Journal: bioRxiv

Article Title: Identification of the cellular transcription factor KLF16 as a novel repressive epigenetic repressor of HIV-1 transcription

doi: 10.64898/2026.05.02.722432

Figure Lengend Snippet: (a) Experimental flowchart. Memory CD4 + T cells were isolated from PBMCs of PWOH were stimulated with α-CD3+α-CD28 antibodies for 3 days and then were nucleofected with non-targeting control (siNT) or KLF16-targeting (siKLF16) small interfering RNA (siRNA). Nucleofected cells were cultured in the presence of rhIL-2 (5 ng/ml) for 24 hours, then infected with VSV-G-pseudotyped HIV-1 encoding GFP (50 ng HIV-p24/10e6 cells) and cultured in the presence of rhIL-2 for an additional 3 days. (b) KLF16 mRNA expression was measured by RT-qPCR 24 hours post-nucleofection. (c) At day 3 post-infection, cells were harvested to quantify the levels of integrated HIV DNA by nested real-time PCR. (d) Levels of HIV-p24 capsid protein were measured by ELISA in cell culture supernatants collected at day 3 post-infection. Results were generated with cells from n=7 PWOH participants. (e)- (g) Memory CD4 + T cells isolated from PBMCs of PWOH (n = 9) or of ART-treated PWH (n = 9) were stimulated with α-CD3+α-CD28 antibodies or left unstimulated for 18 hours and used for the measurement of KLF16 mRNA expression. (e) KLF16 mRNA levels quantified by RT-qPCR in samples from PWOH and PWH ex vivo . (f) KLF16 mRNA levels quantified by RT-qPCR in samples from PWH upon TCR triggering in vitro . (g) KLF16 mRNA levels quantified by RT-qPCR in samples from PWOH upon TCR triggering in vitro . Statistical analysis methods and corresponding p values are indicated above each graph.

Article Snippet: Following isolation, CD4+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies (T Cell TransActTM, Miltenyi Biotec, #130-111-160) for three days and then infected by spinoculation (2h, 800xg, 32°C) with VSV-G-pseudotyped HIVGKO particles at a multiplicity of infection (MOI) of 3000, where GFP and mKO2 expression were used to distinguish between productively-infected and latently-infected cells.

Techniques: Isolation, Control, Small Interfering RNA, Cell Culture, Infection, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Generated, Ex Vivo, In Vitro