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Journal of Virology, August 2007, p. 8009-8015, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.00482-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Memory CD4⁺ T-Lymphocyte Loss and Dysfunction during Primary Simian Immunodeficiency Virus Infection

Yue Sun, Sallie R. Permar, Adam P. Buzby, and Norman L. Letvin^*

Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115

Received 7 March 2007/ Accepted 16 May 2007

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It has long been appreciated that CD4⁺ T lymphocytes are dysfunctional in human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV)-infected individuals, and it has recently been shown that HIV/SIV infections are associated with a dramatic early destruction of memory CD4⁺ T lymphocytes. However, the relative contributions of CD4⁺ T-lymphocyte dysfunction and loss to immune dysregulation during primary HIV/SIV infection have not been fully elucidated. In the current study, we evaluated CD4⁺ T lymphocytes and their functional repertoire during primary SIVmac251 infection in rhesus monkeys. We show that the extent of loss of memory CD4⁺ T lymphocytes and staphylococcal enterotoxin B-stimulated cytokine production by total CD4⁺ T lymphocytes during primary SIVmac251 infection is tightly linked in a cohort of six rhesus monkeys to set point plasma viral RNA levels, with greater loss and dysfunction being associated with higher steady-state viral replication. Moreover, in exploring the mechanism underlying this phenomenon, we demonstrate that the loss of functional CD4⁺ T lymphocytes during primary SIVmac251 infection is associated with both a selective depletion of memory CD4⁺ T cells and a loss of the functional capacity of the memory CD4⁺ T lymphocytes that escape viral destruction.

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CD4⁺ T lymphocytes play an important role in maintaining effective immunity against viral pathogens. They contribute to virus clearance both by providing help to B lymphocytes and by facilitating the generation and activity of cytotoxic T lymphocytes (7). Recent studies suggest that functional CD4⁺ T lymphocytes are also required at the time of immune priming for the development of memory CD8⁺ T lymphocytes (6, 21, 23). Therefore, the mechanisms leading to CD4⁺ T-lymphocyte loss during primary human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection are of central importance in AIDS immunopathogenesis.

Chronic HIV/SIV infection is characterized by a slow, steady loss of CD4⁺ T lymphocytes. In contrast, the loss of CD4⁺ T lymphocytes during acute infection has been considered both moderate and limited. However, recent studies have demonstrated that the intense viral replication that occurs during the first several weeks after SIV/HIV infection is associated with a profound depletion of the memory subset of CD4⁺ T lymphocytes (10-13, 18, 25-27). The degree to which the extent of HIV/SIV replication and memory CD4⁺ T-lymphocyte loss are associated with one another during the period of primary infection remains undefined.

CD4⁺ T lymphocytes of HIV/SIV-infected individuals have a number of well-characterized functional defects, including reduced proliferative capacity and defective production of interleukin-2 (IL-2) (2-5, 8, 14, 15, 17, 22, 24, 28, 29). It is well known that naïve and memory CD4⁺ T lymphocytes differ in their cytokine production capacity. Memory cells have the capacity to produce significant quantities of cytokines, while naïve cells produce little or no cytokine (20). Since a selective loss of memory CD4⁺ T lymphocytes has been demonstrated during the acute phase of HIV/SIV infection, the loss of effective CD4⁺ T-lymphocyte help in this setting may simply reflect the selective destruction of activated memory CD4⁺ T lymphocytes, the cells that have the capacity to proliferate and produce cytokine. However, the depletion of memory CD4⁺ T lymphocytes cannot be the only explanation for the CD4⁺ T-lymphocyte defect in HIV/SIV-infected individuals, since it has been shown that a large number of HIV-specific memory CD4⁺ T cells secrete gamma interferon (IFN-) but are deficient in their ability to produce IL-2 (22, 28). These findings suggest that a functional defect may exist in the HIV/SIV-specific memory CD4⁺ T lymphocytes that escape viral destruction.

In the current study, we evaluated CD4⁺ T lymphocytes and their functional repertoire during primary SIVmac251 infections in six rhesus monkeys. We show that loss of memory CD4⁺ T lymphocytes during primary SIVmac251 infection in this cohort of animals is associated with set point plasma viral RNA levels and that there is a loss of functional capacity in the memory CD4⁺ T lymphocytes that escape viral destruction.

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Animals and viruses. Heparinized blood samples were obtained from Mamu-A*01^– rhesus monkeys (Macaca mulatta). All animals were maintained in accordance with the America Association for Accreditation of Laboratory Animal Care guidelines. The challenge virus used in this study was uncloned pathogenic SIVmac251.

