JAIDS Journal of Acquired Immune Deficiency Syndromes:
Effects of Highly Active Antiretroviral Therapy and Immune Recovery on CD8+ T-Cell-Mediated Inhibition of HIV-1 Transcription
Beaudoin, Greg MSc*; Diker, Bilge MSc*; Angel, Jonathan B MD*†; Copeland, Karen F T PhD*
From the *Molecular Medicine Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada; and †Infectious Diseases Division, The Ottawa Hospital, Ottawa, Ontario, Canada.
Received for publication September 20, 2005; accepted May 30, 2006.
Supported by the AIDS Program Committee of the Ontario Ministry of Health and Long-Term Care (grant PA00103).
J. B. Angel is an Ontario HIV Treatment Network Career Scientist. K. F. T. Copeland is an Ontario HIV Treatment Network Scholar.
Reprints: Karen Copeland, PhD, Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (e-mail: firstname.lastname@example.org).
Summary: To date, the relation between the CD8+ antiviral factor (CAF) and clinical indicators of disease progression in HIV-1 infection (CD4+ T-cell counts and viral load [VL]) is inconclusive. Particularly, the effect of antiretroviral therapy and immune recovery on CAF production remains unclear. Using a transient transfection assay and a reporter gene activated by the HIV-1 long terminal repeat (LTR), we analyzed CAF production in CD8+ T cells of HIV-1-positive individuals divided into 3 groups: patients on protease inhibitor (PI)-based therapy, patients on nonnucleoside reverse transcriptase inhibitor (NNRTI)-based therapy, and patients receiving no therapy. We found that within the untreated group, CAF activity inversely correlated with VL and high CAF was associated with lower VLs over a period of 0.5 to 3 years. Furthermore, patients who were drug-naive demonstrated significantly higher CAF than untreated patients who had previously undergone antiretroviral therapy. CAF activity in treated patients was similar to CAF in drug-naive patients and higher than in off-treatment patients. There seemed to be a trend toward higher CAF in patients on NNRTI-based therapy compared with those on PI-based therapy. These results suggest that immune recovery after highly active antiretroviral therapy (HAART) contributes to the normalization of CAF levels in HIV-1-positive individuals. Furthermore, we have distinguished between CD8+ T-cell-mediated suppression of HIV-1 replication and gene transcription.
CD8+ T cells were first shown to suppress HIV-1 replication almost 2 decades ago,1 yet the protein factor(s) involved in CD8+ antiviral factor (CAF) activity is unknown. CAF suppresses viral replication at the level of transcription,2-7 and it has been shown that CAF activity, particularly the inhibition of long terminal repeat (LTR)-mediated gene expression,8 is not mediated by chemokines.8-11 CAF has also been found not to affect reverse transcription or provirus integration.12
In the context of clinical HIV-1 infection, data are inconclusive as to the effects of infection on CAF activity. Blackbourn et al13 showed that CAF activity in lymphoid tissue-derived CD8+ T cells positively correlated with control of viral replication in the lymphoid tissues, but no such correlation was observed in peripheral blood.13,14 In another study, a negative correlation was found between CAF and viral load (VL) in baboon peripheral blood.15 Others have shown that before antiretroviral therapy, CAF inversely correlates with VL, but that after therapy, the 2 are positively correlated.16 The association between immune status and CAF is also unclear. CAF has been shown to decrease with disease progression,17,18 and other studies have shown a positive19-21 or negative22 correlation between CAF and CD4+ T-cell counts in untreated HIV-1-positive individuals. Improved clinical status was shown not to be a determinant of CAF levels, however.23 This is corroborated by Kootstra et al,24 who showed that there was no correlation between CD4+ T-cell counts and CAF.
