Early reduction of the over-expression of CD40L, OX40 and Fas on T cells in HIV-1 infection during triple anti-retroviral therapy: possible implications for lymphocyte traffic and functional recovery

Abstract

Fas, CD40L and OX40 are members of the tumour necrosis factor (TNF) receptor superfamily with critical roles in T cell activation and death, B cell function, dendritic cell maturation and leucocyte traffic regulation. The aim of this study was to evaluate the effects of anti-retroviral therapy (HAART) on CD40L, OX40 and Fas expression on freshly isolated peripheral blood T cells by three-colour flow cytometry and compare them with lymphoproliferative responses, peripheral blood cell counts and viral load. Fourteen asymptomatic HIV-1+ patients treated with Lamivudine, Stavudine and Nelfinavir were prospectively investigated sequentially for 48 weeks. At baseline, patients exhibited significantly enhanced proportions and counts of CD40L+ and OX40+ cells within the CD4 subset which were corrected by weeks 8-16 of HAART. Interestingly, in the five patients showing viral load rebound during therapy in spite of increasing CD4 counts, the reduction of the levels of these costimulatory molecules was similarly maintained. Therapy induced a decrease in the over-expression of Fas, particularly in the CD4 subset where normal levels were reached at week 8. This reduction occurred in parallel with the major recovery of lymphoproliferative responses. Higher basal levels and lower reduction of Fas were associated with suboptimal suppression of viraemia. In conclusion, this previously undescribed increased expression of CD40L and OX40 may play a role in the HIV-associated pan-immune activation and represent a possible target for immunointervention, as suggested for several immunologically mediated diseases. Moreover, HAART induced an early correction of the over-expression of Fas, CD40L and OX40 in CD4 T cells which could be involved in the recovery of the cell traffic disturbances and in the T cell renewal capacity.

Fig. 1

Longitudinal analysis of viral load and peripheral blood cell populations during anti-retroviral therapy. (a) Kinetics of individual viral load decrease. Note that in nine patients (continuous line) viraemia was kept below the cut off of the test (50 RNA copies/ml) and that in five patients a viral load rebound was documented (dashed line). (b) Mean counts of leucocytes (▴) total lymphocytes (Δ), T cells (CD3⁺) (•), CD4 T cells (CD3⁺CD4⁺) (▪) and CD8 T cells (CD3⁺CD8⁺) (□). Results are shown for the 14 patients at all weeks except at week 2, where absolute counts refer to nine patients. Statistical significance in comparison with baseline values: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2

Recovery of lymphocyte proliferative responses during highly active anti-retroviral therapy (HAART). Peripheral blood mononuclear cells (PBMC) were cultured for 3 days in the absence (a) and in the presence of immobilized anti-CD3 MoAb (b), immobilized anti-CD3 MoAb and soluble anti-CD28 MoAb (c), phytohaemagglutinin (PHA) 20 μg/ml (d), PHA 5 μg/ml (e), pokeweed mitogen (PWM) 1 μg/ml (f) and phorbol myristate acetate (PMA) 50 ng/ml and ionomycin 500 ng/ml (g), and proliferation was assessed by ³H-thymidine incorporation. Results are expressed as mean ct/min of the 14 patients sequentially studied. The mean ct/min values of the lymphoproliferative responses of an healthy control population are shown (○). Statistical significance in comparison with baseline values: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3

Representative flow cytometric analysis of the expression of Fas (CD95), CD40L and OX40 within total T cells at baseline, 4 and 8 weeks of anti-retroviral therapy. Fresh peripheral blood mononuclear cells (PBMC) were directly stained with anti-CD4 FITC, anti-CD3 TC and anti-CD95, CD40L or OX40 conjugated with PE. Analysis was done within a manual setting lymphogate combined with a CD3⁺ cell gate. Thresholds were set according to isotype-matched negative controls. A total of 30 000 events (Fas) or 50 000 events (CD40L and OX40) was analysed.

Fig. 4

Decreased expression of Fas under triple anti-retroviral therapy. (a) Longitudinal analysis of the mean frequency of Fas⁺ cells within the CD4 (▪) and CD8 (□) T cell subsets. (b) Correlation between the reduction in the expression of Fas within the CD4 subset during the first 8 weeks of highly active anti-retroviral therapy (HAART) and the degree of CD4 depletion at baseline (% of CD4 T cells before start of treatment). (c) Comparison between the baseline levels and the kinetics of decrease of Fas expression within the CD4 T cell subset in the group of patients with sustained suppression of viraemia (•) and the group of patients with viral load rebound (○). (d) Similar analysis done within the CD8 T cell subset. Statistical significance in comparison with baseline values: *P < 0.05; **P < 0.01; ***P < 0.001. Statistical significance between the two subgroups of patients according to control of viraemia under therapy: §P < 0.05; §§P < 0.01; §§§P < 0.001.

Fig. 5

Expression of the costimulatory molecules CD40L and OX40 in the peripheral blood CD4 T cells of HIV-1-infected patients at the start of combined anti-retroviral treatment and during the follow up. (a) Individual kinetics of the alteration of proportion of CD40L⁺ cells within the CD4 subset. (b) Individual kinetics of the alteration of the circulating CD40L⁺CD4⁺ T cell absolute counts. (c) Individual kinetics of the alteration of the proportion of OX40⁺ cells within the CD4 subset. (d) Individual kinetics of the alteration of the circulating OX40⁺CD4⁺ T cell absolute counts.