The decrease of regulatory T cells correlates with excessive activation and apoptosis of CD8+ T cells in HIV-1-infected typical progressors, but not in long-term non-progressors

Abstract

Persistent HIV infection results in a decrease in absolute counts of CD4(+) CD25(+) regulatory T cells (Treg). To investigate the role of decreased Treg counts in the regulation of excessive activation and apoptosis of CD8(+) T cells in human immunodeficiency virus (HIV)-1 infection, we characterized Treg in 83 HIV-1-infected individuals, including 19 long-term non-progressors (LTNPs) and 51 typical progressors (TPs) who were treatment-naïve, and 13 AIDS patients on highly active antiretroviral therapy (HAART), of whom nine were complete responders (CRs) and the remaining four were non-responders (NRs) to the treatment. TPs but not LTNPs had a significant decrease in absolute counts of circulating Treg, which was inversely correlated with the activation and apoptosis of CD8(+) T cells. Efficient HAART was found to increase Treg counts in CR patients and temper the excessive activation and apoptosis of CD8(+) T cells. Moreover, isolated Treg significantly inhibited the spontaneous and anti-CD3-induced apoptosis of CD8(+) T cells in a dose-dependent manner in vitro. Thus, our findings indicate that the decrease in Treg closely correlates with the increase in apoptotic CD8(+) T cells and disease progression in chronic HIV-1 infection, and that Treg may play a key role in maintaining the balance between the amount and quality of CD8(+) T cells in HIV-1 infection. Manipulation of Treg function may be a promising strategy for immune therapy of this disease.

Figure 1

Increased frequency of CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells (Treg) and decreased absolute Treg counts in human immunodeficiency virus (HIV)-1-infected patients were both associated with disease progression. (a) Representative plots of CD4⁺ CD25⁺ FoxP3⁺ Treg from individual subjects in the three groups studied (NC, normal control; LTNP, long-term non-progressor; TP, typical progressor). (b) Statistical analysis showing that the frequency of CD4⁺ CD25⁺ FoxP3⁺ Treg in TPs was significantly higher than in LTNPs and NCs. (c) Treg frequency was inversely correlated with CD4 count in TPs. (d) Statistical analysis showing that the absolute counts of CD4⁺ CD25⁺ FoxP3⁺ Treg were significantly decreased in TPs. (e) Absolute counts of CD4⁺ CD25⁺ FoxP3⁺ Treg positively correlated with CD4 counts in TPs. P< 0·001 for multiple comparisons by the non-parametric Kruskal–Wallis H-test. The non-parametric Mann–Whitney U-test was used for comparison between groups. Spearman correlation analysis was performed. Data in (b) and (d) are expressed as box plots, in which the horizontal lines illustrate the 25th, 50th and 75th percentiles. Vertical lines represent the 10th and 90th percentiles. Solid line, linear growth trend; r, correlation coefficient; *P< 0·05; **P< 0·01. IgG, immunoglobulin G.

Figure 2

Typical progressors (TPs) had more activated and apoptotic CD8⁺ T cells than healthy controls and long-term non-progressors (LTNPs), and apoptotic CD8⁺ T cells in vivo were massed in activated subsets. Freshly isolated peripheral blood mononuclear cells (PBMC) were immediately used for CD38, HLA-DR and Annexin-V staining of CD8⁺ T cells in flow cytometric analysis. CD8⁺ T cells in TPs had more CD38 (a) and human leucocyte antigen (HLA)-DR (b) expression than those in LTNPs and normal controls (NCs). (c) Statistical analysis showed that TPs had significantly more apoptotic CD8⁺ T cells than LTNPs and normal controls. Representative plots (d) and statistical analysis (e) show that most apoptotic CD8⁺ T cells expressed CD38 and HLA-DR. The Wilcoxon signed ranks test was used for the two-related-samples test. **P< 0·01.

Figure 3

Apoptosis of regulatory T cells (Treg) was positively correlated with CD8⁺ T-cell activation and apoptosis in vivo. CD38 (a) but not human leucocyte antigen (HLA)-DR (b) expression in CD8⁺ T cells and apoptosis of CD8⁺ T cells (c) were positively correlated with apoptosis of CD4⁺ CD25^high T cells in human immunodeficiency virus (HIV)-1-infected patients. (d) The absolute count of CD4⁺ CD25^high T cells was positively correlated with the percentage of apoptotic CD4⁺ CD25^high T cells. Freshly isolated peripheral blood mononuclear cells (PBMC) were used for CD4⁺ CD25^high Treg apoptosis staining. After CD3, CD4 and CD25 cell surface staining, cells were suspended in Ca²⁺-binding buffer for Annexin-V staining. Treg were defined as CD3⁺ CD4⁺ CD25^high subsets.

Figure 4

Regulatory T cells (Treg) can suppress spontaneous and anti-CD3-induced apoptosis of CD8⁺ T cells. Magnetic bead-purified CD8⁺ T cells obtained by CD8 positive selection were cultured with different doses of magnetic bead-purified CD4⁺ CD25⁺ Treg in the presence or absence of anti-CD3. The cells were then collected for apoptosis analysis by Annexin-V staining. Numbers of apoptotic CD8⁺ T cells were significantly reduced by addition of Treg in both spontaneous and anti-CD3 induced states in a dose-dependent manner in long-term non-progressors (LTNPs) (a) and healthy donors (b). Representative graphs are shown for four normal controls and five LTNPs (CD4 counts from 549 to 735 cells/μl).

Figure 5

Highly active antiretroviral therapy (HAART) elevated counts of peripheral blood CD4⁺ T cells, leading to a simultaneous increase in regulatory T cell (Treg) counts that was significantly associated with reduced activation and apoptosis of CD8⁺ T cells in complete responder (CR) patients, but not in non-responder (NR) patients. (a, b, c) Decreased Treg frequency (a) and apoptosis of Treg (c), and increased Treg counts (b) in CR patients compared with NR patients. (d, e) Lower CD38 (d) and human leucocyte antigen (HLA)-DR (e) expression levels on CD8⁺ T cells in CR patients than in NR patients. (f) Compared with NR patients, CR patients had a lower percentage of apoptotic CD8⁺ T cells.