Blood samples from healthy volunteers were collected into tubes containing Ethylenediamine Tetra-acetic Acid (EDTA) by venipuncture. Samples were diluted with an equal volume of Phosphate Buffered Saline (PBS) and Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll-Hypaque (Amersham Biosciences, Baie d’Urfe, PQ) density centrifugation. Subsequent to density centrifugation, isolated PBMCs were washed twice in PBS. CD8+ and CD4+ T-cell populations were isolated from freshly purified PBMCs through positive selection using anti-CD8 and -CD4 conjugated magnetic beads (Miltenyi, Auburn, CA) respectively, according to the manufacturer’s protocol. Briefly, cells were labeled with the magnetically conjugated antibody of interest for 20 minutes at 4ºC. Subsequent to labeling, cells were placed through a column inserted in the magnetic field of a MACS separator (Miltenyi) and unbound cells were washed with MACS buffer (PBS, 0.5% Bovine Serum Albumin (BSA), 2 mM EDTA, pH 7.2). After washing unbound cells, the column was removed from the separator and the labeled cells were eluted with the addition of MACS buffer. Following elution, the isolated T-cell subsets were washed twice with PBS.

The isolation of CD8+ T-cell subsets was performed using anti-CD8 Multisort conjugated magnetic beads (Miltenyi). In order to isolate the T-cell subsets, a second round of positive selection with magnetic bead-conjugated antibodies to either CD45RO+ or CD45RA+ was performed. Similarly, CD28, CD38 and HLA-DR positive and negative populations were selected using the Multisort conjugated magnetic beads.

PBMCs and T-cell subsets were cultured in RPMI-10 (RPMI 1640 medium supplemented with 10% Fetal Calf Serum (FCS), 100 U/mL penicillin (Invitrogen, Burlington, ON), 100 μg/mL streptomycin (Invitrogen), and 20 U/mL Interleukin (IL)-2 (Sigma-Aldrich, St. Louis, MO)). Cells were incubated at 37(C with 5% CO₂.

The T-cell tropic laboratory strain HIV_IIIB was obtained from Advanced Biotechnologies Incorporated (Columbia, MD). The macrophage tropic laboratory strain, HIV_ADA, was obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (Germantown, MD). The clinical isolates of HIV-1 were generated from the blood of HIV-1 infected individuals. Briefly, PBMCs were isolated from the peripheral blood of infected subjects by Ficoll-Hypaque density centrifugation. The CD8+ T-cell fraction was removed from the isolated PBMCs through the use of anti-CD8 conjugated magnetic microbeads. The remaining cells were subsequently cultured with HIV-negative PHA blastocytes in RPMI-10. The cultures were monitored at days 10 and 15 for HIV-1 p24 production by an Enzyme-Linked Immunosorbent Assay (ELISA) (SAIC-Frederick Inc., Frederick, MD).

The titre of the viral stock was determined by a TCID₅₀ assay. Briefly, 100 µL serial dilutions of the viral stock (approximately 1,500 ng/mL as determined by a HIV-1 p24 ELISA) was used to infect 250,000 MT2 cells (American Type Culture Collection (ATCC), Manassas, VA) in quadruplicate in a 96-well flat bottom plate. Subsequent monitoring for the formation of syncytia was performed over a 7-day culture period. The titre of the viral stock was determined by the Reed-Muench accumulative method. The titre of the HIV_IIIB viral stock used for CD8+ T-cell infections was found to be 1 x 10⁵. Unless otherwise noted, 300 TCID₅₀/mL of the viral inoculum for each of the viral isolates was used to infect isolated cells at a density of 1 x 10⁶ cells/mL during the course of the in vitro experiments.

Immediately after isolation and prior to infection, CD8+ T-cells were stimulated with 2.5 μg/mL of Phytohemagglutinin (PHA) (Sigma-Aldrich) for three hours followed by subsequent washes and a 1 hour incubation with 2 μg/mL polybrene (Sigma-Aldrich) in RPMI-10. Subsequently, the cells were washed, re-cultured in RPMI-10 and infected with 300 TCID₅₀/mL of the appropriate viral strains and isolates. Three days after the infection, the cells were extensively washed to remove any extracellular virus and debris and were re-cultured in RPMI-10 and this time point was considered the zero time point in the infection assays. In order to determine the levels of HIV-1 replication and production, cell-free supernatants were taken at days 5, 10 and 15 (or as indicated in the results and figures) post-infection from the infected cell cultures. Virus replication was assessed by HIV-1 production as measured by an ELISA (SAIC-Frederick Inc.). In order to normalize our results, supernatants were taken from cultures of CD8+ T-cells derived from several different individuals.