The effects of highly active antiretroviral therapy (HAART) on CAF activity within HIV-1-positive individuals have recently been studied, and conflicting results have arisen. In a longitudinal study, CAF was shown to decrease significantly in 76% of patients on HAART but increased or was maintained in untreated patients.25 In addition, CAF from HIV-1-positive individuals undergoing HAART was found to decrease to levels associated with HIV-1-negative individuals in another study.16 Conversely, Kottilil et al26 showed higher CAF in HAART-treated patients compared with untreated controls, and Chun et al27 showed that patients who commenced HAART shortly after acute infection had significantly higher β-chemokine-independent CAF than long-term nonprogressors, untreated patients, and patients who commenced treatment during chronic infection. This is reflective of findings by Oxenius et al,28 who found that HIV-1-specific CD8+ T-cell function was preserved in patients who commenced HAART during primary infection or concurrent to seroconversion; in patients in whom HAART commencement was delayed, HIV-1-specific CD8+ T-cell function was lost. Replication-based assays were used to determine CAF, and most do not control for confounding factors that might inhibit steps of the infection cycle other than that of gene transcription. Our goals were to determine the effects of HAART on CAF in HIV-1-positive individuals and whether there was any association between CAF and clinical profiles using an assay that defines CAF activity as the ability of cell-free supernatants from phytohemagglutinin (PHA)-stimulated CD8+ T cells to suppress LTR-driven gene expression.
Primary CD8+ T cells were cultured in RPMI 1640 (Gibco, Carlsbad, CA) supplemented with 20% fetal calf serum, 200 U/mL of penicillin (Gibco), 200 μg/mL of streptomycin (Gibco), and 20 U/mL of interleukin (IL)-2 (National Institutes of Health [NIH] AIDS Research and Reference Reagent Program) at 37°C and 5% co2. Jurkat T cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum.
HIV-1-positive patients attending the Ottawa Hospital Infectious Diseases Unit provided informed consent to provide blood for this study. Age-matched patients were divided into 3 groups depending on their treatment regimen: protease inhibitor (PI)-containing therapy (n = 12), nonnucleoside reverse transcriptase inhibitor (NNRTI)-containing therapy (n = 13), and an untreated group consisting of patients who had been off treatment for more than 6 months (n = 8) or who were drug naive (n = 8). In both treated groups, selection criteria (to ensure effective treatment) included an undetectable VL (<50 copies/mL) and a CD4+ T-cell count of >200 cells/mm3. T-cell counts and VL data were assessed between 0 and 3 months before CAF analysis.
Isolation of CD8+ Antiviral Factor Containing Cell-Free Supernatants From Primary CD8+ T Cells
Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by Ficoll-Paque PLUS (Pharmacia Fine Chemicals, Piscataway, NJ) gradient separation, washed twice with phosphate-buffered saline (PBS), and cultured at 1 × 106 cells/mL. CD8+ T cells were isolated from cultured PBMCs using magnetic affinity cell sorting (MACS) CD8 microbeads and MS+/RS+ columns (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. Briefly, cells were washed twice with MACS buffer (5% bovine serum albumin and 2 mM of ethylenediaminetetraacetic acid [EDTA] in PBS) and incubated for 20 minutes at 4°C with CD8-specific microbeads. Cells were passed through the magnetic separation column, washed, eluted with MACS buffer, and washed twice with PBS. Cells were then cultured at 1 × 106 cells/mL and activated with 5 μg/mL of PHA for 3 days, washed and resuspended at 1 × 106 cells/mL in fresh media in the absence of PHA, and cultured an additional 3 days. After pelleting the cells, CD8+ T-cell supernatants were removed and their volumes were adjusted with media so the amount of supernatant originating from 0.5 × 106 cells would equal 1 mL. Cell-free supernatants were then heat-inactivated (56°C for 60 minutes), and stored at −80°C.
