HIV-1 infection exerts a differential impact on CD4 and CD8 T cells, causing fundamental changes in the lifecycle of CD4 but not of CD8 T cells [1–4]. This ultimately results in the preferential depletion of the CD4 cell compartment whereas the CD8 T cells keep expanding until late in the course of infection.
Immune reconstitution following highly active antiretroviral therapy (HAART) is characterized by distinct CD4 and CD8 T cell dynamics, often displaying dichotomist trends according to disease stage [5,6]. Therefore, it is intriguing to speculate that CD4 and CD8 T cells posses inherent biological differences in their peculiar regenerative dynamics , which could become manifest under both endogenous and exogenous perturbations of physiological homeostasis [8–10].
Interleukin-2 (IL-2) is the main T cell growth factor, leading to proliferation and differentiation of the whole T lymphocyte compartment. However, the hallmark of immune reconstitution following low-dose IL-2 adjuvant therapy in HIV-positive patients is the selective expansion of CD4 T cells [11–13], ultimately resulting in the relative outgrowth of this T cell subset.
To acquire deeper insight into the effect of IL-2 immunotherapy on T cell homeostasis, the reconstitution dynamics of CD4 and CD8 T cells were compared in patients treated with IL-2 plus HAART and those treated with HAART alone (controls), with particular focus on cell proliferation and neothymic output. A better understanding of the contribution of cell turnover and neothymopoiesis to the shaping of the CD4 and CD8 IL-2-driven rescue will help to delineate the interplay between T lymphocyte subsets in HIV infection, thus providing an innovative framework for the most targeted and clinically advantageous immune-based strategies.
The trial included 15 HIV-positive immunological non-responders with HIV RNA < 50 copies/ml and CD4 T cell count constantly ≤ 200 × 106 cells/l after at least 12 months of stable HAART. The study was approved by the Ethical Committee of ‘Luigi Sacco’ Hospital, Milan, Italy. Eight patients received IL-2 and HAART and seven received HAART only. After three low-dose cycles of IL-2 (one cycle included 3 × 106 IU daily subcutaneously at days 1–5 and 8–12), for an overall duration of 10 weeks, all the patients were followed over 48 weeks . CD4, CD8, naive CD4+45RA+62L+, memory CD4+45RA− and activated CD8+CD38+ T lymphocytes were determined longitudinally by flow cytometry (Elite ESP, Coulter, Florida, USA) using CD4–peridinin chlorophyll protein, CD8–fluorescein isothiocyanate (FITC), CD45RA–FITC, CD62L–phycoerithrin (PE) and CD38–PE. After immunomagnetic sorting of CD4 and CD8 T cells from peripheral blood mononuclear cells (purity > 95%; CD4 and CD8 Positive isolation kit; Oxoid, Milan, Italy), neothymopoiesis was evaluated by measuring the VαJα coding joint in T cell receptor excision circles (TREC) with a polymerase chain reaction enzyme-linked immunosorbent assay . In order to minimize the dilution effect of cell turnover on TREC frequency, total TREC (expressed as TREC/μl blood) was calculated as (TREC/CD4 T cells) × (absolute number CD4 T cells). To take the effect of cell turnover into further account, cellular proliferation rate was evaluated by flow cytometric measurement of the expression of Ki67 nuclear antigen (clone MIB-1, Immunotech, Westbrook, Maine, USA) on purified CD4 and CD8 T cells, as previously described . All the parameters were analysed at four time points: before study initiation (week 0), at the end of IL-2 administration (week 10) and after 34 (week 34) and 48 weeks (week 48). Statistical analysis was performed by Wilcoxon signed rank test.
Figure 1 shows the schedule of dosing with low- dose prolonged intermittent IL-2 immunotherapy and HAART. There were no baseline differences between the eight patients receiving IL-2 and the seven receiving only HAART in clinical–epidemiological features , CD4 and CD8 counts, turnover and TREC (Fig. 2). In particular, both groups had comparable median age: IL-2 patients 36 years (range, 30–55) and HAART controls 37 years (range, 26–40).
Table 1 shows CD4/CD8 ratio, CD8+CD38+ activated T lymphocytes, naive and memory immunophenotype, in IL-2-receiving patients and in HAART-alone controls during the whole study period.
