Blocking PD-1 engagement rapidly upregulates innate antiviral and immune functions that associate with HIV reservoir decay [scRNA-seq]

The HIV reservoir, a small pool of PD-1+CD4+ T cells that carry integrated HIV genomes, remains a formidable obstacle to HIV cure. The clinical use of PD-1 inhibitors has shown promising results in curbing the HIV reservoir in people with HIV (PWH) and cancer, but the underlying mechanisms are unknown. Here, we demonstrate that anti-PD-1 therapy leads to a decay in the frequency of cells harboring HIV DNA at the end of a two-year follow-up in 9 of 14 PWH receiving pembrolizumab within the CITN-12 clinical trial (NCT02595866). This reduction correlates with an innate immune response, characterized by increased monocyte frequencies and enhanced myeloid cell-specific transcription of interferon-stimulated genes (ISGs), chemokines, antiviral restriction factors, and TLR signaling. These changes emerge as early as 24 hours post-treatment and persist until the end of therapy. Within 24 hours of treatment, we also observe an increase in proliferating effector HIV-specific CD8+ T cells, increased expression of effector T cell genes, and a decline in plasma TGF-β levels. We validate the generalizability of these ISG expression modules across >1,000 publicly available scRNA-seq samples, revealing a consistent association between high ISG expression in peripheral blood mononuclear cells (PBMCs) and lower expression of WNT pathway and Treg gene sets - pathways known to regulate immune quiescence and persistence of the HIV reservoir - in PBMCs from uninfected controls, as well as in samples from individuals with cancer or infections. Furthermore, we demonstrate that ISGs triggered by TLR ligands protect CD4+ T cells from HIV infection in vitro. These findings support a mechanism by which ISGs restrict the HIV reservoir in vivo and define a set of immune signaling pathways that can be used to identify PWH in whom the HIV reservoir may decay upon anti-PD-1 therapy. Single cell RNA sequencing was performed on Peripheral blood mononuclear cells (PBMCs) on a subset of participants that had bulk RNA sequencing data, at the following timepoints: C01D01 (pre-treatment), C01D02 (hours after the first treatment cycle ), and one week after the first cycle (C01D08).

HIV储存库（HIV reservoir）是携带整合型HIV基因组的一小群PD-1+CD4+ T细胞，仍是实现HIV治愈的重大障碍。临床使用PD-1抑制剂在HIV感染者（people with HIV, PWH）合并癌症的患者中抑制HIV储存库已展现出可喜成果，但其背后的分子机制尚不明确。本研究证实，在CITN-12临床试验（NCT02595866）中接受帕博利珠单抗（pembrolizumab）的14名HIV感染者里，有9名在为期2年的随访终点时，其携带HIV DNA的细胞频率出现下降。该细胞频率的降低与先天免疫应答相关，其特征为单核细胞频率升高，以及干扰素刺激基因（interferon-stimulated genes, ISGs）、趋化因子、抗病毒限制因子和TLR信号通路的髓系细胞特异性转录增强。这类变化最早可在治疗后24小时出现，并持续至治疗结束。在治疗后的24小时内，我们还观察到增殖的效应性HIV特异性CD8+ T细胞增多、效应性T细胞基因表达上调，以及血浆转化生长因子-β（transforming growth factor-β, TGF-β）水平下降。我们在超过1000份公开的单细胞RNA测序（single cell RNA sequencing, scRNA-seq）样本中验证了这些ISG表达模块的通用性，结果显示，在未感染个体的外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）以及癌症或感染患者的样本中，外周血单个核细胞内高ISG表达与WNT通路和调节性T细胞（regulatory T cell, Treg）基因集的低表达存在一致关联——这两类通路已知可调控免疫静息状态与HIV储存库的维持。此外，本研究证实，TLR配体诱导的ISGs可在体外保护CD4+ T细胞免受HIV感染。上述发现支持了ISGs在体内限制HIV储存库的作用机制，并确定了一组免疫信号通路，可用于识别那些接受抗PD-1治疗后HIV储存库可能出现衰减的HIV感染者。我们对部分具备批量RNA测序数据的参与者采集了外周血单个核细胞样本，在以下时间点开展了单细胞RNA测序：C01D01（治疗前）、C01D02（首次治疗周期后数小时）以及首次治疗周期后一周（C01D08）。