Diethylaminoethyl-Dextran Transient Transfection of Jurkat T Cells
Jurkat T cells were grown to confluence, resuspended at 1 × 106 cells/mL in fresh media for 18 hours, and then pelleted and resuspended in an equal volume of fresh media for 2 to 3 hours. Cells were washed twice in serum-free RPMI 1640 and resuspended at 6 × 106 cells/mL in transfection buffer containing 250 μg/mL of diethylaminoethyl (DEAE)-dextran (Sigma, Oakville, Ontario, Canada) and 50 mM of Tris, pH 7.3, in serum-free RPMI 1640. We then added 1 μg/mL of a vector in which the LTR of HIV-1 drives the expression of chloramphenicol acetyltranferase (pLTRCAT)29 with an LTR sequence of a clinical isolate (HIV-1BRU) to position +77, and 0.5 μg/mL of a vector in which Tat expression is controlled by the SV40 promoter (pSVtat)30 to activate the LTR. Cells were incubated in a 37°C water bath with shaking at 50 rpm for 45 minutes. Transfected cells were pelleted, washed twice with serum-free RPMI 1640, and cultured at 3.75 × 106 cells/mL. Each transfection was performed on a maximum of 30 × 106 cells. Freshly transfected Jurkat T cells were aliquoted in 12-well tissue culture plates (1.875 × 106 cells per well), and 1 mL of CAF-containing cell-free CD8+ T-cell supernatants was added to each well. Cells were cultured for 24 hours and then activated with 25 ng/mL of PMA (Sigma) and 2 μM of ionomycin (Sigma) to enhance baseline reporter gene expression further. After 18 hours of activation, cells were washed twice with PBS and resuspended in 0.5 M of Tris. Cells were lysed using 3 freeze-thaw cycles, and lysates were cleared of cell debris by centrifuging at 14,000g for 15 minutes. Protein concentrations in cleared lysates were determined using the BioRad Protein Reagent (BioRad, Mississauga, Ontario, Canada). For CAF determination, 10 μg of total lysate protein was loaded into a CAT enzyme-linked immunoassay (ELISA) plate (Roche, Laval, Quebec, Canada), and volumes were adjusted to normalize total protein concentration to 50 μg/mL. CAF activity for each CD8+ T-cell supernatant was determined as percent suppression of CAT expression relative to a media-treated control.
Determination of Interferon-γ and Interleukin-16
Interferon (IFN)-γ and IL-16 concentrations in cell-free supernatants were determined using ELISA kits (IFN-γ ELISA; BD Biosciences, Mississauga, Ontario, Canada; IL-16 ELISA: Biosource International, Camarillo, CA), according to the manufacturer's instructions.
HIV-1 Infection of Jurkat T Cells
Jurkat T cells were activated with 25 μg/mL of PMA and then treated with 2 μg/mL of polybrene for 1 hour. The cells were washed and then infected with a 300 tissue culture infectious dose (TCID50) of HIVIIIB) of HIVIIIB) correct as written? Please revise to clarify meaning. -->, a β-chemokine insensitive strain. After 5 days, Jurkat cells were resuspended at 5 × 106 cells/mL in fresh media and aliquoted in a 96-well round-bottom tissue culture plate (0.75 × 106 cells per well) for determination of HIV-1 suppressive activity.
Determination of HIV-1 Suppressive Activity
Cell-free CD8+ T-cell supernatants, with volumes normalized as discussed previously, were added to HIV-1-infected Jurkat T cells, and cells were cultured for 24 hours. Jurkat supernatants were then measured for p24 concentrations using a p24 Antigen Capture Assay Kit (SAIC-Frederick, Inc., Frederick, MD). HIV-1 suppressive activity was determined with the same method as CAF determination, as percent suppression of viral replication relative to a media-treated control.
Significant differences between groups were determined using the Student t test or the Mann-Whitney rank-sum test. Correlations between CAF activity and various immunologic or virologic parameters were determined using the Spearman rank-order correlation test. Calculations were performed using SigmaStat 3.0 software (SPSS, Inc., Chicago, IL).
We found no significant differences in T-cell counts between the PI and NNRTI groups at the time of sample acquisition (Table 1) or before treatment commenced (data not shown). Mean pretreatment VLs of each group were compared, and the NNRTI group demonstrated a lower log10 VL (4.2) than the PI group (5.0). This difference was statistically significant (P = 0.009).
