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The Journal of Immunology, 2008, 180, 5907 -5915
Copyright © 2008 by The American Association of Immunologists, Inc.
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HIV-1-Specific T Cell Precursors with High Proliferative Capacity Correlate with Low Viremia and High CD4 Counts in Untreated Individuals1

Sandra A. Calarota*, Andrea Foli{dagger}, Renato Maserati{ddagger}, Fausto Baldanti§, Stefania Paolucci§, Mary A. Young, Christos M. Tsoukas||, Julianna Lisziewicz# and Franco Lori2,*,{dagger}

* Research Institute for Genetic and Human Therapy, {dagger} Laboratori di Biotecnologie e Tecnologie Biomediche, {ddagger} Malattie Infettive, and § Servizio di Virologia, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico, Policlinico S. Matteo, Pavia, Italy; Georgetown University Medical Center, Washington, DC 20007; || McGill University Health Center, Montréal, Québec, Canada; and # Genetic Immunity, Budapest, Hungary


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Evidences have recently suggested that the preservation of vaccine-induced memory rather than effector T cells is essential for better outcome and survival following pathogenic SIV challenge in macaques. However, an equivalent demonstration in humans is missing, and the immune correlates of HIV-1 control have been only partially characterized. We focused on the quantification of Ag-specific T cell precursors with high proliferative capacity (PHPC) using a peptide-based cultured IFN-{gamma} ELISPOT assay (PHPC assay), which has been shown to identify expandable memory T cells. To determine which responses correlate with viral suppression and positive immunologic outcome, PBMC from 32 chronically untreated HIV-1-infected individuals were evaluated in response to peptide pools, representing the complete HIV-1 Gag, Nef, and Rev proteins, by PHPC and IFN-{gamma} ELISPOT assay, which instead identifies effector T cells with low proliferative capacity. High magnitude of Gag-specific PHPC, but not ELISPOT, responses significantly correlated with low plasma viremia, due to responses directed toward p17 and p15 subunits. Only Gag p17-specific PHPC response significantly correlated with high CD4 counts. Analysis of 20 additional PBMC samples from an independent cohort of chronically untreated HIV-1-infected individuals confirmed the correlation between Gag p17-specific PHPC response and either plasma viremia (inverse correlation) or CD4 counts (direct correlation). Our results indicate that the PHPC assay is quantitatively and qualitatively different from the ELISPOT assay, supporting that different T cell populations are being evaluated. The PHPC assay might be an attractive option for individual patient management and for the design and testing of therapeutic and prophylactic vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although several lines of evidence suggest that Ag-specific CD8+ T cell responses play a critical role in controlling HIV-1 infection in humans (1, 2, 3, 4) and SIV in the macaque model (5, 6), the precise immune correlates of HIV-1 control remain to be identified. Recent studies in nonhuman primate models of AIDS indicate that the preservation of vaccine-induced Ag-specific central memory CD4+ T cells is essential for better outcome and survival following pathogenic SIV challenge (7, 8), and that vaccine-induced virus-specific central memory CD8+ T cells inversely correlate with virus levels in challenged macaques (9, 10). However, the association of Ag-specific central memory T cells with viral load level and CD4 counts in HIV-1 infection have not been established. A distinct feature of central memory T cells is their ability to proliferate after Ag re-exposure (11). After proliferation, central memory T cells acquire effector functions, such as IFN-{gamma} secretion. In contrast, effector memory T cells retain the ability to secrete IFN-{gamma} but have reduced ability to proliferate (11). Therefore, assays that quantify Ag-specific circulating T cell precursors with high proliferative capacity (PHPC)3 are promising candidates to evaluate correlates of viral control and positive immunologic outcome in natural HIV-1 infection.