IL-2 immunotherapy resulted in a significant and sustained rise in both absolute and percentage CD4 T cells (weeks 10 and 48; P < 0.01) and CD4/CD8 cell ratio (weeks 10 and 48; P < 0.05), whereas CD8 count remained stable up to 48 weeks (P = 0.05) (Table 1; Fig. 2a–d). At week 48, CD4 T cells displayed a more relevant recovery than CD8 T cells in percentage terms (P < 0.05), with a tendency toward higher rescue also in absolute count (P = 0.069) (Fig. 2a–d). Conversely, HAART controls displayed a constrained yet parallel CD4 and CD8 recovery, reaching significance only in absolute value at 48 weeks (CD4 cells P < 0.01; CD8 cells P < 0.05), with no significant changes in CD4/CD8 ratio at all time points (P > 0.05) (Fig. 2a–d). At week 48, IL-2 patients experienced significantly higher recovery than the HAART-alone controls in CD4 cells (P < 0.01), but not in CD8 cells (P > 0.05) (Fig. 2a–d).
Since peripheral T cell pool is maintained by both proliferation and renewal, the contribution of cell turnover and neothymopoiesis to both subsets was examined. At baseline, overall Ki67 expression was significantly higher in CD8 cells (4%) than in CD4 cells (1.6%) (P < 0.01), confirming data describing relatively higher turnover rates within the CD8 subset [15,16] as a possible on-going response to low-level viral replication despite successful HAART [17,18]. IL-2 induced a significant rise in Ki67+CD4+ at week 10, from 2.1% to 3.4% (P < 0.05), but not in Ki67+CD8+ (P > 0.05). At IL-2 discontinuation, both CD4 and CD8 T cell turnover progressively dropped to below pretreatment values (Fig. 2e) . However, in HAART-alone controls, the frequency of proliferating CD4 and CD8 were closely matched at all time points (Fig. 2f).
To evaluate the role played by IL-2 on CD8 T cell dynamics further, the CD8 T cell turnover data was integrated by studying the CD8+CD38+ (activated) lymphocytes . At baseline, IL-2-receiving patients and controls displayed comparable proportions (P > 0.05) (Table 1). Similar to the effect on CD8 cell turnover, IL-2 did not induce an elevation in the percentage of CD8+CD38+ T lymphocytes (week 10; P > 0.05), which indeed progressively decreased, reaching significance by week 34 (week 34 P < 0.05; week 48 P < 0.01) [21,22]; no changes were observed in the HAART controls (week 48; P > 0.05) (Table 1).
Analysis of individual CD4 subsets showed a discrepant pattern of recovery between IL-2-patients and HAART-alone controls. In particular, IL-2 immunotherapy induced a significant and sustained expansion of both CD4+CD45RA+CD62L+ (naive) (week 10 and 48; P < 0.05) and CD4+CD45RA− (memory) (week 10 and 48; P < 0.05) subsets (Table 1), whereas HAART alone resulted in a preferential rescue of memory CD4 cells (week 48; P < 0.05) with no significant changes in the naive compartment (week 48; P > 0.05) (Table 1). At week 48, IL-2 patients experienced higher naive and memory CD4 recovery compared with controls (P < 0.01) (Table 1).
Having demonstrated that IL-2 immunotherapy induces a preferential expansion of CD4 T cells [11-13], a large proportion of which bear a naive phenotype, the impact of neothymic output on both CD4 and CD8 subsets was analysed. As with other parameters, CD4 and CD8 T cells displayed a dichotomy in TREC content, with a temporary tendency to increase at week 10 in CD4 cells (from 4.1 × 106 to 6.5 × 106 copies/μl; P = 0.08) and no changes within CD8 pool (P > 0.05) (Fig. 2g). As TREC signal is affected by proliferative dilution [23,24], the concurrent rise in CD4 TREC and turnover strongly suggests a more substantial actual increase in CD4 neothymopoiesis. No significant CD4 and CD8 TREC changes were seen in HAART controls at all time points (P > 0.05) (Fig. 2h). Overall, at week 10, IL-2-receiving patients experienced a more relevant CD4 TREC increase than did HAART controls (P < 0.05), whereas no significant differences were shown in CD8 TREC (P > 0.05) (Fig. 2g,h).
