Study Design
Eight 4-6 year-old, male and female pathogen-free (SPF) rhesus monkeys (Macaca mulatta) were housed and cared for in accordance with the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animal Science and the recommendations of the Weatherall report for the use of non-human primates in research (http://www.acmedsci.ac.uk/more/news/the-use-of-non-human-primates-in-research/) in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility. Macaques were intravenously infected with 100 tissue culture infective doses (TCID50) of SIVmac239 as described 19. All animal procedures and experiments were performed according to protocols approved by the IACUC of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (No. XJ19005). All animals were anesthetized with ketamine hydrochloride (10 mg/kg) prior to sample collection and experiments were performed in a biosafety level 3 laboratory.
ART was initiated 98 days after infection and continued for three months. The ART regimen consisting of two reverse transcriptase inhibitors, 5 mg/ml tenofovir disoproxil fumarate (TDF) and 40 mg/ml emtricitabine (FTC), plus 2.5 mg/ml of the integrase inhibitor dolutegravir (DTG), was subcutaneously administered once daily at 1 ml/kg body weight 20. The eight monkeys were followed up three months after discontinuation of ART. As part of the longitudinal observation, the effect of SIV RNA and total viral DNA on CD4 T-cell counts, immunocyte subsets and cytokines was measured at the indicated time points (Fig. 1).
Quantification of SIV RNA and total SIV DNA
Viral RNA (vRNA) was isolated from plasma using a QIAamp viral RNA mini kit (Qiagen, Valencia, CA, United States). Total viral DNA (vDNA) was extracted from monkey peripheral blood mononuclear cells (PBMCs) using a QIAamp blood DNA mini kit (Qiagen, Valencia, CA, United States) as previously reported 19. Viral RNA was subjected to quantitative real-time reverse transcription-PCR (qRT-PCR) on an ABI 9700 real-time PCR system (Applied Biosystems) using the following primers and probe: Gag91 forward primer: 5¢–GCA GAG GAG GAA ATT ACC CAG TAC–3¢; Gag91 reverse primer: 5¢–CAA TTT TAC CCA GGC ATT TAA TGT T–3¢; Probe: 5’-(FAM)-ACC TGC CAT TAA GCC CGA—(MGB)-3’. The copy numbers were estimated by comparison to a pGEM-SIV gag477 standard curve. The limits of detection were 100 copy equivalents of RNA or DNA per ml of plasma. Triplicate test reactions were performed for each sample.
Flow cytometry
Aliquots (50 μl) of EDTA-treated whole blood were stained with monoclonal antibodies to CD3 PerCP (SP34-2, BD Biosciences, 552851), CD4 FITC (OKT-4, Biolegend, 317408), and CD8 PE (RPA-T8, BD Biosciences, 555367). CD4+ T-cell counts were determined with BD Trucount tubes according to the manufacturer′s instructions (BD Biosciences, CA, USA). PBMCs were isolated using conventional Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Uppsala, Sweden). Polychromatic flow cytometry was performed to quantitate activated CD4+ or CD8+ T lymphocytes (Suppl. Fig. 1) and CD4+ or CD8+ memory T lymphocyte subsets (Suppl. Fig. 2). Activated or memory T lymphocyte subsets (Table 1) from 1×106 PBMCs were stained with anti-CD3 BV605 (SP34-2, BD Biosciences, 562994), anti-CD4 BV711 (OKT-4, Biolegend, 317440), anti-CD8 PE (RPA-T8, BD Biosciences, 557086), anti-CCR7 BV421 (G043H7, Biolegend, 352208), anti-CD45RA APC (5H9, BD Biosciences, 561210), anti-CD38 FITC (AT-1, Stemcell, 60131FI), anti-HLA-DR BV510 (G46-6, BD Biosciences, 563083), and anti-PD-1 PerCP-cy5.5 (EH12.2H7, Biolegend, 329914) monoclonal antibodies. Cells were resuspended in 1% paraformaldehyde, subjected to flow cytometry within 24 h on a FACSAriaII (BD Biosciences, CA, U.S.) and analyzed using FlowJo V10 software.
Multiplex analysis using Luminex
Blood samples were centrifuged for 10 min at 600 ×g, and serum was immediately aliquoted and stored at ˗80°C. The following 11 cytokines were measured with a Luminex kit according to the manufacturer's instructions: IL-1β, IL-2, IL-6, IL-8, IL-10, IFN-γ, MCP-1, MIP-1β, TNF-α (Merck Millipore, Billerica, Massachusetts, USA, PRCYTOMAG-40K-09C), TGF-β (Merck Millipore, Billerica, Massachusetts, USA, PRCYTOMAG-40K-09C), and IP-10 (EPX01A-40284-901, Carlsbad, CA, USA). After thawing the samples on ice and sufficient mixing, 25 μl of supernatant was loaded into each well of a 96-well plate and mixed with 25 μl of assay buffer and 25 μl of magnetic beads. The plates were incubated with agitation overnight at 4°C. After washing, 25 μl of detection antibody was added to each well and the plate was incubated 1 h at room temperature (RT). Then, 25 μl of streptavidin-PE was added to each well and incubated for 30 min at RT. Next, 150 μl sheath fluid was added to each well after washing. Plates were read on a Luminex® 200 (Bio-Rad, CA, USA) and the data analyzed for median fluorescent intensity (MFI) using a five-parameter logistic method for calculating analyte concentration.
Statistical analysis
Comparisons between the two groups were determined using paired t-tests. Comparison of quantitative variables was assessed with Friedman’s test. The Spearman rank test was used to determine correlations. All data were analyzed using GraphPad Prism 9.0 software (GraphPad Software Inc., San Diego, CA, United States). Significance was set at * p<0.05, ** p<0.01, and *** p<0.001.