The IFN-{gamma} ELISPOT assay is currently used to identify HIV-1-specific T cell responses in humans. This assay quantifies T cells secreting IFN-{gamma} within 18–24 h of Ag stimulation, that is, short-lived effector T cells. Conversely, the cultured IFN-{gamma} ELISPOT assay quantifies Ag-specific T cell PHPC (PHPC assay), likely representing memory T cells (12, 13, 14, 15, 16). In this assay, cells are first cultured with the Ag for 12 days to allow precursor T cells to expand in response to Ag and to acquire effector function. In the meantime, the Ag-stimulated effector T cells should undergo apoptosis (17, 18). It has been reported that Ag-specific T cell responses measured as PHPC correlated with protection against malaria (14, 15, 16) and with suppression of viral rebound in chronic hepatitis B carriers (19). However, evaluation of T cell responses by PHPC assay in HIV-1-infected individuals has not been described previously.

In the present manuscript, Ag-specific T cells quantified by ELISPOT and PHPC assays were compared in order to discern which responses correlate with viral control and preservation of CD4+ T cells in chronically HIV-1-infected individuals naive to antiretroviral treatment. We focused on immune responses to Gag and Nef proteins because they are the most frequently recognized by HIV-1-infected individuals (20, 21, 22, 23) and to Rev protein because it is expressed early in the virus life cycle and Rev-specific immune response is present in long-term asymptomatic HIV-1-infected patients (24, 25). Subsequently, we analyzed the relationship between Ag-specific IFN-{gamma} production, measured by both ELISPOT and PHPC assays, and plasma viral load or CD4 cell counts and found that Ag-specific PHPC (particularly against Gag p17), but not ELISPOT, response is associated with low plasma viremia and high CD4 cell counts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Individual characteristics and samples analyzed

A total of 32 chronically HIV-1-infected individuals naive to antiretroviral treatment and 5 uninfected control subjects were analyzed. All samples from HIV-1-infected individuals were obtained from stored frozen PBMC: 28 samples were from the HIV/AIDS Outpatient Clinic, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo, Pavia, Italy, and 4 samples were from the McGill University Health Center, Montreal, Canada. Table I provides the viral load and CD4+ T cell counts for the 32 HIV-1-infected individuals. The 5 uninfected subjects were recruited from the blood bank at the Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo, Pavia, Italy. Twenty additional samples from chronically untreated HIV-1-infected individuals were obtained from the Women’s Interagency HIV Study (WIHS) cohort, Georgetown University Medical Center participating site. At the time of PHPC analysis, the mean plasma viral load was 11,528 ± 21,527 HIV-1 RNA copies/ml (range: 80–71,000) and the mean CD4 counts was 595 ± 370 cells/mm3 blood (range: 230-1537). PBMC were isolated by standard Ficoll-Hypaque centrifugation and cryopreserved in FBS (Life Technologies) containing 10% DMSO (Sigma-Aldrich) and kept in liquid nitrogen until use. PBMC were thawed, washed, and rested overnight at 37°C, in 5% CO2 atmosphere, in RPMI 1640 medium (Eurobio) containing 2 mM L-glutamine (Eurobio) and supplemented with 10% heat-inactivated FBS (Life Technologies), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Eurobio) (complete culture medium). One day later, cell viability determined by trypan blue exclusion was ≥85%.


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  		Table I. Viral load and CD4 counts in chronically HIV-1-infected individuals naive to antiretroviral treatment

 
Synthetic peptides

Peptides used in this study were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Peptides (15 aa in length with 11-aa overlap) corresponded to the complete sequence of HIV-1 Consensus B Gag, Nef, and Rev. Gag peptides (n = 123) were divided into three pools (41 peptides per pool): Gag p17 pool spanned all p17 and the first 43 amino acids of p24, Gag p24 pool spanned mainly p24, and Gag p15 pool spanned the last 35 amino acids of p24 and all p15. Nef peptides (n = 49) were used as a single pool as well as Rev (n = 27). Peptides were prepared as the corresponding pool at a concentration of circa 100 mg/ml in DMSO, aliquoted, and stored at –20°C.