Relation Between Patient Clinical Profiles and CD8+ T-Cell-Mediated Long Terminal Repeat Suppression
To determine CAF activity, cell-free supernatants of PHA-activated CD8+ T cells of HIV-1-positive patients on HAART and untreated controls were assessed for their relative abilities to suppress LTR-driven gene expression. After assessing CAF activity in all patient samples, we determined whether there was any association between CAF and T-cell levels, VL, and HAART profiles. Clinical data from all patients are shown in Table 1. We found no correlation between CAF activity and CD4+ or CD8+ T-cell counts. Furthermore, we found no association between CAF and the specific drug regimen or the amount of time an individual was treated (Table 2). We also found no correlation between CAF and pretreatment T-cell counts or VL (data not shown).
In the untreated group, a negative correlation was observed between CAF and VL (P = 0.004, r = 0.848; Table 3). When the untreated group was subdivided into patients that were drug naive (NAIVE, n = 8) or were off treatment for at least 6 months (OFF, n = 8), we observed that the NAIVE group demonstrated significantly higher CAF activity than the OFF group (30.0% and 9.3%, respectively; P = 0.027; Fig. 1A). We examined VL over time (up to 3 years before and/or after sample acquisition) for each of the patients in the untreated group to determine whether individuals with a higher CAF at the time of our study were able to maintain lower VLs over a long period in the absence of antiretroviral treatment. Some of the untreated patients began HAART shortly after we collected their blood samples, and thus were not included in this analysis. We found that individuals in the NAIVE group with the highest CAF activity were able to maintain lower VLs over a period of 0.5 to 3 years compared with 5 of 8 individuals in the OFF group. Also, NAIVE individuals with a lower CAF (patients C7 and C11) had VLs similar to the 6 OFF patients with the highest VLs (Fig. 2). Average VLs below and above a log10 value of 4 were compared with each other, and all noted differences were significant (P ≤ 0.001). CAF activity, average VLs, and the amount of time over which data were collected are given in Table 3. CD4 counts before the resumption of treatment for the OFF group and prior treatment of these subjects are summarized in Table 4.
CD8+ T-Cell-Mediated Long Terminal Repeat Suppression in Patients on HAART Is Similar to That of Drug-Naive Patients
We compared CAF activity from patients in each of the study groups to determine whether HAART had any effect on CAF levels. The median values of CAT suppression in each group were: 22.5% (range: 0%-55.6%) for the PI group, 46.0% (range: 0%-90.2%) for the NNRTI group, 24.9% (range: 0%-65.6%) for the NAIVE group, and 6.75% (range: 0%-27.2%) for the OFF group (see Fig. 1A). As with NAIVE group, CAF from NNRTI group was significantly higher than that of the OFF group (P = 0.018). Results were further interpreted to reflect only individuals who effectively suppressed LTR activity (>50% suppression), similar to interpretations by others.31 We found that a higher proportion of the NNRTI group (5 of 13 persons) effectively suppressed LTR activity compared with the other groups (1 of 12 persons in the PI group, 2 of 8 persons in the NAIVE group, and 0 of 8 persons in the OFF group; see Fig. 1B). This trend was also observed when using 20% as the lower limit of effective suppression. IFNγ and IL-16 suppress LTR activation, and concentrations of these cytokines were assessed in CD8+ T-cell supernatants by ELISA to determine whether the LTR suppression observed in our system could be attributed to one or both of these cytokines. No correlation between CAF and IFNγ levels was observed. A positive correlation was observed between CAF and IL-16 levels in the PI group only (P = 0.007, r = 0.718); however, the highest of the IL-16 concentrations in our system was approximately 1000-fold lower than required for HIV-1 inhibition32 and likely did not contribute to LTR suppression here.
