Previous studies have shown that significant numbers of HIV-1-specific CD8 cytotoxic T lymphocytes (CTL) exist in the peripheral blood of chronically HIV-1-infected patients  and yet proliferation assays using 3H-thymidine incorporation could barely detect the activity of HIV-1-specific CD4 T cells . It has been suggested that exhaustion of HIV-1-specific CD4 T cells may be the key for the failure to eliminate HIV-1 from patients despite the existence of significant numbers of HIV-1-specific CD8 CTL. To understand the dynamics of HIV-1-specific T cells, an intracellular cytokine staining method was used to detect CD4 and CD8 T cells specific to HIV-1 or cytomegaovirus (CMV) in the same patients.
Materials and methods
Sixty-three HIV-1-infected patients took part in the study. Thirty patients were untreated (CD4 T cell count 444 (± 165) × 106 cells/l; viral load 19 602 ± 25 517 copies/ml; age range 24–67 years), and 33 patients were taking highly active antiretroviral therapy (HAART) including protease inhibitor(s) for varying periods: 3–6 months (CD4 T cell count 280 (± 122) × 106 cells/l; age range 25–41 years), 6–18 months (CD4 T cell count 485 (± 229) × 106 cells/l; age range 27–58 years) or > 18 months (CD4 T cell count 313 (± 210) × 106 cells/l; age range 24–64 years). All treated patients had undetectable viral loads. Twenty-nine uninfected controls were included in the study (CD4 T cell count 513 (± 98) × 106 cells/l; age range 25–41 years); seven of these individuals were documented as CMV seronegative. Each subject was analyzed once (cross-sectional study). All subjects gave informed consent for the study.
Monoclonal antibodies against CD4, CD28, interferon γ (IFN-γ) and interleukin 4 were obtained from Becton Dickinson Immunocytometry Systems (BDIS, San Jose, California, USA). Monoclonal antibodies against CD8, perforin, and tumor-induced attack-1 (TIA-1) were obtained from Coulter (Miami, Florida, USA), Dr Eckhard R. Podack (University of Miami, Miami, Florida, USA) and Dr Paul Anderson (Dana-Farber Cancer Institute, Boston, Massachusetts), respectively. Ethidium monoazide bromide (EMA) was obtained from Molecular Probes (Eugene, Origon, USA). Monensin and saponin were obtained from Sigma Chemical Company (St Louis, Missouri, USA).
CMV antigen preparation was obtained from BioWhittaker (Walkersville, Maryland, USA). Heat-inactivated HIV-1 virions were prepared in our laboratory using CD4 T cell line (MT2) infected with HIV-1NL4−3. HIV-1 p24 levels of these viral preparations were determined by HIV-1 p24 antigen ELISA kit (Cellular Products, Buffalo, New York, USA).
Cell preparation and antigen stimulation
Peripheral blood mononuclear cells were isolated by centrifuging heparinized blood over Ficoll-Hypaque (Pharmacia Biotech AB, Uppsala, Sweden). The peripheral blood mononuclear cells were placed in tissue culture tubes containing complete medium with titred CMV or HIV-1 antigen preparations and anti-CD28 monoclonal antibody, and incubated at 37°C in a humidified 5% CO2 atmosphere for 16 h in the presence of 2 μmol/l monensin.
Immunofluorescent staining and flow cytometric analysis
After staining with EMA and anti-CD4 or CD8 antibodies, cells were fixed with formaldehyde and resuspended in permeabilization buffer containing 0.5% saponin. Antibodies against intracellular antigens were added to the cell suspension and incubated for 30 min. Cells were washed, suspended in phosphate-buffered saline containing bovine serum albumin and sodium azide and then analyzed on a FACSCalibur flow cytometer (BDIS) using CELLQuest software (BDIS). Samples from the same healthy volunteers were measured at each analysis to standardize the frequencies and specific mean fluorescence intensities. The mean numbers of HIV-1-specific CD4 and CD8 T cells in 29 healthy controls were 116 (± 48) × 106 and 58 (± 35) × 106 cells/l, respectively, the threshold for positivity of HIV-1-specific CD4 and CD8 T cells was defined as 250 and 100 × 106 cells/l, respectively.