IFN-{gamma} ELISPOT assay

A human IFN-{gamma} ELISPOT kit (Diaclone) was used. MultiScreen-IP 96-well plates (Millipore) were coated with capture mAb diluted 1/100 in PBS and incubated overnight at 4°C. After several washes with PBS, plates were blocked for 2 h at room temperature with complete culture medium. PBMC were added in duplicate at an input cell number of 1 x 105 cells per well in 100 µl complete culture medium. HIV-1 peptide pools were diluted 1/200 in complete culture medium and 100 µl was added to each well. PHA (5 µg/ml; Sigma-Aldrich) was used as a positive control. Cells resuspended only in complete culture medium served as a negative control. After an incubation of 24 h at 37°C 5% CO2 atmosphere, plates were washed with PBS supplemented with 0.1% Tween 20 (Sigma-Aldrich) (wash buffer) followed by an overnight incubation at 4°C with 100 µl per well biotinylated detection mAb, diluted 1/100 in PBS supplemented with 1% BSA (Diaclone) (PBS + 1% BSA). Plates were washed with wash buffer and 100 µl per well streptavidin-alkaline phosphatase conjugated, diluted 1/1000 in PBS + 1% BSA, was added and incubated at 37°C 5% CO2 atmosphere for 1 h. The wells were then washed with wash buffer, and 100 µl substrate buffer (5-bromo-4-chloro-3-indolyl phosphate/NBT; Diaclone) was added per well. The colorimetric reaction was terminated after 10 min at room temperature by washing several times with tap water. Plates were air-dried and the spots counted using an automated ELISPOT reader system (A-EL-Vis). The mean background medium control response was 2.3 (± 3.4) IFN-{gamma} spots per well. The mean number of IFN-{gamma} spots per well in response to PHA stimulation was 334.2 (± 186).

T cell PHPC assay

PBMC (5 x 105 per ml) were plated in each well in a 24-well flat-bottom tissue culture plate. Cells were stimulated with HIV-1 peptide pools (diluted 1/400, one pool per well), or PHA (5 µg/ml) or complete culture medium (control wells) and cultured at 37°C 5% CO2 atmosphere for 12 days. On days 3 and 7, 500 µl of supernatant per well were removed and replaced with fresh complete culture medium supplemented with 10 IU/ml recombinant human IL-2 (R&D Systems). On day 11, cells from each well were counted and, on day 12, cells were washed three times with complete culture medium and tested at 100 µl per well (2.5 x 104 cells, in duplicate or triplicate) in the same way as the ELISPOT in response to the corresponding Ag used for stimulation. Spots were counted using the same automated ELISPOT reader system and set-up parameters as for the ELISPOT assay. The mean number of IFN-{gamma} spots per well in response to control medium was 10.3 (± 14.1). A mean number of 103.3 (± 92.5) IFN-{gamma} spots per well in response to PHA stimulation was detected.

Data analysis

The mean number of spots from duplicate or triplicate wells was adjusted to 1 x 106 PBMC. Data are presented as ELISPOT counts and PHPC counts. The PHPC counts (net spots/million PBMC) were calculated as follows: (mean number of spots/million PBMC in wells from each pool of peptides minus mean number of spots/million PBMC in wells with control medium) x proliferation index. The proliferation index was calculated as number of Ag-stimulated cells after 12 days of culture divided by the number of control (medium-stimulated) cells after 12 days of culture, to account for the fact that the PHPC assay allows for the expansion of T cells during the culture. ELISPOT and PHPC counts in response to Gag p17, Gag p24, and Gag p15 pools were summed to calculate total Gag response (denoted as Gag). Statistical analysis and scatterplot graphic representations were performed using Statistica software (Statistica for Windows version 7.1). Student’s t test was used to assess differences in net spots/million PBMC detected by ELISPOT and PHPC assays. Correlations between Ag-specific responses determined by ELISPOT and PHPC assays with plasma viral load and CD4 counts were determined by Pearson’s test. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Detection of HIV-1-specific IFN-{gamma} production by ELISPOT and PHPC assays