CD4⁺ T-lymphocyte counts and plasma viral RNA levels. Peripheral blood CD4⁺ T-lymphocyte counts were calculated by multiplying the total lymphocyte count by the percentage of CD3⁺CD4⁺ T cells determined by monoclonal antibody (MAb) staining and flow cytometric analysis. Plasma viral RNA levels were measured by an ultrasensitive branched DNA amplification assay with a detection limit of 125 copies per milliliter (Bayer Diagnostics, Berkeley, CA).

Antibodies. The antibodies used in this study were directly coupled to fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-Texas Red (ECD), peridinium chlorophyll protein-Cy5.5 (PerCP-Cy5.5), PE-Cy7, AmCyan, Pacific Blue, allophycocyanin (APC), and Alexa Fluor 700. All reagents were validated and titrated using rhesus monkey peripheral blood mononuclear cells (PBMCs). The following MAbs were used: anti-CCR7-FITC (150503; R&D Systems), anti-CD95-PE (DX2; BD Biosciences), anti-CD45RA-ECD (2H4; Beckman Coulter), anti-CD28-PerCP-Cy5.5 (L293; BD Biosciences), anti-IFN--PE-Cy7 (B27; BD Biosciences), anti-CD3-Pacific Blue (SP34-2; BD Biosciences), anti-CD4-AmCyan (L200; BD Biosciences), anti-IL-2-APC (MQ1-17H12; BD Biosciences), anti-CD8-Alexa Fluor 700 (RPA-T8; BD Biosciences), anti-Ki-67-FITC (B56; BD Biosciences), anti-perforin-FITC (Pf-344; Mabtech), and anti-granzyme B-Alexa Fluor 700 (GB11; BD Biosciences).

PBMC stimulation and intracellular cytokine staining. PBMC were incubated at 37°C in a 5% CO₂ environment for 6 h in the presence of RPMI 1640/10% fetal calf serum medium alone (unstimulated), a pool of 15-mer Gag peptides (2 µg/ml of each peptide), or staphylococcal enterotoxin B (SEB) (5 µg/ml; Sigma-Aldrich) as a positive control. All cultures contained monensin (GolgiStop; BD Biosciences) as well as 1 µg/ml anti-CD49d (BD Biosciences). The cultured cells were stained with MAbs specific for cell surface molecules, including CD3, CD4, CD8, CD28, and CD95. After being fixed with Cytofix/Cytoperm solution (BD Biosciences), cells were permeabilized and stained with antibodies specific for IFN- and IL-2. Labeled cells were fixed in 1% formaldehyde-phosphate-buffered saline. Samples were collected with a LSR II instrument (BD Biosciences) and analyzed using FlowJo software (Tree Star). Approximately 500,000 to 1,000,000 events were collected per sample. The background level of cytokine staining varied from sample to sample but was typically <0.01% of the CD4⁺ T cells and <0.05% of the CD8⁺ T cells. The only samples that were considered positive were those for which the percentages of cytokine-staining cells were at least twice those of the background or for which there was a distinct population of cytokine bright-positive cells.

Statistical analyses. Statistical analyses and graphical presentations were computed with GraphPad Prism. The Spearman correlation test was performed to analyze the association between the different parameters.

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Plasma viral RNA levels during primary SIVmac251 infection. Six Mamu-A*01^– rhesus monkeys were inoculated by the intravenous route with SIVmac251 and monitored for 140 days to evaluate their clinical, immunologic, and virological status. Plasma virus was initially detected at day 7 after infection and was maximal at day 14 (Fig. 1A). Variation was observed between monkeys in their set point plasma viral RNA levels on day 140. Two animals had relatively low levels of viral replication (approximately 10⁴ copies per ml of plasma), and two animals had moderate levels of viral replication (approximately 10⁶ copies per ml of plasma). Two additional animals had very high levels of viral replication, with set point plasma viral RNA levels of approximately 10⁸ copies per ml of plasma, and these animals died by day 150 postchallenge. A similar variation between monkeys was also observed when we compared the area under the curve of the log viral loads as a function of time for these animals (Fig. 1B).

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FIG. 1. Plasma viral RNA levels during primary SIVmac251 infection. (A) Dynamics of plasma viral RNA levels. (B) Area under the curve of viral RNA levels through day 140 postinfection.

Set point plasma viral RNA levels are associated with the loss of peripheral blood central memory and effector memory CD4⁺ T lymphocytes during primary SIVmac251 infection. The peripheral blood absolute CD4⁺ T-cell counts of the monkeys remained relatively stable during the period of acute SIVmac251 infection (Fig. 2A), and no correlation was observed in these monkeys between the proportion of absolute CD4⁺ T-cell counts lost, defined as (Day 0 – Day 140)/Day 0 (where Day 0 is the value on the day of infection and Day 140 is the value on day 140 postinfection), and the set point plasma viral RNA levels during primary infection (Fig. 2B).