In order to assess productive infection, the reverse transcriptase inhibitor, Azidothymidine (AZT) (NIH AIDS Research and Reference Reagent Program) was used to treat the cells at a concentration of 10 μM for one hour prior to infection and was not used further throughout the time course.

Total RNA was isolated from 3 x 10⁶ cells using the GeneElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). First strand cDNA was reverse transcribed with a pd(N)₆ primer and Moloney Murine Leukaemia Virus (MMLV) reverse transcriptase using the First-Strand cDNA synthesis kit (Amersham Biosciences) according to the manufacturer’s protocol. RT-PCR reactions were set up for each cDNA towards the gag region of HIV-1 (p24 forward: 5’-ATAGAGGAAGAGCAAAACAAAA-3’ p24 reverse: 5’-CAAAATTACCCTATAGTGCA-3’). Each reaction was conducted in a total volume of 25 µL containing 2.5 µL of 2 mM dNTP (Amersham Biosciences), 1.25 µL of 50 mM MgCl₂ (Amersham Biosciences), 1 µL of each primer (from a 100 mM stock) and 1 U of Taq DNA polymerase (Amersham Biosciences). A total of 5 µL of each cDNA was used in the RT-PCR analysis. RT-PCR amplification was performed for 35 cycles of: denaturing at 94ºC for 60 seconds, annealing at 55ºC for 60 seconds and extension at 72ºC for 60 seconds. This was followed by a final extension for 10 minutes at 72ºC. RT-PCR amplicons were run at 80 V on a 1% agarose gel containing 0.5 µg/mL of ethidium bromide and visualized under Ultraviolet (UV) light.

Flow cytometric analysis was gated on the live cell populations. Measurement of the levels of intracellular HIV-1 staining was performed as follows: 1 x 10⁶ cells were permeabilized with the Fix & Perm cell permeabilization kit (Caltag Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Cells were labeled with 10 µL of the appropriate fluorochrome-conjugated monoclonal antibodies CD8-PC5 (Serotec, Raleigh, NC), CD4-PC5 (Serotec) and HIV-1_gag-FITC (Coulter clone KC57, Beckman Coulter, Fullerton, CA) prior to flow cytometry analysis. The KC57 antibody identifies HIV-1 core antigens (p55, p39, p33 and p24). Results were assessed on an Epics ALTRA system (Beckman Coulter) after gating on lymphocytes based on their forward and side scatter properties. Analysis was performed using Expo32 software (Applied Cytometry Systems, Sacramento, CA) and was based on a minimum of 10, 000 events. Values are expressed as the mean fluorescence intensity.

For the analysis of the purity of the isolated populations of CD8+ T-cells, the following fluorochrome-conjugated monoclonal antibodies were used: CD8-PE (Serotec), CD4-FITC (Serotec), CXCR4-PE (BD Biosciences, San Diego, CA), CCR5-PE (BD Biosciences), IgG-FITC (BD Biosciences), CD3-FITC (BD Biosciences), CD56/16-PE (BD Biosciences). For flow cytometric analysis, the cells of interest were washed twice in PBS and once in flow binding buffer (PBS, 0.1% NaN₃, 2% BSA). The cells were subsequently resuspended at a concentration of 1 x 10⁶ cells per 100 μL of flow resuspension buffer and were labeled with 10 μL of the appropriate antibody for 30 minutes at room temperature. Prior to flow cytometric analysis, the cells were fixed in 200 μL of PBS/4% paraformaldehyde at 4ºC for 5 minutes. Cell-surface molecule expression during the course of HIV-1 infection was examined in positively selected cells using CD8-PE (Serotec), CD4-FITC (Serotec), CXCR4-PE (BD Biosciences), CCR5-PE (BD Biosciences), CD45RA-PE (BD Biosciences),CD45RO-FITC (Immunotech, Fullerton, CA), CD28-FITC (BD Biosciences), CD38-FITC (BD Biosciences) and HLA-DR-FITC (BD Biosciences) conjugated monoclonal antibodies. In order to determine the surface expression of both CD45RA and CD45RO in the CD8+CD4- and CD8+CD4+ populations, cells were examined by flow cytometry at day 5 post-infection after staining with antibodies directly conjugated to FITC.

