Publication Date:
2016-12-02
Description:
Introduction:Potent antiretroviral drugs block the progression of HIV infection and inhibit virus integration into the host DNA. Response to therapy is typically monitored by counting CD4+ T cells and measuring plasma viral load, which becomes undetectable in most patients. However, at present HIV cannot be eradicated and persists in the host. Robust data are needed to understand the importance and clinical meaning of the residual activity of the virus, and it is crucial to investigate the role of intracellular HIV reservoirs in patients with undetectable viremia. Since HIV establishes latent infection at different degrees within central memory (CM), effector memory (EM) or naïve (TN) CD4+ T cells, measuring the content of HIV-DNA in different lymphocyte populations is a novel approach to follow the infection. Similarly, the residual capacity of the host to reconstitute the immune system can be accurately monitored by measuring: i) the amount of cells expressing signal-joint (sj) T cell receptor (TCR) rearrangement excision circles (sjTREC), that are a marker of homeostatic proliferation, and ii) telomere length, for evaluating cell senescence. Thus, using flow cytometry and cell sorting along with a molecular biology approach based on droplet digital PCR (ddPCR), we have quantified proviral HIV DNA, the amount of sjTREC+ cells and telomere length in different subsets of CD4+ T cells, such as TN, CM and EM. Methods:According to the Declaration of Helsinki, after informed consent and approval by the local Ethical Committee, we enrolled 32 HIV+ patients (mean age 49.0±7.2 years, 11 females) successfully treated for 〉2 years, with a CD4+ T cell count 〉500 cells/uL and plasma viremia undetectable from at least one year. After staining with fluorochrome-labeled mAbs, TN, CM and EM CD4+ T cells were sorted with a S3e sorter (Bio-Rad, CA, USA) equipped with a specifically designed biosafety containment hood (Biobubble, UK). Cell purity was always 〉95%. Proviral HIV-DNA and sjTREC were quantified in each lymphocyte subset with QX200 droplet digital PCR (Bio-Rad); telomere length, expressed as relative T/S (the ratio between telomere length and single gene copy, related to CD178, i.e., Fas Ligand) was quantified with CFX9600 Real time PCR (Bio-Rad). Viro-immunological parameters such as CD4+ and CD8+ T cell counts and percentages, and viral load were collected for each withdrawal together with the clinical characteristics of patients. Results:Considering all patients, HIV proviral DNA, measured as LTR copies/1,000 cells, was significantly lower in TN cells (mean±SEM: 0.77±0.23) compared to CM (2.42±0.38) or to EM (2.34±0.33), with p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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