HIV infection is characterized by progressive CD4 depletion, associated with persistently high levels of T-cell turnover . HIV-2 infection, as compared to HIV-1 infection, is associated with a much slower CD4 decline and, consequently, prolonged disease length . Nevertheless, when matched for the degree of CD4 depletion, HIV-2 infected (HIV-2+) and HIV-1-infected (HIV-1+) individuals show similar levels of T-cell activation and cycling [3,4]. In HIV-1+ patients there is evidence of extensive CD8 replicative history with telomere shortening [5–7], though data on CD4 T cells remain controversial [8–10].
We investigated, for the first time, whether increased CD4 turnover and prolonged chronic inflammation in HIV-2+ individuals lead to significant telomere attrition. CD4 telomere length was estimated by three-colour flow-fluorescence in-situ hybridization (FISH) in thawed peripheral blood mononuclear cells (PBMCs), as previously described . Blood samples were obtained after written consent and the study was approved by the Ethical Board of the Faculty of Medicine of the University of Lisbon.
HIV-2 and HIV-1 cohorts featured comparable CD4 depletion (HIV-2: 647 ± 84 cells/μl, 28.3 ± 2.8%; HIV-1: 530 ± 94 cells/μl, 21.3 ± 2.5%; and seronegatives: 950 ± 71 cells/μl; 42.7 ± 1.9%; P < 0.01 for both HIV cohorts as compared to seronegatives) and hyper-immune activation (HIV-2: 4.4 ± 0.9%, HIV-1: 7.4 ± 1.1%, and seronegatives: 1.6 ± 0.2% for frequency of HLA-DR+CD38+ in CD4; HIV-2: 16.1 ± 2.3%, HIV-1: 19.5 ± 2.8%, and seronegatives: 5.4 ± 1.1% for frequency of HLA-DR+CD38+ in CD8; P < 0.01 for both HIV cohorts as compared to seronegatives), but viraemia was strikingly lower in HIV-2+ patients, who included 11 antiretroviral therapy (ART)-naive individuals with undetectable viraemia (HIV-2: 5.8 × 103 ± 1.5 × 103 and HIV-1: 6.6 × 105 ± 6.5 × 105 RNA copies/ml).
We found no significant differences in telomere length of circulating CD4 from HIV-2+ and seronegative individuals (Fig. 1a). As expected , and validating the assay, memory CD4 showed significantly shorter telomeres than naive in both cohorts (Fig. 1a; P < 0.0001). Of note, telomere length of naive and memory CD4 did not differ between HIV-2 and seronegative cohorts. Thus, despite the prolonged length of HIV-2 infection  and the evidence of increased immune activation, no major CD4 telomere attrition was found.
Conversely, HIV-1+ individuals featured significantly decreased CD4 telomere length, as compared to seronegative controls (Fig. 1a), in close association with the frequency of circulating CD4 T cells (r = 0.4320; P = 0.0447).
HIV-2+ individuals tended to be older (HIV-2: 49 ± 2 years; HIV-1: 44 ± 3 years; seronegatives: 43 ± 3 years; not statistically different), a factor known to be associated with shorter telomeres , stressing the relevance of our finding of preserved telomere length in the HIV-2 cohort. Additionally, and in agreement with epidemiological trends , our HIV-2 cohort was enriched in non-Caucasians (12/26; HIV-1: 1/22; seronegatives: 1/23) and females (13/26; HIV-1: 5/22; seronegatives: 18/23). As ethnicity and sex have also been described to impact on telomere length, although with conflicting results [15–17], linear regression and one-way analysis of variance were performed, which indicated a lack of significant effects of age, ethnicity and/or sex on the results obtained for telomere length in our HIV-2 cohort (P > 0.05).
Of note, the overall reduction of CD4 telomere length in HIV-1 infection seemed to be related to preferential telomere erosion in the naive subset (Fig. 1a), with no differences being observed regarding memory CD4.
