Study design and participants
This cross-sectional study was conducted at the acquired immune deficiency syndrome (AIDS) clinical care centre of Beijing Ditan Hospital from March 2018 to June 2019. To be eligible to participate in the study, MSM had to be male, aged 20-40 years, have had sexual contact with men in the previous 12 months and were willing to provide written informed consent. All participants had a confirmed serological diagnosis of HIV and about to commence antiretroviral therapy (ART-naive). Using frequency-matching by sex and 5-year age categories, healthy volunteers were recruited as control group (HCs) from participants confirmed to be HIV-seronegative attending an HIV prevention trial.
The exclusion criteria consisted of acute or chronic HBV or HCV infection, immune inflammatory diseases, advanced stages of severe chronic diseases, such as atherosclerotic disease, congestive heart failure, poorly controlled diabetes mellitus, renal or hepatic disease, chronic obstructive pulmonary disease, as well as individuals under immunosuppressive therapy. This study was approved by the Ethics Committee of the Beijing Ditan Hospital. Written informed consent was obtained from all participants.
Fasting venous blood samples were collected. CD4 and CD8 T cell counts (cells/ul) were determined in all subjects, while HIV-1 viral load (HIV-RNA, copies/ml) were determined only in HIV-infected subjects. Absolute CD4 and CD8 T cell counts were determined using the flow cytometer-500 (Beckman Coulter Inc CA), according to the manufacturer instructions. Plasma HIV-1 RNA levels were determined using the COBAS Taqman HIV-1 test (Roche, Branchburg, New Jersey, USA) according to the manufacturer’s instructions. The detection limit was 40 HIV RNA copies/mL.
Flow Cytometry analysis
All analyses were performed using fresh peripheral blood mononuclear cell (PBMC) samples, isolated by density centrifugation using Ficoll-Hypaque (Amersham Biosciences, Amersham, Buckinghamshire, United Kingdom) from 20 ml EDTA venous blood. The following monoclonal antibodies were used for T cell immunophenotyping: CD4-APC-CY7, CD8-FITC, CD28-APC, CD45RA-PE-CY7, CCR7-PERCP-CY5.5, CD27-AmCyan, HLA-DR-Pacific Blue, and CD57-PE(BD Biosciences, San Jose, CA). Combinations of CD45RA and CD27 are commonly employed to define four different T cell subsets: 1) CD45RA+CD27+ [naive T cells (TN)]; 2) CD45RA-CD27+[central memory T cells (TCM)]; 3) CD45RA-CD27-[effector memory T cells (TEM)]; and 4) CD45RA+CD27-[terminally differentiated memory T cells (TemRA)]. Immune activation on CD4 and CD8 cell subsets was characterized by by HLA-DR expression. Senescence on CD4 and CD8 cell subsets was assessed by CD57 and CD28 expression (CD57+CD28- measuring senescent T lymphocytes). Fluorescence was measured with a FACS Canto II (BD Biosciences, Breda, the Netherlands). A total of 100,000 events were collected in the lymphocyte gate using morphological parameters (forward and side-scatter). The raw data were analysed using a fluorescence activated cell sorter (FACS) Diva version 5 (BD Biosciences, Heidelberg, Germany) and FlowJo software (TreeStar Inc., Ashland, OR, USA).
Isolation of naive and memory CD8 T cells
CD8 T cells were obtained by MACS selection from fresh PBMCs using negative selection MACS-kits according to manufacturer’s instructions (CD8 T cell Isolation Kit; Miltenyi). In a second step, CD45RA+ and CD45RO+ T cells were obtained via positive selection using CD45RA or CD45RO Microbeads (Miltenyi). The purity of the naive CD8 (CD45RA+) and memory CD8(CD45RO+）was ≥95% as analyzed by flow cytometry. Isolated CD8 T cell subpopulations from both the patients and controls were frozen in 90% FCS/10% DMSO in the vapor phase oliquid nitrogen until future use.
DNA extraction and measurement of telomere length by quantitative real-time PCR
After isolating naive and memory CD8 T cells, DNA was extracted using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturers’ instructions. The concentration and purity of the DNA was quantified using a Nanodrop Spectrophotometer (ThermoFisher Scientific, Waltham, Massachusetts, USA). The telomere length was determined with the widely used quantitative real-time PCR(qPCR) method. The relative telomere length was measured as the ratio of standard DNA quantities for telomere template (T) over single copy gene(SCG), human beta-globin. The telomere and SCG gene primers were as follows: (Tel-forward,5’-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3’; Tel reverse, 5’-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3’) for telomere PCR and (SCG-forward, 5’-GCTTCTGACACAACTGTGTTCACTAGC-3’; SCG-reverse, 5’-CACCAACTTCATCCACGTTC-ACC-3’) for the SCG PCR. All samples were blindly and consecutively run in triplicate together with reference samples. The inter assay coefficient of variation was <5%. The formula to convert T/S ratio to base pairs was 3,274 + 2,413*(T/S). The conversion from T/S ratio to bp was calculated based on a comparison of telomeric restriction fragment length from Southern blot analysis and T/S ratios using DNA samples from the human diploid fibroblast cell line IMR90 at different population doublings.
All continuous variables were expressed as the median with an interquartile range(IQR) or mean with standard deviation(SD), as appropriate. All groups were tested for the normal distribution with a Kolmogorov-Smirnov test. Differences between groups were analyzed by Student’s t-test or the Mann-Whitney U test according to data distribution. All statistical analysis was carried out using Graphpad Prism version 5 (GraphPad, La Jolla, CA, USA) and SPSS 22.0 (College Station, TX, USA) software. In the graphs, the P values are indicated as follows: * < 0.05; ** < 0.01; *** < 0.001.