Using PBMC specimens from 32 chronically HIV-1-infected individuals naive to antiretroviral treatment and 5 uninfected control subjects, ELISPOT and PHPC counts were evaluated in response to peptide pools representing the complete HIV-1 Consensus B Gag, Nef, and Rev proteins. Based on the available number of PBMC from HIV-1-infected individuals, T cell responses to Gag was evaluated in all 32 samples, while Nef and Rev responses were evaluated in 24 and 20 samples, respectively; 18 samples were tested for all three proteins. In HIV-1-infected individuals, the mean number of ELISPOT counts (net spots/million PBMC) in response to Gag, Nef, and Rev was 913.4 (SD: ± 1021.7), 1031 (± 770.7), and 105.8 (± 141.5), respectively (Fig. 1, A–C). The mean number of PHPC counts (net spots/million PBMC) in response to Gag (6005.2 ± 9519.1 spots/million PBMC), Nef (2747.3 ± 5878.6 spots/million PBMC), and Rev (2386.7 ± 5715.2 spots/million PBMC) was 7-fold (p = 0.0053), 3-fold (p = 0.1658), and 23-fold (p = 0.0947) higher than those detected by the ELISPOT assay, respectively (Fig. 1, A–C). The PHPC results did not simply reflect a proportional increase of the ELISPOT counts. In fact, in some patients, the number of spots obtained by PHPC assay was lower than that obtained by ELISPOT assay. To confirm that the PHPC assay was not just an "ELISPOT assay with increased sensitivity," we examined the relationship between the two assays. No correlation was found between ELISPOT and PHPC counts in response to Gag, Nef, and Rev (p = 0.8977, p = 0.3698, and p = 0.3219, respectively) (Fig. 1, D–F). Restricting the analysis to those 18 samples that have been tested for all three proteins gave similar results (data not shown). These results suggest that the PHPC assay is not only quantitatively but also qualitatively different from the ELISPOT assay, confirming that different T cell populations are being evaluated.


Figure 1
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  		FIGURE 1. HIV-1-specific T cell responses determined by IFN-{gamma} ELISPOT and PHPC assays. PBMC from uninfected individuals (n = 5) and chronically untreated HIV-1-infected individuals in response to peptide pools representing HIV-1 Gag (n = 32 samples) (A), Nef (n = 24 samples) (B), and Rev (n = 20 samples) (C) proteins were evaluated. Results are shown as ELISPOT and PHPC counts (net spots/million PBMC). Gag-specific ELISPOT and PHPC counts represent the summed value of net spots formed in response to each of the three HIV-1 Gag pools used for stimulation. Data from each subject were plotted individually, and the horizontal line represents the respective mean number. Correlations between ELISPOT and PHPC counts (net spots/million PBMC) in response to Gag (D), Nef (E), and Rev (F) in PBMC from chronically untreated HIV-1-infected individuals are shown. Linear regression lines, correlation coefficients, and p values are given in the graph.

 
Gag-specific PHPC response correlates with low viremia and high CD4 counts

We then investigated the association between Ag-specific ELISPOT and PHPC responses and the level of plasma viremia in chronically untreated HIV-1-infected individuals. As shown in Fig. 2A, no statistically significant association was found between Gag-, Nef-, and Rev-specific ELISPOT counts and plasma viral load. Conversely, a significant negative correlation between Gag-specific PHPC counts and plasma viral load was found (p = 0.0238) (Fig. 2B).