CD8+ T-Cell-Mediated Long Terminal Repeat Suppression Does Not Correlate With CD8+ T-Cell-Mediated Inhibition of HIV-1 IIIB Replication
CAF activity inhibits HIV-1 replication at the level of transcription, but it is not clear whether CD8+ T-cell-mediated inhibition of viral replication in an acute infection assay accurately reflects suppression of the LTR by CAF. We sought to determine whether levels of suppression observed in our transient transfection system were mirrored in an acute infection assay. Jurkat T cells were infected with HIV-1 IIIB and treated with the same cell-free CD8+ T-cell supernatants as in the transcription-based assay or with media as a control. After 24 hours, viral replication was measured by p24 ELISA and replication suppression was assessed. We performed Spearman rank-order correlation tests on each subgroup and found no correlation between levels of CAF and levels of suppression of HIV-1 replication (Fig. 3).
The relation between CAF and disease progression, in terms of T-cell counts and VLs, in HIV-1-positive individuals is not clear. Using a transcription-based system to evaluate CAF, we have shown that within each group studied, there is no correlation between CD4+ T-cell counts and CD8+ T-cell-mediated LTR suppression, in agreement with a previous study.24 Others have shown a positive19-21 or negative22 correlation between CD4+ T-cell counts and CD8+ T-cell-mediated suppression of viral replication in acute infection assays. In addition, no correlation was observed between CD8+ T-cell counts and CAF. We have also demonstrated that within untreated HIV-1-positive individuals, there is an inverse correlation between VL and CD8+ T-cell-mediated LTR suppression. This is in agreement with a study of baboon-derived CD8+ T-cell-mediated suppression of viral replication.15 This contradicts other findings, however, where no correlation was seen between blood VL and CAF.13,14
CAF activity in the drug-naive subgroup (NAIVE) was significantly higher than that of the off-treatment subgroup (OFF). This associated strongly with relative VLs for the 2 groups, such that NAIVE patients with the highest CAF activity were able to maintain lower VLs continually than most OFF patients, without antiretroviral treatment, over a span of 0.5 to 3 years. Furthermore, NAIVE individuals with lower CAF demonstrated higher VLs in the same range as OFF individuals. This indicates a possible relation between CAF and disease progression in vivo. There are few data available that describe an actual antiviral role for CAF in vivo, with the exception of high CAF levels seen regularly in long-term nonprogressors21,33-35 and exposed uninfected individuals.36-40 Our data show that in untreated HIV-1-positive individuals, higher levels of CAF can be associated with lower VLs for at least 0.5 to 3 years. This suggests that CAF may be partly responsible for suppression of viral replication in vivo. Nevertheless, we cannot exclude the reverse situation, where VL and disease progression have a negative effect on CAF levels in HIV-1 infection. Certain immunologic factors, such as CD28 costimulation, IL-2, and IL-15 derived from mature dendritic cells, are known to be involved in CAF production and have also been shown to be decreased in advanced HIV-1 infection.41-46 It is therefore possible that general immune dysregulation associated with higher VLs and advanced disease progression might subsequently diminish an individual's ability to produce CAF. This notion is supported by the fact that drug-naive individuals demonstrating lower CAF tend to have VLs in the same range as the OFF group, suggesting that VL may be an important determinant of CAF activity. CAF activity in the naive group may be higher for several reasons, such as better general health or higher CD4 counts. In addition, lower CAF production in the OFF group may result from irreversible changes that may have been induced by prior therapy, which could affect CAF production. For example, antiretroviral therapy has been demonstrated to induce irreversible changes in metabolism in HIV-infected individuals.47,48
The effects of antiretroviral therapy on CAF production are not well established. Others have shown CAF to be increased26,27 or decreased16,25 in patients on HAART, using replication inhibition-based systems to determine CAF activity. In our transcription-based system, we have shown that there is no significant difference in CAF-mediated suppression of the LTR between treated individuals and those in the NAIVE group and that both are higher than in the OFF group (only the NNRTI group was significantly higher). This suggests that HAART might return CAF to a level similar to that of untreated individuals capable of maintaining lower VLs. CAF has been shown by some to decrease with disease progression.17,18,21 It is therefore possible that by reducing VL and subsequently restoring immune functions, HAART reverses this trend.
