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<0.0001 in both cases. Conversely, TN cells contained a higher number of sjTREC copies/1,000 cells (11.62±1.54) compared to CM (0.99±0.23) or to EM (1.26±0.44); p<0.0001 in both comparisons. No appreciable changes were observed in T/S ratio among CD4+ subsets. The stratification of patients revealed that HIV content was significantly higher in TN and CM T cells of patients with shorter time of treatment. Similarly, stratifying for CD4/CD8 ratio revealed that those with lower ratio had more intracellular HIV. A similar trend was observed also in EM cells, even if it was not statistically significant. The CD4+ T cell count pre-therapy or CD4+ T cell nadir did not influence the HIV reservoir.

Conclusions: The importance of recovering a good CD4/CD8 ratio after successful antiretroviral therapy is further underlined by the fact that different amounts of virus were present and well measurable in different CD4+ T cell populations from patients with high or low ratio. The length of treatment was also significantly correlated to a better virological outcome. It is to note that in our patients homeostatic proliferation or cell senescence likely did not affect HIV reservoir. Linking highly purified cell sorting to a molecular biology approach based on ddPCR is crucial to understand what happens to the HIV reservoir when plasma viremia is suppressed. The measure of HIV DNA in selected lymphocyte populations not only will lead to a better comprehension of the features of HIV reservoir and its monitoring, but could also be useful for the development of eradication strategies.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.

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