Statistical comparisons of distributions were performed by the nonparametric two-sample Wilcoxon rank test.
Quantitative analyses on virus-specific T cells before and after therapy
Mean numbers of HIV-1-specific CD4 and CD8 T cells in 30 untreated HIV-1-infected patients were 546 (± 441) and 2855 (± 3502) × 106 cells/l, respectively, while those of CMV-specific CD4 and CD8 T cells were 7611 (± 7440) and 7443 (± 13 769) × 106 cells/l, respectively (Fig. 1). The absolute count for HIV-1-specific CD8 T cells were almost at the same level as those of CMV-specific CD8 T cells (P = 0.67), whereas the HIV-1-specific CD4 T cells were less than one tenth of CMV-specific CD4 T cells (P < 0.0001).
In patients treated with HAART for < 0.5 years or for 0.5–1.5 years, HIV-1-specific CD4and CD8T cell counts were significantly lower than those in untreated patients (< 0.5 years: CD4 cell count 216 (± 122) × 106 cells/l; n = 5, P = 0.05; CD8 cell count 1220 (± 1243) × 106 cells/l; n = 5; P = 0.27; for 0.5–1.5 years: CD4 cell count 218 (± 135) × 106 cells/l; n = 19; P = 0.002; CD8 cell count 425 (± 484) × 106 cells/l; n = 15; P = 0.002). Interestingly, HIV-1-specific CD4 T cell counts in patients treated with HAART for > 1.5 years were higher than those in patients treated for 0.5–1.5 years (CD4 cell count 469 (± 341) × 106 cells/l; n = 9; P = 0.03; CD8 cell count 441 (± 297) × 106 cells/l; n = 8; P = 0.44). However, there was no significant difference in the number of CMV-specific T cells between untreated patients and patients treated with HAART.
Qualitative analyses of virus-specific T cells in untreated HIV-1-infected patients
Figure 2 shows a representative result for intracellular TIA-1 and perforin expression in antigen-specific CD8 T cells in an untreated patient. Specific mean fluorescence intensity of IFN-γ staining in HIV-1-specific CD8 T cells was significantly lower than that in CMV-specific CD8 T cells. The majority of IFN-γ-producing CD8 T cells expressed TIA-1 in both HIV-1- and CMV-specific CD8 T cells (Fig. 2a,c). However, most of the IFN-γ-producing CD8 T cells did not express perforin in HIV-1-specific CD8 T cells in spite of significant expression of perforin in CMV-specific CD8 cells (Fig. 2b,d).
In untreated HIV-1-infected patients, HIV-1-specific T cells had quantitative and qualitative abnormalities. Quantitatively, HIV-1-specific CD4 T cell counts were low despite the significant numbers of HIV-1-specific CD8 T cells. It has been suggested that vigorous cytopathic HIV-1 infection of CD4 T cells occurs through contact with dendritic cells exposed to HIV-1 . This may explain the quantitative abnormality in HIV-1-specific T cells that we observed.
Qualitative abnormalities observed were the decreased expression of IFN-γ and perforin in HIV-1-specific CD8 T cells. TIA-1 is a cytotoxic granule protein of CTL that induces apoptosis in permeabilized target cells in vitro, and unsynchronized perforin and granzyme A expression in CD8 T cells in lymph nodes of chronically HIV-1-infected patients has been reported . This is similar to the findings reported here. Since most HIV-1-infected patients lose the HIV-1-specific CD4 T cell response early in the course of infection , the impairment of perforin expression in CD8 T cells may be secondary to the impairment of HIV-1-specific CD4 T cells. CTL responses are thought to play a critical role in the control of viral production and disease progression , and the perforin/granzyme system is most important for killing HIV-1-infected targets . Consequently, impaired expression of perforin in CTL may be related to failure to eliminate HIV-1. One therapeutic approach for HIV-1 infection would involve the development of strategies to recover CTL function and for this the mechanism of the qualitative abnormality described here in HIV-1-specific T cells must be clarified.
We thank David Chao for his comments on drafts of the manuscript.
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