Activation, survival and apoptosis of CD45RO+ and CD45RO- T cells of human immunodeficiency virus-infected individuals: effects of interleukin-15 and comparison with interleukin-2

Abstract

HIV infection is associated with increased representation of T cells bearing an activated, memory (CD45RO+) phenotype. Although administration of antiretroviral agents and interleukin-2 (IL-2) augment depleted CD4+ T-cell numbers, such therapies have been preferentially beneficial for CD45RO+ T cells. Interleukin-15 (IL-15) exhibits many biological activities in common with IL-2, including promoting T-cell survival and proliferation. The present study found that these two cytokines differed in their ability to induce proliferation, enhance survival, and control apoptosis of CD45RO+ and CD45RO- T-cell populations of human immunodeficiency- (HIV) infected individuals. When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 in activating and stimulating proliferation of CD4+CD45RO+, CD8+CD45RO+ and CD8+CD45RO- cells, but failed to be more effective than IL-2 in reducing apoptosis. Poor activation of CD4+CD45RO- cells by IL-15 and to IL-2 appeared to be attributable to low expression of the beta receptor chain utilized by both cytokines. However, IL-15 was more effective than IL-2 in enhancing survival of the CD4+CD45RO- population, suggesting a greater protective effect of IL-15 for naive CD4+ T cells, which are preferentially lost in HIV-infected individuals.

Figure 1

Activation of CD45RO⁺ and CD45RO⁻ T-cell populations of HIV-infected individuals. PBMCs of 16 patients were incubated at 10⁶ cells/ml in RPMI-1640 medium (Sigma, St Louis, MO) supplemented as previously described¹⁹ without and with the addition of IL-2 and of IL-15 (R & D Systems, Abingdon, UK) at a final concentration of 10 ng/ml. As assessed by the CTLL-2 proliferation assay, the activity of IL-2 was 2·4 10³ U/μg and its ED₅₀ (O.2 ng/ml) was twofold that of IL-15. At Day 1, cells were stained as previously described¹⁹ with monoclonal antibodies (mAbs) against CD69 (IgG₁, clone L78), CD45RO (IgG₁, clone UCHL1), and CD4 (IgG₁, clone SK3) or CD8 (IgG₁, clone SK1) (Becton Dickinson, San Jose, CA). Three-colour immunofluorescence analysis was performed using a FACScan flow cytometer equipped with Cell Quest software (Becton Dickinson). Shown in (a) are representative FACScan profiles indicating percentages of CD69⁺ cells within CD45RO⁺ and CD45RO⁻ populations, gated on CD4⁺ and CD8⁺ subsets of one patient. Percentages of CD69⁺ cells within each population of the patient group are shown in (b). Statistical significance was assessed by the Wilcoxon’s signed-rank test for paired data. Horizontal bars depict median values.

Figure 2

Altered pool sizes of non-blastic, blastic and apoptotic cells in CD45RO⁺ and CD45RO⁻ populations. PBMCs were incubated for 3 days in the absence of exogenous cytokines, and with the addition of IL-2 and of IL-15 (10 ng/ml), followed by staining with anti-CD45RO mAbs and mAbs against CD4 and CD8. Shown are typical forward scatter (size) versus side scatter (granularity) profiles of cells gated on the indicated populations. Apoptotic cells (a) are defined by their small size and high granularity. Viable cells in cultures stimulated with IL-2 and with IL-15 included non-blastic cells (NB) and distinctly larger blastic cells (B), the latter being absent in cultures incubated without exogenous cytokines. The percentages of blastic cells within each gated population of the patient group (n = 16) are indicated (mean±SD).

Figure 3

Differential expression of the β and γ receptor chains. PBMCs of HIV-infected individuals were incubated overnight in the absence of exogenous cytokines and then stained with mAbs against the β-chain (IgG_2a, clone MIK-β) (CLB, Amsterdam, The Netherlands), CD45RO and either CD4 or CD8. Freshly isolated PBMCs from the same individuals were stained with mAbs against the γ-chain (IgG_2b, clone TUGh4) (Pharmingen, San Diego, CA), CD45RO and either CD4 or CD8. Shown are the percentages of cells expressing the β- and γ-chains within CD45RO⁺ and CD45RO⁻ populations of the CD4⁺ and CD8⁺ subsets. Statistical significance was assessed by the Wilcoxon’s signed-rank test for paired data. Horizontal bars depict median values. P > 0·05 was considered not significant (n.s.)

Figure 4

Protective effect of IL-15 and IL-2 against apoptosis. PBMCs were incubated without or with the addition of IL-15 or IL-2 and at Day 3 were stained with 7-AAD as previously described²⁰ and with mAbs against CD45RO and either CD4 or CD8. Shown in A are representative FACScan profiles indicating the percentages of 7-AAD-stained cells within the CD45RO⁺ and CD45RO⁻ populations gated on the CD4⁺ and CD8⁺ subsets of one patient. Percentages of apoptotic cells within each population of the patient group are shown in 9b). Statistical significance was assessed by the Wilcoxon’s signed-rank test for paired data. Horizontal bars depict median values. P > 0·05 was considered not significant (n.s.).