Lymphocyte proliferation was measured by the XTT based colorimetric assay (Roche Laboratories, Meylan, France). In the memory CD8+ T-cell studies, CD8+CD45RO+ T-cells were positively isolated from the PBMCs of a healthy volunteer by magnetic labeling. Cells were either left uninfected or infected with 300 TCID₅₀/mL of HIV_IIIB. For proliferation tests, approximately 450,000 cells were seeded in triplicate in 100 μL of culture medium (RPMI-10 supplemented with 20 U/mL of IL-2) per well of a 96-well flat bottom plate. Cells were incubated in the presence of each of the following mitogens: 3 µg/mL αCD3/CD28 (Sigma-Aldrich); 10 ng/mL Phorbol Myristate Acetate (PMA) (Sigma-Aldrich); 2 U/mL Tetanus Toxoid (TT) (Sigma-Aldrich); and 5 µg/mL PHA. On days 2-3 post-mitogenic stimulation, cells were labeled with 50 μL of the XTT labeling reagent as per the manufacturer’s protocol. The proliferation index was assessed by measuring the absorbance at 492 nm (with a reference wavelength at 690 nm) 18 hours post-labeling.

Data are expressed as the mean ± SEM. Differences in measured variables between experimental and control groups were assessedusing the Student’s t test. Statistical significance was accepted at p < 0.05.

After establishing the purity of the selected CD8+ T-cell population and demonstrating the absence of any contaminating CD4+ T-cells or Natural Killer (NK) cells (data not shown), the ability of CD8+ T-cells to support HIV-1 infection was ascertained. in vitro infection of primary CD8+ T-cells was performed with T-cell and macrophage tropic strains of HIV-1, along with Non-Syncytia Inducing (NSI) and Syncytia Inducing (SI) clinical isolates of the virus. The CD8+ T-cells were infected with an equal amount of virus, allowing the infection sensitivity of the CD8+ T-cells for the viral isolates and strains to be compared. Fig. (1A) shows a 15 day time course of virus replication of the two laboratory strains and the two clinical isolates of HIV-1 in positively selected CD8+ T-cells. As measured by a HIV-1 p24 ELISA, the T-cell tropic strain, HIV_IIIB, maintained the highest levels of viral production in the CD8+ T-cell population and the levels of virus replication for the HIV_IIIB strain reached a peak at day 15 post-infection (Fig. 1A). The replication of the T-cell tropic clinical isolate was also supported in CD8+ T-cells, although to lower levels than the laboratory strain. Similarly, CD8+ T-cells also supported moderate levels of replication of the macrophage tropic laboratory strain, HIV_ADA, but replication of the macrophage tropic clinical isolate was poor.

Fig. (1). Infection of CD8+ T-cells by HIV-1. (A) Selected CD8+ T-cells were infected with 300 TCID50/mL of the T-cell tropic and macrophage tropic laboratory strains and clinical isolates of HIV-1. HIV-1 p24 production was measured by an ELISA at the indicated time points with cell-free supernatants from cultures. Results show the mean HIV-1 production and SEM of three independent experiments. (B) CXCR4 receptor expression on CD8+ T-cells as measured by flow cytometry. One representative of three experiments is shown. The mean fluores-cence intensity of CXCR4 receptor and SEM of three independent experiments is 55.9% ± 2.1. (C) CCR5 receptor expression on CD8+ T-cells. One representative of three experiments is shown. The mean fluorescence intensity of CCR5 receptor and SEM of three independent experiments is 11.6 ± 1.0. CD8+ T-cells were also stained with negative controls (isotype control, dashed line). (D) Positively selected CD4+ and CD8+ T-cells were infected with 300 TCID50/mL of HIVIIIB. HIV-1 production was measured by an ELISA at the indicated time points from cell-free supernatants

Fig. (2). Productive HIV-1 infection of CD8+ T-cells in vitro. (A) RT-PCR analysis of HIV-gag transcripts in the uninfected and infected populations of isolated CD4+ and CD8+ T-cells. Isolated lymphocyte populations were left uninfected or infected with 300 TCID50/mL of HIVIIIB for 7 days prior to RT-PCR analysis. Detection of β-actin was run as a loading control. Parallel samples were run without reverse transcriptase as a control for DNA contamination of the samples (data not shown). (B) Infection of CD8+ T-cells is sensitive to AZT. Infection was performed in the absence and presence of AZT and HIV-1 production was measured by an ELISA for the HIV-1 gag protein, p24. Cells were treated with 10 µM of AZT for one hour prior to infection. CD8+ T-cells from two uninfected individuals were infected with 300 TCID50/mL of HIVIIIB laboratory strain. The results shown are the mean and SEM of three independent experiments.

