HIV-2 infection features a much slower course than HIV-1 infection, often asymptomatic for over 20 years, without antiretroviral therapy (ART). Nevertheless, CD4 T cells progressively decline, in direct correlation with immune activation and cell cycling. We report, for the first time, preserved telomere length within naive and memory CD4 subsets in prolonged HIV-2 infection despite the increased CD4 turnover.
aUnidade de Imunologia Clínica, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa
bCentro de Investigação Interdisciplinar Egas Moniz (CiiEM), Instituto Superior de Ciências da Saúde Egas Moniz, Caparica
cLaboratório de Biologia Molecular, Serviço de Medicina Transfusional, Centro Hospitalar Lisboa Ocidental, Hospital Egas Moniz
dCentro de Malária e Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
Correspondence to Ana E. Sousa, MD, PhD, Unidade de Imunologia Clínica, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal. Tel: +351 21 799 95 25; fax: +351 21 799 95 27; e-mail: firstname.lastname@example.org
Received 17 July, 2012
Revised 20 September, 2012
Accepted 24 September, 2012
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.
1. Grossman Z, Meier-Schellersheim M, Sousa AE, Victorino RM, Paul WE. CD4+ T-cell depletion in HIV infection: are we closer to understanding the cause?
. Nat Med
2. Drylewicz J, Matheron S, Lazaro E, Damond F, Bonnet F, Simon F, et al. Comparison of viro-immunological marker changes between HIV-1 and HIV-2-infected patients in France
3. Foxall RB, Albuquerque AS, Soares RS, Baptista AP, Cavaleiro R, Tendeiro R, et al. Memory and naive-like regulatory CD4+ T cells expand during HIV-2 infection in direct association with CD4+ T-cell depletion irrespectively of viremia
4. Sousa AE, Carneiro J, Meier-Schellersheim M, Grossman Z, Victorino RM. CD4 T cell depletion is linked directly to immune activation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viral load
. J Immunol
5. Effros RB, Allsopp R, Chiu CP, Hausner MA, Hirji K, Wang L, et al. Shortened telomeres in the expanded CD28-CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis
6. Pommier JP, Gauthier L, Livartowski J, Galanaud P, Boue F, Dulioust A, et al. Immunosenescence in HIV pathogenesis
7. Appay V, Rowland-Jones SL. Premature ageing of the immune system: the cause of AIDS?
. Trends Immunol
8. Nichols WS, Schneider S, Chan RC, Farthing CF, Daar ES. Increased CD4+ T-lymphocyte senescence fraction in advanced human immunodeficiency virus type 1 infection
. Scand J Immunol
9. Palmer LD, Weng N, Levine BL, June CH, Lane HC, Hodes RJ. Telomere length, telomerase activity, and replicative potential in HIV infection: analysis of CD4+ and CD8+ T cells from HIV-discordant monozygotic twins
. J Exp Med
10. Wolthers KC, Bea G, Wisman A, Otto SA, de Roda Husman AM, Schaft N, et al. T cell telomere length in HIV-1 infection: no evidence for increased CD4+ T cell turnover
11. Plunkett FJ, Soares MV, Annels N, Hislop A, Ivory K, Lowdell M, et al. The flow cytometric analysis of telomere length in antigen-specific CD8+ T cells during acute Epstein-Barr virus infection
12. Wolthers KC, Noest AJ, Otto SA, Miedema F, De Boer RJ. Normal telomere lengths in naive and memory CD4+ T cells in HIV type 1 infection: a mathematical interpretation
. AIDS Res Hum Retroviruses
13. Bekaert S, De Meyer T, Van Oostveldt P. Telomere attrition as ageing biomarker
. Anticancer Res
14. Carvalho AC, Valadas E, Franca L, Carvalho C, Aleixo MJ, Mendez J, et al. Population mobility and the changing epidemics of HIV-2 in Portugal
. HIV Med
15. Barrett EL, Richardson DS. Sex differences in telomeres and lifespan
. Aging Cell
16. Diez Roux AV, Ranjit N, Jenny NS, Shea S, Cushman M, Fitzpatrick A, Seeman T. Race/ethnicity and telomere length in the Multi-Ethnic Study of Atherosclerosis
. Aging Cell
17. Hunt SC, Chen W, Gardner JP, Kimura M, Srinivasan SR, Eckfeldt JH, et al. Leukocyte telomeres are longer in African Americans than in whites: the National Heart, Lung, and Blood Institute Family Heart Study and the Bogalusa Heart Study
. Aging Cell
18. Albuquerque AS, Foxall RB, Cortesao CS, Soares RS, Doroana M, Ribeiro A, et al. Low CD4 T-cell counts despite low levels of circulating HIV: insights from the comparison of HIV-1 infected patients with a discordant response to antiretroviral therapy to patients with untreated advanced HIV-2 disease
. Clin Immunol
19. Li T, Wu N, Dai Y, Qiu Z, Han Y, Xie J, et al. Reduced thymic output is a major mechanism of immune reconstitution failure in HIV-infected patients after long-term antiretroviral therapy
. Clin Infect Dis
20. Teixeira L, Valdez H, McCune JM, Koup RA, Badley AD, Hellerstein MK, et al. Poor CD4 T cell restoration after suppression of HIV-1 replication may reflect lower thymic function
21. Gautier D, Beq S, Cortesao CS, Sousa AE, Cheynier R. Efficient thymopoiesis contributes to the maintenance of peripheral CD4 T cells during chronic human immunodeficiency virus type 2 infection
. J Virol
22. Soares RS, Tendeiro R, Foxall RB, Baptista AP, Cavaleiro R, Gomes P, et al. Cell-associated viral burden provides evidence of ongoing viral replication in aviremic HIV-2-infected patients
. J Virol
23. Chiara M, Rony Z, Homa M, Bhanumati V, Ladomirska J, Manzi M, et al. Characteristics, immunological response & treatment outcomes of HIV-2 compared with HIV-1 & dual infections (HIV 1/2) in Mumbai
. Indian J Med Res
24. van der Ende ME, Prins JM, Brinkman K, Keuter M, Veenstra J, Danner SA, et al. Clinical, immunological and virological response to different antiretroviral regimens in a cohort of HIV-2-infected patients
2003; 17 (Suppl 3):S55–S61.
25. Azevedo RI, Soares MV, Barata JT, Tendeiro R, Serra-Caetano A, Victorino RM, Sousa AE. IL-7 sustains CD31 expression in human naive CD4+ T cells and preferentially expands the CD31+ subset in a PI3K-dependent manner
26. Albuquerque AS, Cortesao CS, Foxall RB, Soares RS, Victorino RM, Sousa AE. Rate of increase in circulating IL-7 and loss of IL-7Ralpha expression differ in HIV-1 and HIV-2 infections: two lymphopenic diseases with similar hyperimmune activation but distinct outcomes
. J Immunol
27. Soares MV, Borthwick NJ, Maini MK, Janossy G, Salmon M, Akbar AN. IL-7-dependent extrathymic expansion of CD45RA+ T cells enables preservation of a naive repertoire
. J Immunol