This study investigated the long-term kinetics of CD4 and CD8 T cells in a cohort of HIV-positive patients treated with low-dose prolonged intermittent IL-2 immunotherapy compared with patients receiving HAART alone. The results show different IL-2-driven dynamics in CD4 and CD8 subsets, clearly demonstrating that IL-2 has a differential impact on these T lymphocyte subsets, with a preferential action on CD4 T cells. In particular, we confirm previous data pointing to enhanced proliferation of existing cells as the mechanism responsible for the IL-2-driven CD4 reconstitution [25,26]. Moreover, our data suggest a possible temporary boost of neothymopoiesis: the actual increase may be masked somewhat here by the increased proliferation. The disproportionate rise of proliferating CD4 versus CD8 cells  could be explained by both in vitro higher expression of IL-2 receptor mRNA in CD4 than in CD8 T cells upon activation , and the more significant in vivo IL-2-driven upregulation of IL-2 receptor α-chain (CD25) on CD4 [28,29]. By showing a preferential impact of IL-2 on CD4 neothymic synthesis, our data add to the evidence that successful CD4 reconstitution is dependent upon neothymopoiesis [7,8] and suggests a possible role of IL-2 in selectively reconstituting T helper activity to neoantigens , given that TREC increases have indeed been correlated with improved neoantigen immune function . Moreover, our data show that IL-2 does not affect CD8 cell turnover and thymopoiesis, thus implying that CD8 T cells follow different homeostatic dynamics [7,8], possibly peripheral redistribution. The broad examination of T lymphocyte dynamics during IL-2 immunotherapy provides a more detailed definition of the protean interactions between specific T lymphocyte subsets during immune reconstitution; this will be of great value in the design of immune-based strategies in the treatment of HIV infection.
We are grateful to Shane Fodgen, Greta Di Felice and Anna Cristina Sacchetta for critical reading of the manuscript and valuable advice; Maura Mezzetti (Istituto dei Metodi Quantitativi, Università ‘Luigi Bocconi', Milan) for assistance with statistical analysis; Chiron Italia for valuable advice; Bianca Ghisi for excellent typing assistance; all patients participating in the study; and Patrizia Franza, Maria Tomminello and all the staff at the Institute of Infectious Diseases and Tropical Medicine, ‘L. Sacco’ Hospital, who cared for the patients.
1. Wolthers K, Bea G, Wisman A, Otto S, de Roda Husman A, Schaft N, et al
. T cell telomere length in HIV-1 infection: no evidence for increased CD4+ T cell turnover. Science
2. Wolthers K, Noest A, Otto S, Miedema F, de Boer R. Normal telomere lengths in naive and memory CD4+ T cells in HIV type 1 infection: a mathematical interpretation. AIDS Res Hum Retroviruses
3. Grossman Z, Heberman R, Vatnik N, Intrator N. Conservation of total T-cell counts during HIV infection: alternative hypotheses and implications. J Acquir Immune Defic Syndr Hum Retrovirol
4. Ribeiro R, Mohri H, Ho D, Perelson A. In vivo dynamics of T cell activation, proliferation and death in HIV-1 infection: why are CD4+ but not CD8+ T cells depleted? Proc Natl Acad Sci USA
5. Autran B, Carcelain G, Li T, Blanc C, Mathez D, Tubiana R, et al
. Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science
6. Stuart J, Hamann D, Borleffs J, Roos M, Miedema F, Boucher C, et al
. Reconstitution of naive T cells during antiretroviral treatment of HIV-infected adults is dependent on age. AIDS
7. Douek D, Picker L, Koup R. T cell dynamics in HIV-1 infection. Annu Rev Immunol
8. Mackall C, Fleisher T, Brown M, Andrich M, Chen C, Feuerstein I, et al
. Distinctions between CD8+ and CD4+ T-cell regenerative pathways result in prolonged T-cell subset imbalance after intensive chemotherapy. Blood
9. Homann D, Teyton L, Oldstone M. Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory. Nat Med
10. Foulds K, Zenewicz L, Shedlock D, Jiang J, Troy A, Shen H. Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J Immunol
11. Arno A, Ruiz L, Juan M, Jou A, Balague M, Zayat M, et al
. Efficacy of low-dose subcutaneous interleukin-2 to treat advanced human immunodeficiency virus type 1 in persons with ≤ 250/μl CD4 T cells and undetectable plasma virus load. J Infect Dis
12. Marchetti G, Meroni L, Varchetta S, Terzieva V, Bandera A, Manganaro D, et al