Fig. (3). Detection of intracellular HIV-1 expression in CD8+ T-cells. CD8+ T-cells were infected with 300 TCID50/mL of HIVIIIBor left uninfected. (A) Intracellular p24 was examined on the CD8+ T-cell population by flow cytometry at day 15 post-infection with antibodies to HIV-1gag (p24-RD1) and CD8. In the infected population, the number in the upper right quadrant represents the number of cells found in the circle as to minimize any false-positives. (B) To determine the contribution of CD4+ cells to in vitro CD8+ T-cell infection, intracellular HIV-1 expression was examined in the CD8+ T-cell population by flow cytometry at day 15 post-infection with antibodies to HIV-1gag and CD4 only. Numbers in the quadrants represent the percentage of gated cells in each quadrant. A minimum of 10, 000 events was assessed for each analysis.

Fig. (4). CD4 cell-surface molecule up-regulation on the surface of CD8+ T-cells. Uninfected and HIV-1 infected CD8+ T-cells were examined at the indicated time points by flow cytometry for the expression of the CD8 and CD4 cell-surface molecules. CD8+ T-cells were infected with 300 TCID50/mL of IIIB and cell-surface expression of CD8 and CD4 was assessed at days 3, 9 and 12 by flow cytometry. Numbers in the quadrants represent the percentage of gated cells in each quadrant. A minimum of 10, 000 events were assessed for each analysis.

Fig. (5). HIV-1 replication in CD8+ T-cell subsets. CD28, CD38 and HLA-DR positive and negative populations were infected with HIV-1 and cultured. Cell-free supernatant samples were taken post-infection at the indicated time points for measurements of HIV-1 production by an ELISA. Results are the mean and SEM of three independent experiments.

Fig. (6). HIV-1 preferentially replicates in the CD8+CD45RO+ memory T-cells. (A) CD45RO positive and CD45RA positive populations were infected with 300 TCID50/mL of IIIB and cultured. Supernatants were taken at days 5 and 10 post-infection for p24 measurement by ELISA. Results are representative of two independent experiments. (B) Uninfected (□) and infected (■) memory and naïve T-cells were ex-amined for CD45RO, CD45RA, CD4 and CD8 receptor expression by flow cytometry at day 5 post-infection.

Fig. (7). Proliferative response of HIV-1 infected CD8+CD45RO+ memory T-cells in response to antigenic stimulation. (A) Positively selected CD8+ T-cells were stained with CD45RO-FITC and CD45RA-PE to determine the percentage of memory cells in the isolated CD8+ T-cell population. (B) The purity of the selected CD8+CD45RO+ memory T-cells was assessed by flow cytometry and staining with CD45RO-FITC, CD45RA-PE (right histogram) and CD8-PE (left histogram). Numbers in the quadrants represent the percentage of gated cells in each quadrant. (C) Expression of the activation markers CD28, CD38 and HLA-DR in uninfected (□) and infected (■) CD8+CD45RO+ memory T-cells. Results are the mean percentage and SEM of three independent experiments as measured by flow cytometry. Statistical significance of p < 0.01 is denoted by an (*). (D) Lymphocyte proliferative ability of uninfected (□) and infected (■) CD8+CD45RO+ memory T-cells as measured by the XTT based colorimetric assay. Memory T-cells were infected (or left uninfected) for 7 days prior to antigen stimulation. Cells were stimulated with αCD3/CD28, Phytohemagglutinin (PHA), Phorbol Myristate Acetate (PMA), Tetanus Toxoid (TT) or left unstimulated (-). Proliferation was measured at days 2-3 post-antigenic stimulation by labeling with an XTT reagent during the last 18 hours of culture. Results are the mean and SEM of three independent experiments. Data is expressed as the mean absorbance (A492nm – A690nm) and SEM.

