Immune Activation and Exhaustion Marker Expression on T-cell Subsets in Perinatal HIV-1 Infection as Correlates of Viral Persistence

HIV-1 infection of resting memory CD4+ T cells forms a barrier to curing HIV-1. Identification of immune biomarkers that correlate with HIV-1 reservoir size could aid with assessing efficacy of HIV-1 eradication strategies, especially in pediatric infections where blood sampling is limited. In adults, the immune exhaustion marker PD-1 on central memory CD4+ T cells (Tcm) correlates with HIV-1 reservoir size. Immune correlates of HIV-1 reservoir size are less defined in perinatal infection. Using multi-parameter flow cytometry, we examined immune activation (CD69, CD25, HLA-DR), and exhaustion (PD-1, TIGIT, LAG-3 and TIM-3) markers on CD4+ T cell subsets (naïve (Tn), central memory (Tcm), and a combination (Ttem) of transitional (Ttm) and effector memory (Tem) in 10 children living with HIV-1 (median age 15.9 years; median duration of virologic suppression 7.0 years), in whom HIV-1 reservoir size was determined with both the Intact Proviral HIV-1 DNA assay (IPDA) and the Tat/Rev limiting dilution assay (TILDA). Total HIV-1 DNA in CD4+ T cells was also measured. Correlations between immune activation, and exhaustion markers on T cell subsets with the various markers of proviral reservoir size, and baseline CD4+ T cell transcriptomes were examined. The median total HIV-1 DNA concentration was 211.9 copies per million CD4+ T cells, with a median intact proviral load in individuals with HIV-1 subtype B of 8.0 copies per million CD4+ T cells. Levels of HLA-DR and TIGIT on Ttem were strongly correlated with total HIV-1 DNA (r=0.758, p=0.015) and (r=0.721, p=0.023), respectively, but not with intact proviral load or inducible reservoir size. HIV-1 DNA load was also positively correlated with transcriptional clusters associated with HLA-DR. In contrast, PD-1 expression on Tcm was inversely correlated with both total HIV-1 DNA (r=-0.67, p=0.039) and HLA-DR in Ttem (r=-0.89, p=0.060). Gene expression profiles for HLA-DR and PD-1 were also inversely correlated. In conclusion, with virologically suppressed perinatal HIV-1 infection, HLA-DR and TIGIT on Ttem CD4+ T cells correlate with total HIV-1 DNA, and may serve as immune biomarkers for transcriptionally active proviruses, including defectives persisting on ART. RNA-seq from CD4+ T cells, from individuals with perinatally-acquired HIV-1.

静息记忆CD4+ T细胞的HIV-1感染是阻碍HIV-1治愈的关键障碍。识别与HIV-1储存库大小相关的免疫生物标志物，有助于评估HIV-1根除策略的有效性，尤其适用于血液采样受限的儿童感染人群。在成人中，中枢记忆CD4+ T细胞（central memory CD4+ T cells, Tcm）表面的免疫耗竭标志物PD-1与HIV-1储存库大小呈相关性，但围产期感染中，与HIV-1储存库大小相关的免疫因素尚不清楚。本研究采用多参数流式细胞术，对10名HIV-1感染儿童（中位年龄15.9岁；中位病毒学抑制时长7.0年）进行了检测，分析了CD4+ T细胞亚群——初始T细胞（naïve T cells, Tn）、中枢记忆T细胞（central memory CD4+ T cells, Tcm），以及过渡性T细胞（transitional T cells, Ttm）与效应记忆T细胞（effector memory T cells, Tem）的混合亚群（Ttem）——表面的免疫活化标志物（CD69、CD25、HLA-DR）及耗竭标志物（PD-1、TIGIT、LAG-3、TIM-3）的表达水平。上述受试者的HIV-1储存库大小同时通过完整前病毒HIV-1 DNA检测法（Intact Proviral HIV-1 DNA assay, IPDA）与Tat/Rev极限稀释试验（Tat/Rev limiting dilution assay, TILDA）进行了测定，同时还检测了CD4+ T细胞中的总HIV-1 DNA水平。研究分析了T细胞亚群表面的免疫活化、耗竭标志物与前病毒储存库大小的各项指标，以及基线CD4+ T细胞转录组之间的相关性。结果显示，CD4+ T细胞中的总HIV-1 DNA中位浓度为每百万CD4+ T细胞211.9拷贝；HIV-1 B亚型感染者的完整前病毒载量中位值为每百万CD4+ T细胞8.0拷贝。Ttem亚群表面的HLA-DR与TIGIT水平分别与总HIV-1 DNA呈强正相关（r=0.758, p=0.015）与（r=0.721, p=0.023），但与完整前病毒载量或可诱导储存库大小无显著相关性。HIV-1 DNA载量还与HLA-DR相关的转录簇呈正相关。与之相反，Tcm亚群表面的PD-1表达与总HIV-1 DNA（r=-0.67, p=0.039）及Ttem亚群表面的HLA-DR表达水平（r=-0.89, p=0.060）均呈负相关。HLA-DR与PD-1的基因表达谱也呈负相关。综上，在病毒学抑制的围产期HIV-1感染者中，Ttem CD4+ T细胞表面的HLA-DR与TIGIT表达水平与总HIV-1 DNA相关，可作为转录活性前病毒（包括抗逆转录病毒治疗（antiretroviral therapy, ART）持续存在的缺陷型前病毒）的免疫生物标志物。本数据集包含围产期获得性HIV-1感染者CD4+ T细胞的RNA测序（RNA-seq）数据。