Figure 2
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  		FIGURE 2. Relationship between Ag-specific IFN-{gamma} ELISPOT or PHPC responses with plasma viral load or CD4 counts in chronically untreated HIV-1-infected individuals. Analysis was performed between plasma viremia and ELISPOT (A) or PHPC assay (B) and between CD4 counts and ELISPOT (C) or PHPC assay (D) in response to peptide pools representing HIV-1 Gag, Nef, and Rev proteins. Data are shown for log10 HIV-1 RNA copies/ml plasma, CD4 cells/mm3 blood, and ELISPOT and PHPC counts (net spots/million PBMC). Gag-specific ELISPOT and PHPC counts represent the summed value of net spots formed in response to each of the three HIV-1 Gag pools used for stimulation. Linear regression lines, correlation coefficients, and p values are given in each graph. Significant correlation (p < 0.05) is in boldface.

 
Next, the association between Ag-specific ELISPOT and PHPC responses and CD4 counts in chronically untreated HIV-1-infected individuals was analyzed. No statistically significant association was found between Gag-, Nef-, and Rev-specific ELISPOT counts and CD4 counts (Fig. 2C); however, a positive trend was observed between Gag-specific PHPC counts and CD4 counts (Fig. 2D). Altogether, the results demonstrate that Gag-specific T cell PHPC are associated with low viral replication and increase of CD4 counts.

The importance of Ag-specific helper CD4 T cells in vivo for sustained memory CD8 T cell responses during chronic infections has been pointed out previously (26). To determine whether CD4 help is also required for optimal responses in the PHPC assay and to establish whether the correlation between the PHPC assay and CD4 counts simply reflected reduced CD4 help in the assay, we compared Gag-specific PHPC response in total and CD4-depleted PBMC from three chronically untreated HIV-1-infected individuals. We found comparable responses upon CD4 cell depletion, suggesting that Gag-specific T cell response detected by PHPC assay is likely independent from CD4 help (data not shown).

ELISPOT and PHPC responses to HIV-1 Gag protein subunits

To further characterize the HIV-1-specific Gag response, we dissected ELISPOT and PHPC responses to the three HIV-1 Gag peptide pools, spanning mainly p17, p24, and p15 (Fig. 3A). Gag-specific ELISPOT and PHPC responses (Fig. 3, B and C, respectively) did not mirror each other in either magnitude or distribution; some subjects who responded in the PHPC assay did not respond in the ELISPOT assay and vice versa, further illustrating that these assays are measuring different T cell responses with distinct specificities.


Figure 3
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  		FIGURE 3. IFN-{gamma} ELISPOT and PHPC responses to HIV-1 Gag protein subunits in PBMC from 32 chronically untreated HIV-1-infected individuals. Schematic representation of the three HIV-1 Gag peptide pools in the complete Gag protein is shown (A). ELISPOT (B) and PHPC (C) responses to the three HIV-1 Gag peptide pools. Data are shown for ELISPOT and PHPC counts (net spots/million PBMC). Error bars represent the SD of the mean.

 
No statistically significant correlation was found between ELISPOT counts in response to the three HIV-1 Gag peptide pools and plasma viremia (Fig. 4A); in contrast, p17- and p15-specific PHPC counts significantly correlated with low plasma viral load (p = 0.0007 and p = 0.0097, respectively) (Fig. 4B).


Figure 4
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  		FIGURE 4. Relationship between IFN-{gamma} ELISPOT or PHPC responses to HIV-1 Gag subunits with plasma viral load or CD4 counts. Correlations between plasma viral load and ELISPOT (A) or PHPC assay (B) in response to Gag 17, Gag 24, and Gag 15 pools as well as between CD4 counts and ELISPOT (C) or PHPC assay (D) in response to Gag 17, Gag 24, and Gag 15 pools in PBMC from 32 chronically untreated HIV-1-infected individuals are shown. Correlations are shown for log10 HIV-1 RNA copies/ml plasma, CD4 cells/mm3 blood, and ELISPOT and PHPC counts (net spots/million PBMC). Linear regression lines, correlation coefficients, and p values are given in the graph. Significant correlations (p < 0.05) are in boldface.