There seems to be a trend towards higher CAF in patients on NNRTI-based therapy. A slight but nonsignificant difference in CAF (P = 0.074) was observed between the PI and NNRTI groups. The lack of significance was attributable to a high degree of variability within each group. Analysis of the proportions of individuals in each group who effectively suppressed LTR activity (>50% suppression) showed that 38.5% of individuals in the NNRTI group demonstrated effective suppression compared with 7.7% of individuals in the PI group. A similar trend was seen when the limit of effective suppression was set at 20%, showing that the observed differences were not attributable to suppression levels in the PI group falling just below 50%. Interestingly, we observed that the NNRTI group had significantly lower pretreatment VLs than the PI group, and, as previously mentioned, we found an association between lower VL and higher CAF in untreated individuals. There was no correlation between lower VL and high CAF within the NNRTI group, however; thus, it is unlikely that lower pretreatment VLs had any effect on posttreatment CAF levels. It is not clear why the NNRTI group demonstrates a higher proportion of effective suppressors, because little is known about the comparative effects of PI- and NNRTI-based therapy on CD8+ T-cell function. Smith et al49 showed that CD8+ T cells from NNRTI-treated patients expressed higher levels of CD38 and human leukocyte antigen-D-related (HLA-DR) than PI-treated patients. An activated phenotype involving elevated expression levels of CD38 and/or HLA-DR has been associated with higher CAF activity.18,50 Therefore, this might explain why a higher proportion of the NNRTI group in our system demonstrated effective suppression of LTR activity. This contrasts with a study in which patients switching from PI-based to NNRTI-based HAART experienced a decrease in CD38+ CD8+ T cells.51 Moreover, others have found that there is no difference in CD8+ T-cell activation in either type of treatment52,53 and that when patients switch from PI- to NNRTI-based therapy, there is no change in CD8+ T-cell activation.54
CAF activity is mediated by at least 1 unknown CD8+ T-cell-derived soluble factor that suppresses viral replication by inhibiting LTR activation. The LTR suppression observed in our system can be attributed to CAF activity, because we excluded the possibility of interference by IL-16 and IFNγ, which are known to inhibit LTR activation.55,56 Furthermore, β-chemokines (macrophage inflammatory protein-1α [MIP-1α], MIP-1β, and regulated on activation, normal T-cell expressed and secreted [RANTES]) that block viral entry57,58 do not inhibit LTR-mediated gene expression;8 therefore, controls for these chemokines were not included. Taking these controls into consideration, it is most likely that the LTR suppression seen in our system was the direct result of CAF activity.
We investigated whether the data collected in our LTR suppression system would be mirrored in an acute infection assay. We showed a different pattern of inhibition of viral replication compared with that of LTR suppression. No correlation was found between LTR suppression and HIV-1 suppression in our system. Furthermore, no correlations were observed between HIV-1 suppression and IFNγ and IL-16. Taken together, these findings indicate that CD8+ T-cell-mediated suppression of viral replication in vitro could occur independently of chemokines and known LTR suppressors and that these levels of replication suppression do not always reflect CAF-mediated LTR suppression.
Previous studies of the effects of HAART on CD8+ T-cell noncytotoxic antiviral activity were performed using replication inhibition-based assays.16,25-27 Although these studies describe CD8+ T-cell-mediated inhibition of viral replication, they do not specifically demonstrate the effects of HAART on CAF-mediated LTR suppression. We have shown that cell-free supernatants derived from activated CD8+ T cells differentially modulate HIV-1 gene transcription and viral replication. Taken together with previous published data, this lends support to the possibility that more than 1 type of CD8+ T-cell antiviral activity exists. To date, CAF-mediated LTR suppression is the best characterized of these activities, whereas the mechanisms of action of other potential antiviral activities remain to be determined.
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CD8+ T cell; chemokine; HIV-1; replication; transcription
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