In contrast to the viral kinetics of the T-cell and macrophage tropic laboratory strains and the T-cell tropic clinical isolate, the levels of HIV-1 production for the macrophage tropic clinical isolate declined over the 15 day time course. Input virus was not the cause of the low levels of viral production observed by this clinical isolate as CD8+ T-cells were extensively washed 3 days post-infection and day 0 of the infection assay was denoted at this point. Based on the above results, the HIV_IIIB strain was used in subsequent experiments due to the ability of CD8+ T-cells to support high levels of replication of this virus.

Most HIV-1 strains require CXCR4 or CCR5 as a co-receptor for viral entry, along with CD4 expression. The surface expression of the chemokine receptors on primary CD8+ T-cells was examined by flow cytometry. It was observed that the CXCR4 chemokine receptor was significantly expressed on the surface of the CD8+ T-cells (55.9% ± 2.1, Fig. 1B). The expression of the CCR5 chemokine receptor was only moderately expressed on the CD8+ T-cell-surface (11.6% ± 1.0, Fig. 1C). Comparison of viral replication kinetics was assessed between CD8+ and CD4+ T-cells over a 15 day time course (Fig. 1D). CD8+ T-cells were found to support approximately 10-fold lower levels of HIV-1 replication than CD4+ T-cells in vitro. Fig. (1D &amp 1Aa) illustrate differences in CD8+ T-cell infection and replication kinetics of viral production. A caveat of in vitro infection is the observation of these variances due to differences in CD8+ T-cell populations used for the studies and the requirement for cell culture activation through cytokines/antigens for productive infection.

Productive infection was demonstrated by RT-PCR analysis. Only HIV-1 infected CD8+ and CD4+ T-cells, showed that viral infection was productive as both T-cell populations showed a HIV-1 gag-specific band when RT-PCR analysis was performed (Fig. 2A). Productive infection was also demonstrated in Fig. (2B) where the presence of AZT abrogated HIV-1 replication in CD8+ T-cells isolated from two volunteers. Differences in the levels of viral production were also observed between the two subjects. Subject 2 supported lower levels of HIV-1 production in their isolated CD8+ T-cells in the absence of AZT, indicating differences in viral replication supported by CD8+ T-cells from various individuals.

To further determine whether CD8+ T-cells could support HIV-1 infection, flow cytometry was performed to detect the presence of HIV-1 p24 (HIV-1_gag) intracellularly in this population. As shown in Fig. (3A, circle, upper right quadrant), 6.5% of the total CD8+ T-cell population were infected intracellularly with HIV-1_gag. As a control for the experiment, the contribution of CD8+CD4+ cells to the 6.5% of total infected cells was determined. The isolated and infected CD8+ T-cells were stained additionally with a CD4 antibody and intracellular HIV-1 expression was again measured by flow cytometry (Fig. 3B). From the results, approximately 1.6% of the CD8+CD4+ cells contributed to the total levels of the CD8+ T-cell infection obtained in Fig. (3A). They did not contribute significantly to the total levels of infection due to the low mean fluorescence intensity of this population. Two distinct populations of CD8+ T-cells were observed in the flow cytometric analysis based upon the expression levels of the CD8 molecule. These populations, that have been observed by others, differ in their surface expression of the CD8 cell-surface molecule and have been aptly termed CD8^high and CD8^low expressing populations [24, 25]. A similar experiment illustrated that approximately 20 - 25% of CD4+ T-cells stained intracellularly for HIV-1 (data not shown).

To examine the possibility of CD4 up-regulation in the in vitro infection system used throughout the studies, CD8+ T-cells were infected and monitored for CD4 expression by flow cytometry over a 12 day time course (Fig. 4). Interpretation of the flow cytometry data showed very distinct populations. Analysis of both the uninfected and infected CD8+ T-cell populations showed that CD4 expression on these cells reached a maximum of 0.4% and 0.5%, respectively, on day 12 of culture. This suggests that cell culture conditions, not HIV-1 infection per se, resulted in this observed up-regulation of CD4 on the surface of CD8+ T-cells. The overall majority of CD8+ T-cells were of the single-positive phenotype and the contribution of Double Positive (DP) cells was found to be minimal.