 
No statistically significant association was observed between ELISPOT responses to the three HIV-1 Gag peptide pools and CD4 counts (Fig. 4C). However, when PHPC responses to the three Gag peptide pools were plotted against CD4 counts, only p17-specific PHPC and CD4 counts significantly and directly correlated (p = 0.0173) (Fig. 4D).

Because the association between p17-specific PHPC response and plasma viral load was the most significant one, we evaluated p17-specific PHPC response in 20 additional samples from a separate cohort of chronically untreated HIV-1-infected individuals participating in the WIHS cohort (Georgetown University participating site). The inverse correlation between p17-specific PHPC response and plasma viremia was confirmed (r = –0.5807, p = 0.0073; Fig. 5A). The correlation between p17-specific PHPC response and CD4 counts was also confirmed (r = 0.5942, p = 0.0057; Fig. 5B).


Figure 5
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  		FIGURE 5. Relationship between PHPC response to HIV-1 Gag p17 subunit with plasma viral load or CD4 counts. Correlations between plasma viral load (A and C) or CD4 counts (B and D) and PHPC assay in response to Gag 17 pool in 20 PBMC samples from chronically untreated HIV-1-infected individuals in the WIHS cohort (A and B) or 52 samples (C and D) combining the WIHS cohort and the cohort described in Fig. 4 are shown. Correlations are shown for log10 HIV-1 RNA copies/ml plasma, CD4 cells/mm3 blood, and PHPC counts (net spots/million PBMC). Linear regression lines, correlation coefficients, and p values are given in the graph. Significant correlations (p < 0.05) are in boldface.

 
Combining the two cohorts (total samples = 52) further increased the statistical significance of the inverse correlation between plasma viremia and p17-specific PHPC responses (p = 0.00002; Fig. 5C) and the direct correlation between CD4 counts and p17-specific PHPC responses (p = 0.00009; Fig. 5D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study is the first report demonstrating that Gag-specific responses, measured by PHPC, but not ELISPOT, assay inversely correlated with the magnitude of plasma viral load and directly correlated with CD4+ T cell counts in chronically HIV-1 infected individuals naive to antiretroviral treatment. The results indicate that the presence of Ag-specific T cell precursors with high proliferative capacity is associated with HIV-1 control and preservation of CD4 counts, hallmarks of slow progression to AIDS, similar to what has been recently observed in a nonhuman primate model (7, 8).

HIV-1 Gag p17 contains many overlapping CTL epitopes restricted by several HLA molecules (27). The significant association between Gag PHPC response directed toward the p17 subunit and low viral load is consistent with the observations that HLA-A2-restricted CD8+ T cell responses against an epitope in p17 (aa 77–85, SLYNTVATL), which is presented in high abundance in chronically HIV-1-infected cells (28), inversely correlate with viral load, as measured by tetramer binding staining (4), and has been associated with long-term control of HIV-1 (29). Results from a recent study provides evidence that Gag-derived epitopes are the first to be presented in infected lymphocytes, and that the early presentation of Gag-derived epitopes does not require de novo protein synthesis (30). Because HIV-1 products are translated sequentially as p17, p24, and p15, it can be hypothesized that p17 is presented earlier than the other Gag subunits. Additionally, here we report a significant association between Gag p17-specific PHPC counts and high CD4+ T cell counts. Altogether, our data suggest an important role of Gag p17-specific PHPC responses in control of viremia.

The finding that the mean Ag-specific IFN-{gamma} responses detected by the PHPC assay was higher than those detected by the ELISPOT assay suggests a higher sensitivity of the PHPC assay; however, we did not find any significant correlation between both IFN-{gamma} ELISPOT assays in response to Gag, Nef, and Rev, in agreement with other investigators (12, 31, 32). These results indicate that different IFN-{gamma} producing cell populations are being measured, reflecting the different nature of these two assays. In fact, during the 12-day culture period, T cell precursors expand in response to the Ag and differentiate into effectors cells, while the immediate effector cells present in the culture undergo apoptosis (17, 18).

