TGF-beta blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo [RNA-seq]

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-beta blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on suppressive ART were treated with 4 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption(ATI). One macaque did not rebound, while the remaining rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with the 64Cu-DOTA-F(ab')2-p7D3-probe. Half to 1 Log decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk and single-cell RNAseq revealed a shift toward an effector phenotype in T and NK cells. To understand early and later effect of galunisertib in the context of SIV infection, we performed bulk RNAseq of PBMCs isolated from before cycle 1 (24hrs) and 1hrs after the first dose of galunisertib or after the last dose of cycle 1 We also performed bulk RNAseq of rectal biopsy specimens collected before cycle 1 (24hrs) and after the last dose of cycle 1. 7 Macaques, 3 time points for PBMCs, 2 time points for Rectal Biopsies

抗逆转录病毒治疗（Antiretroviral Therapy, ART）期间，HIV-1持续感染的核心机制是静息免疫细胞中建立了长期存活的病毒储库。本研究采用带条形码标记的SIVmac239（猴免疫缺陷病毒mac239株，Simian Immunodeficiency Virus mac239, SIVmac239）静脉感染非人灵长类（Non-Human Primate, NHP）模型，并对抗TGFBR1抑制剂（anti-TGFBR1 inhibitor）galunisertib（LY2157299）实施治疗性给药，证实了体内TGF-β阻断可发挥潜伏期逆转作用，能够减少病毒储库并激活免疫应答。8只接受抑制性ART的SIV感染猕猴接受了共4个、每个周期时长2周的galunisertib给药方案。末次给药3周后停止ART，并在抗逆转录病毒治疗中断（Antiretroviral Therapy Interruption, ATI）后6周对所有猕猴实施安乐死。其中1只猕猴未出现病毒反弹，其余7只在ATI后第2周至第6周期间发生病毒反弹。血浆病毒载量变化以及使用64Cu-DOTA-F(ab')2-p7D3探针的免疫PET/CT成像结果证实，galunisertib可诱导病毒激活。在淋巴结、肠道及外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）中，检测到细胞相关（Cell-Associated, CA-）SIV DNA水平下降了0.5至1个对数级；而PBMC中的完整前病毒数量下降了3倍。未观察到炎症细胞因子的全身性升高。高维流式细胞术、批量RNA测序及单细胞RNA测序结果显示，T细胞与自然杀伤（Natural Killer, NK）细胞向效应表型发生了显著偏移。为阐明galunisertib在SIV感染情境下的早期与后期作用，我们对第1给药周期前（24小时）、首次给药后1小时及第1给药周期末次给药后的分离PBMC进行了批量RNA测序。本研究同时对第1给药周期前（24小时）及末次给药后采集的直肠活检标本开展了批量RNA测序。本研究共纳入7只猕猴，其PBMC样本设置3个时间点，直肠活检标本设置2个时间点。