Subsets of CD8+ T-cells have been shown to be significant in the control of HIV-1 infection and disease progression. It has been postulated that higher levels of CD8+ CD38+ T-cells may be associated with poor prognosis and were found to be elevated in individuals who have progressed to AIDS [26, 27]. In contrast, high levels of CD8+HLA-DR+ cells were observed in asymptomatic individuals [28]. In studies with CD8+ CTLs, HLA-DR expression was found to correlate with proliferation and CD28 expression [29]. Based on the possibility that HIV-1 infection may be involved in hindering the function and activities of various key CD8+ T-cell subsets, the susceptibility of the aforementioned subsets to in vitro HIV-1 infection was examined. Viral production was measured in the CD8+ T-cell subset cultures over a 10 day time course by a HIV-1 p24 ELISA. The negative fractions obtained from the subset isolation were also assessed for HIV-1 production. Interestingly, the CD8+CD28+, CD8+CD38+ and CD8+HLA-DR+ subsets all supported very low levels of HIV-1 replication (Fig. 5A). In fact, the levels of viral production decreased over the course of in vitro infection in these subsets. Fig. (5A) also showed that 2.5 -4 fold higher levels of replication were supported in the CD8+CD28-, CD8+CD38- and CD8+HLA-DR- populations, compared to the positive fractions.

Susceptibility of the memory and naïve CD8+ T-cell populations was also examined in response to HIV-1 infection with the HIV_IIIB strain. The CD8+CD45RA- and CD8+CD45RO+ memory cell populations both supported approximately 5 – 10 times higher levels of HIV-1 replication than the CD8+CD45RA+ and CD8+CD45RO- naïve populations (Fig. 6A). The p24 levels obtained in these experiments were lower than the levels found in previous experiments, as these infections were performed using lower levels of PHA stimulation to minimize any possible up-regulation of receptors. We examined receptor expression during the course of infection (Fig. 6B) and confirmed by flow cytometry that there was no alteration in receptor expression during the course of in vitro infection within the memory and naïve CD8+ T-cell populations.

HIV-1 infection has been reported to affect the frequency and function of circulating CD8+ T-cells, thereby hindering the ability of these cells to eradicate viral infection and maintain T-cell mediated immunity. In order to better define the effect of HIV-1 infection on the function and abilities of the CD8+ T-cell memory subset, CD8+CD45RO+ T-cell proliferation in response to stimulation was measured. As assessed by flow cytometry and shown in Fig. (7A), the majority of the isolated CD8+ T-cells were of the naive subset (82.5% CD8+CD45RA+ naive T-cells; 14.5% CD8+CD45RO+ memory T-cells). The purity of our selected CD8+CD45RO+ memory T-cell population was also assessed. The selected memory T-cell population was devoid of any contaminating CD8+CD45RA+ naïve T-cells (Fig. 7B, right histogram) and was composed of approximately 97% pure cells of the CD8+CD45RO+ phenotype (Fig. 7B, left histogram).

Prior to mitogen stimulation, the CD8+CD45RO+ memory T-cells were infected (or left uninfected) for 7 days. Before measuring proliferation, the activation state of the memory cells was assessed before and after infection (Fig. 7C). Based upon the flow cytometry results, the mean fluorescence of CD28 expression nearly doubled (uninfected,

27.3% ± 0.3 infected, 52.3% ± 2.4) during the course of HIV-1 infection and this was found to be significant (p-value = 0.0026). There were marginal increases in CD38 and HLA-DR expression in response to infection. To determine the proliferative capacity of the memory subset, cells were stimulated for 2-3 days with various mitogens both in the presence and absence of HIV-1 infection. Based upon the results, it was found that minimal differences in in vitro lymphocyte proliferation between infected and uninfected CD8+CD45RO+ memory T-cells in response to mitogenic stimulation (Fig. 7D) were observed. Overall, HIV-1 infection resulted in a slightly reduced rate of proliferation in the CD8+CD45RO+ memory subset. Mitogenic stimulation with PHA resulted in a greater than 2-fold increase in proliferation in the uninfected memory T-cell population. However, the presence of PHA resulted in an approximately 30% reduction in the cleavage of XTT in the HIV-1 infected population, indicating a decrease in proliferation. Conversely, αCD3/CD28 stimulation resulted in a nearly 3-fold increase in the absorbance values in both the uninfected and infected CD8+CD45RO+ memory T-cell populations.