The ELISPOT assay is widely used to identify Ag-specific T cell responses in both natural HIV-1 infection and following immunization with vaccine candidates. Concordant with studies published by others, we found that the mean Gag- and Nef-specific IFN-{gamma} responses were higher than the mean Rev-specific response by using the ELISPOT assay (20, 21, 22, 23). However, several evidences indicate that Ag-specific responses measured by the ELISPOT assay have no direct effect on plasma viral load or CD4 counts (20, 22, 23, 33, 34). Only the broadness of the ELISPOT response inversely correlated with plasma viremia in the setting of HIV-1 infection; however, this demonstration required the observation of large cohorts (35, 36). It is therefore noteworthy that a small number of patients was sufficient to demonstrate a robust inverse correlation between the immune responses measured by PHPC assay and plasma viral load.

An inverse correlation between HIV-specific CTL responses and plasma viremia had been obtained in the past by the measurement of Ag-specific CTL activity of in vitro expanded PBMC by the chromium-release assay (37, 38, 39, 40). The PHPC assay follows basic principles similar to those of the classical chromium-release CTL assay. The PHPC method not only magnifies the immune response by allowing the Ag-specific T cells present to divide, but also enables the resting memory T cells to differentiate into effector cells. We demonstrated in our study that cells with high proliferative capacity are associated with suppression of viral replication. As such, the PHPC assay provides an alternative to the laborious in vitro re-stimulation CTL assay, allowing the evaluation of Ag-specific precursor T cell responses in an ELISPOT format.

In conclusion, this study represents the first evaluation of HIV-1-specific memory T cells with high proliferative capacity quantified by the PHPC assay in HIV-1-infected individuals naive to antiretroviral treatment. The presence of T cells with high proliferative capacity is associated with low plasma viremia and high CD4+ T cell counts. The PHPC assay might prove useful to evaluate the status of the immune system and the recovery of its function in patients treated with antiretroviral drugs, as well as to assess the immunogenicity of prophylactic and therapeutic vaccines.


   Acknowledgments
 
We thank Valentina La Rosa for technical assistance, Michael Stevens for critical reading of the manuscript, and Sylva Petrocchi and Gabriela Barbieri for editorial assistance. Part of the data in this manuscript was collected by the WIHS Collaborative Study Group with centers (Principal Investigators) located at New York City/Bronx Consortium (Kathryn Anastos), Brooklyn, NY (Howard Minkoff), Washington DC Metropolitan Consortium (Mary Young), The Connie Wofsy Study Consortium of Northern California (Ruth Greenblatt), Los Angeles County/Southern California Consortium (Alexandra Levine), Chicago Consortium (Mardge Cohen), and Data Analysis Center (Stephen Gange).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo (Grant RCR 08038400 2001 to F.L.). The Women’s Interagency HIV Study is funded by the National Institute of Allergy and Infectious Diseases with supplemental funding from the National Cancer Institute, and the National Institute on Drug Abuse (U01-AI-35004, U01-AI-31834, U01-AI-34994, U01-AI-34989, U01-AI-34993, and U01-AI-42590). Funding was also provided by the National Institute of Child Health and Human Development (UO1-HD-32632) and the National Center for Research Resources (M01-RR-00071, M01-RR-00079, and M01-RR-00083). Back

2 Address correspondence and reprint requests to Dr. Franco Lori, Research Institute for Genetic and Human Therapy, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico, Policlinico San Matteo, P. le Golgi 2, 27100 Pavia, Italy. E-mail address: rightpv{at}tin.it Back

3 Abbreviations used in this paper: PHPC, precursors with high proliferative capacity; WIHS, Women’s Interagency HIV Study. Back

Received for publication July 18, 2007. Accepted for publication February 27, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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