HIV infection is associated with progressive naive CD4 loss. The ability to sustain this subset is thought to affect the size and diversity of the overall CD4 pool, possibly contributing to slower HIV disease progression. Both HIV cohorts exhibited a decline of naive cells within total CD4, which was particularly marked in advanced HIV-1 infection (<300 CD4 T cells/μl) (Fig. 1b). Accordingly, in HIV-1+ individuals, the proportion of naive cells was inversely associated with CD4 depletion (CD4 cell counts: r = 0.7134, P = 0.0002; %CD4: r = 0.8080, P < 0.0001) and T-cell activation (% of HLA-DR+CD38+ in CD4: r = −0.8512, P < 0.0001; and in CD8: r = −0.4704, P = 0.0272), which did not occur in the HIV-2+ cohort (P > 0.05 for all the aforementioned parameters).
Notably, HIV-1+ individuals in advanced disease stage (<300 CD4 T cells/μl) featured the lowest naive CD4 telomere length (Fig. 1c). All but one of these patients were on long-term ART (44 ± 8months, n = 8), with undetectable viraemia, despite poor immunological recovery, and have been included in a previously described ART-discordant cohort [3,18]. Our findings are in agreement with the reported impairment of thymic function that has been linked with discordant responses to ART in HIV-1 infection [19,20]. On the contrary, our data suggest that, in advanced HIV-2 infection, there is a better maintenance of naive CD4 telomere length (Fig. 1c). The sustainability of the naive compartment may be related to a relatively preserved thymic activity during HIV-2 infection, as we reported . Of note, the HIV-2 cohort also included 12 ART-treated patients, that, as previously described , showed significant CD4 depletion (360 ± 69 cells/μl; 6/12 patients with <300 CD4 T cells/μl) despite low viremia (undetectable in 9/12 patients), in agreement with the poor immunological recovery upon ART usually observed in HIV-2 infection [2,22–24].
Naive CD4 homeostasis also relies on peripheral homeostatic proliferation . The frequency of cycling cells within the naive CD4 pool, estimated by the cell cycle marker Ki-67 as previously described , was markedly increased in both HIV cohorts, in relation to seronegatives (Fig. 1d), although significantly lower than those observed within memory CD4 cells (HIV-2: 18.1 ± 2.1%; HIV-1: 18.2 ± 2.8%; seronegatives: 8.9 ± 0.7%; P < 0.0001 for the comparison between frequency of cycling naive and memory CD4 in all cohorts; P < 0.001 for the comparison of proportion of cycling memory cells between both HIV and seronegative cohorts). The proportion of cycling cells within naive CD4 was more noticeably elevated in patients with advanced HIV-1 infection (<300 CD4 T cells/μl), reaching statistical significance both in comparison with seronegatives and corresponding early-stage disease group (Fig. 1d).
Thus, in HIV-1+ individuals with advanced disease, maintenance of naive CD4 seemed to critically depend upon homeostatic peripheral expansion. In contrast, no significant increase in the frequency of cycling cells was observed in advanced HIV-2 disease with similar levels of immune activation .
Our data further support a contribution of thymopoiesis throughout the course of HIV-2 infection that may help limit the degree of telomere attrition in the naive CD4 compartment. The latter could also be related to maintained telomerase activity in HIV-2 infection. This possibility warrants further investigation in HIV-2+ patients, given the evidence indicating their preserved responsiveness to IL-7 , an important telomerase inducer .
Overall, this study revealed that prolonged HIV-2 infection is associated with preserved CD4 telomere length, despite increased immune activation and lymphocyte turnover.
We gratefully acknowledge the collaboration of the following colleagues: Emília Valadas, Francisco Antunes, Manuela Doroana, Margarida Lucas, Sara Sousa and Inês Filipa Martins.
Author contributions: R.T. designed and performed experiments, analysed data and wrote the paper; A.S.A., R.B.F., R.C., R.S.S., A.P.B., M.V.D.S. and P.G. performed experiments; A.E.S. designed the research and wrote the paper.
Financial support: This work was supported by grant (PTDC/SAU-MIC/109786/2009) from ‘Fundação para a Ciência e a Tecnologia’ (FCT) and by ‘Programa Operacional Ciência e Inovação 2010’ (POCI2010), as well as by Fundação Calouste Gulbenkian to A.E.S. R.T., A.S.A., R.B.F., R.C. and R.S.S. received scholarships from FCT.
Conflicts of interest
Disclosures: The authors have no conflicting financial interests.
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