. Low-dose prolonged intemittent interleukin-2 adjuvant therapy: results of a randomized trial among human immunodeficiency-virus positive patients with advanced immune impairment. J Infect Dis
13. Katlama C, Carcelain G, Duvivier C, Chouquet C, Tubiana R, De Sa M, et al
. Interleukin-2 accelerates CD4 cell reconstitution in HIV-infected patients with severe immunosuppression despite highly active antiretroviral therapy: the ILSTIM study – ANRS 082. AIDS
14. Al-Harthi L, Marchetti G, Steffens C, Poulin J, Sekaly R, Landay A. Detection of T cell receptor circles (TRECs) as biomarkers for de novo T cell synthesis using a quantitative polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). J Immun Meth
15. Sachsenberg N, Perelson A, Yerly S, Schockmel G, Leduc D, Hirschel B, et al
. Turnover of CD4+ and CD8+ T lymphocytes in HIV-1 infection as measured by Ki-67 antigen. J Exp Med
16. Fleury S, Rizzardi G, Chapuis A, Tambussi G, Knabenhans C, Simeoni E, et al
. Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy. Proc Natl Acad Sci USA
17. Zaunders J, Cunningham P, Kelleher A, Kaufmann G, Jaramillo A, Wright R, et al
. Potent antiretroviral therapy of primary human immunodeficiency virus type-1 (HIV-1) infection: partial normalization of T lymphocyte subsets and limited reduction of HIV-1 DNA despite clearance of plasma viremia. J Infect Dis
18. Dyrhol-Riise A, Voltersvik P, Olofsson J, Asjö B. Activation of CD8 T cells normalizes and correlates with the level of infectious provirus in tonsils during highly active antiretroviral therapy in early HIV-1 infection. AIDS
19. Caggiari L, Zanussi S, Crepaldi C, Bortolin M, Caffau C, D'Andrea M, et al
. Different rates of CD4+ and CD8+ T-cell proliferation in interleukin-2–treated human immunodeficiency virus-positive subjects. Cytometry
20. Giorgi J, Liu Z, Hultin L, Cumberland W, Hennessey K, Detels R. Elevated levels of CD38+ and CD8+ T cells in HIV infection add to the prognostic value of low CD4+ T cell levels: results of 6 years of follow-up. The Los Angeles Center, Multicenter AIDS Cohort Study. J Acquir Immune Defic Syndr
21. Hecht F, Hare C, McGrath M, Liu L, Gascon R, Kahn J, et al
. Interleukin-2 in conjunction with HAART in early HIV infection increases naive and memory CD4 cells and lowers activation markers. Tenth Conference on Retroviruses and Opportunistic Infections
. Boston, February 2003 [abstract 649].
22. Sullivan A, Hardy G, Burton C, Pires A, Nelson M, Gotch F, et al
. Effect of IL-2 therapy on T-cell phenotype, activation, and IL-2 receptor expression. Tenth Conference on Retroviruses and Opportunistic Infections
. Boston, February 2003 [abstract 651].
23. Hazenberg M, Otto S, Cohen Stuart J, Verschuren M, Borleffs J, Boucher C, et al
. Increased cell division but not thymic disfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection. Nat Med
24. Hazenberg M, Stuart J, Otto S, Borleffs J, Boucher C, de Boer R, et al
. T-cell division in human immunodeficiency virus (HIV)-1 infection is mainly due to immune activation: a longitudinal analysis in patients before and during highly active antiretroviral therapy (HAART). Blood
25. De Paoli P, Bortolin M, Zanussi S, Monzoni A, Pratesi C, Giacca M. Changes in thymic function in HIV-positive patients treated with highly active antiretroviral therapy and interleukin-2. Clin Exp Immunol
26. Natarajan V, Lempicki RA, Sereti I, Badralmaa Y, Adelsberger JW, Metcalf JA, et al
. Increased peripheral expansion of naive CD4+ T cells in vivo after IL-2 treatment of patients with HIV infection. Proc Natl Acad Sci USA
27. Leung J, Lai C, Chui Y, Ho R, Chan C, Lai K. Characterization of cytokine gene expression in CD4+ and CD8+ T cells after activation with phorbol myristate acetate and phytohaemagglutinin. Clin Exp Immunol
28. David D, Bani L, Moreau J, Demaison C, Sun K, Salvucci O, et al
. Further analysis of interleukin-2 receptor subunit expression on the different human peripheral blood mononuclear cell subsets. Blood
29. Sereti I, Herpin B, Metcalf J, Stevens R, Baseler M, Hallahan C, et al
. CD4 T cell expansions are associated with increased apoptosis rates of T lymphocytes during IL-2 cycles in HIV infected patients. AIDS
30. Blattman J, Grayson J, Wherry E, Kaech S, Smith K, Ahmed R. Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo. Nat Med
31. Markert ML, Hicks CB, Bartlett JA, Harmon JL, Hale LP, Greenberg ML, et al
. Effect of highly active antiretroviral therapy and thymic transplantation on immunoreconstitution in HIV infection. AIDS Res Hum Retroviruses