All clinical study protocols were reviewed and approved by the Ethics Review Committee of China Medical University (CMU), Shenyang, P. R. China, and written informed consent from each participant in the study was obtained. We initially enrolled and evaluated 200 HIV-1–infected patients from the HIV patient cohort of the Key Laboratory of AIDS Immunology of the National Health and Family Planning Commission in The First Affiliated Hospital of CMU. Among the HIV-infected patients, 98 had no previous or current ART at the time of the p-cofilin profiling, and 102 had ongoing ART for over a year, but 4 of the ART-treated patients had a viral load greater than 1000 copies/ml and were excluded from the study for possible drug resistance. The CD4 T cell count and viral load of these subjects were measured every 3 months. One hundred age- and sex-matched HC were enrolled from the HIV voluntary counseling and testing center of CMU. A summary of the subjects is listed in tables S1 and S2. Of the ART-naïve patients, 65 eventually received ART at around 6 months after the p-cofilin profiling and were treated for more than a year. All of these patients receiving ART reached undetectable plasma HIV-1 RNA. ART-treated patients were further evaluated and categorized into IRs and INRs. Both IRs and INRs were treated with ART for more than 1 year. IRs were those who had a CD4 T cell recovery greater than 20% and a CD4 T cell count more than 500 cells/μl; INRs had a CD4 T cell recovery less than 20% or a CD4 T cell count less than 200 cells/μl. For isolating blood resting CD4 T cells from study subjects, peripheral blood mononuclear cells (PBMCs) were freshly obtained from the subjects and purified by Ficoll-Hypaque density gradient centrifugation, followed by negative isolation of resting CD4 T cells as previously described (9, 49). Briefly, monoclonal antibodies against human CD14, CD56, HLA-DR, CD8, CD11b, and CD19 (BD Biosciences, San Jose, CA) were used. Antibody-bound cells were depleted using Dynabeads Pan Mouse IgG (Thermo Fisher Scientific). Purified cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). One million resting CD4 T cells from each blood donor were lysed in 40 μl of SDS/T-PER extraction buffer [Novex Tris-Glycine SDS Sample Buffer, T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific), and 2.5% 2-mercaptoethanol (Sigma-Aldrich)]. Cell lysates were heated at 100°C for 8 min, immediately frozen and stored at −80°C, and then transported on dry ice to Theranostics Health (Gaithersburg, MD, USA) for p-cofilin reverse-phase protein microarray (RPPA) analyses. A total of 296 coded cell lysates were printed onto the microarrays and profiled; 3 lysates did not generate readable signals and were excluded from data analyses.

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