and generating better effector cells is important but it is also critical to block PD-1 inhibitory signals at the target site for optimal immunotherapy. Finally, we show that CD25 engagement with IL-2 plays an important and essential role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind CD25 but still binds CD122/132 almost completely abrogated the synergistic effects seen after PD-1/IL-2 combination therapy. There is currently considerable interest in PD-1/IL-2 combination therapy for cancer patients and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies. Overall design: Cell sorting was performed byFACS Aria II (BD Biosciences).For scRNA-seq (10X Genomics), LCMV chronically infectedmice (> day 40 p.i.) were untreated or treated with various therapeutic modalities for 2 weeks, and DbGP33+CD8+T cells in spleens were sorted from pooled spleens. Naïve (CD44lo) CD8+T cells were sorted from pooled spleens from uninfected mice (n=2).

本研究探讨了慢性淋巴细胞性脉络丛脑膜炎病毒（Lymphocytic Choriomeningitis Virus, LCMV）感染过程中，细胞因子白细胞介素-2（Interleukin-2, IL-2）与程序性死亡受体-1（Programmed Cell Death-1, PD-1）靶向免疫治疗的协同作用机制。在该严苛的终身慢性感染伴不可逆T细胞耗竭的LCMV小鼠模型中，PD-1阻断联合IL-2治疗是最为有效的联合治疗方案之一。本研究得出以下核心结论：其一，相较于PD-1单药治疗，PD-1/IL-2联合治疗可实现更高效的病毒控制，该效应由CD8+ T细胞应答所介导。其二，本研究鉴定出了可增殖并响应联合治疗的病毒特异性CD8+ T细胞，并证实这群细胞即为驻留淋巴组织的PD-1+TCF-1（T cell factor 1, TCF-1）+干细胞样CD8+ T细胞：这类细胞作为储备细胞，在慢性感染过程中维持CD8+ T细胞应答，同时也可响应PD-1阻断治疗。然而，联合治疗可显著改变这类慢性感染储备CD8+ T细胞的分化程序，最终生成转录组与表观遗传组均具有独特特征的效应性CD8+ T细胞，这类细胞与急性病毒感染后出现的高功能效应性CD8+ T细胞表型相似。与之相比，PD-1单药治疗并不会改变这类细胞的分化程序：虽可获得更多病毒特异性CD8+ T细胞，但这群细胞的转录组与表观遗传组特征与未接受治疗的慢性感染小鼠体内的细胞并无二致。耗竭性CD8+ T细胞的这种表观遗传可塑性缺失，是PD-1治疗的潜在障碍；而联合治疗在慢性感染过程中可重塑病毒特异性CD8+ T细胞的表观遗传特征，这或许是IL-2治疗与PD-1阻断治疗产生显著协同效应的关键决定因素。本研究同时强调，在靶点部位阻断PD-1/PD-L1（Programmed Cell Death Ligand 1, PD-L1）抑制通路，对实现有效的病毒控制至关重要。扩增CD8+ T细胞群体并生成功能更优的效应细胞固然重要，但在靶点部位阻断PD-1抑制信号以实现最优免疫治疗，同样不可或缺。最后，本研究证实，IL-2与CD25（cluster of differentiation 25, CD25）的结合在IL-2细胞因子与PD-1阻断治疗的协同效应中发挥了至关重要的作用。无论是使用抗体阻断CD25，还是采用无法结合CD25但仍可结合CD122/CD132的突变型IL-2，均可几乎完全消除PD-1/IL-2联合治疗所产生的协同效应。当前，PD-1/IL-2联合治疗在癌症患者中的应用备受关注，本研究阐明了IL-2与PD-1阻断治疗协同作用的潜在机制，可为相关人类转化研究提供重要参考。实验整体设计如下：细胞分选采用FACS Aria II（BD Biosciences）完成。对于单细胞RNA测序（single-cell RNA sequencing, scRNA-seq，10X Genomics平台），慢性LCMV感染小鼠（感染后天数>40天）分为未处理组与不同治疗方案处理组，处理周期为2周；随后从混合脾脏中分选脾脏内的DbGP33特异性CD8+ T细胞。从未感染小鼠的混合脾脏中分选初始态（CD44lo）CD8+ T细胞，每组样本量n=2。