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FIG. 2. Set point plasma viral RNA levels are associated with the loss of peripheral blood central memory and effector memory CD4+ T lymphocytes. Six Mamu-A*01– rhesus monkeys were infected with SIVmac251 by the intravenous route and monitored for 140 days. Absolute CD4+ T-cell counts were assessed at various times during the course of the primary infection. Data from individual monkeys are shown from left to right in increasing order of the animals' set point plasma viral RNA levels (A). No correlation was observed between the proportion of absolute CD4+ T-cell counts lost, defined as (Day 0 – Day 140)/Day 0, and the set point plasma viral RNA levels through day 140 postinfection (B). CD4+ T lymphocytes were then divided into naïve, central memory, and effector memory subpopulations based on their expression of CD28 and CD95. The percentage of total CD4+ T lymphocytes represented by each of these three subsets was assessed at various times during the course of the primary infection (C). A positive correlation was observed for these animals between their loss of central memory and effector memory CD4+ T lymphocytes, defined as (Day 0 – Day 140)/Day 0, and their set point plasma viral RNA levels (D). Naïve CD4+ T cells showed a negative correlation. (C and D).

To examine the loss of memory CD4⁺ T cells during acute infection, total peripheral blood CD4⁺ T lymphocytes were divided into naïve, central memory, and effector memory subpopulations based on their expression of CD28 and CD95. The relative representation of each CD4⁺ T-cell subset was assessed at regular intervals during the course of primary infection, and the association of the preservation of each subset with plasma viral RNA levels was evaluated. Central memory CD4⁺ T lymphocytes were preserved in the animals with low set point viral loads. In contrast, this population of cells was selectively depleted during the initial phase of intensive viral replication in the animals with high set point viral loads (Fig. 2C). In fact, a positive correlation was apparent between the loss of central memory CD4⁺ T lymphocytes, defined as (Day 0 – Day 140)/Day 0, and the set point plasma viral RNA levels in these animals (Fig. 2D). Peripheral blood effector memory CD4⁺ T lymphocytes were transiently increased at day 14 and then declined in monkeys with high set point viral loads (Fig. 2C). A similar positive correlation was observed between the loss of effector memory CD4⁺ T lymphocytes and set point viral loads in these animals (Fig. 2D). The relative representation of naïve CD4⁺ T lymphocytes showed the expected inverse changes (Fig. 2C and D).

Memory CD4⁺ T-lymphocyte subsets defined by different antibody combinations showed similar dynamics following SIVmac251 infection. Several different phenotypic profiling strategies have been advanced for differentiating naïve, central memory, and effector memory CD4⁺ T-lymphocyte subsets (1, 13, 19). We sought to determine whether these strategies differ in their power to define the subset of memory CD4⁺ T lymphocytes that is lost in rhesus monkeys following SIVmac251 infection. We therefore divided total peripheral blood CD4⁺ T lymphocytes into naïve, central memory, and effector memory subpopulations based on their expression of CD28/CD95, CD45RA/CD95, CCR7/CD28/CD95, or CCR7/CD45RA, and the fraction of each CD4⁺ T-lymphocyte subset was quantitated at fixed intervals during the course of primary infection. Loss of both central memory and effector memory cells was observed for these six monkeys during the first 150 days following infection, and the loss of CD4⁺ T-lymphocyte subsets, defined by the various antibody combinations, had similar associations with set point viral loads in these monkeys (Fig. 3). Therefore, the peripheral blood memory CD4⁺ T-lymphocyte subsets defined by these different antibody combinations had similar dynamics following SIVmac251 infection.

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FIG. 3. Loss of memory CD4+ T-lymphocyte subsets defined by various antibody combinations had similar correlations with set point plasma viral RNA levels. Total CD4+ T lymphocytes were divided into naïve, central memory, effector memory, and intermediate memory subpopulations based on their expression of CD28/CD95, CD45RA/CD95, CD28/CCR7/CD95, or CCR7/CD45RA. The percentage of total CD4+ T lymphocytes represented by each of these subsets was assessed at various times during the course of primary infection. Comparable positive correlations were observed between the loss of memory CD4+ T-lymphocyte subsets defined by these different antibody combinations and the set point plasma viral RNA levels in these animals.

Low frequency of Gag-specific T-cell responses following SIVmac251 infection. To assess the changes that occurred in antigen-specific CD4⁺ and CD8⁺ T-lymphocyte function, we prospectively evaluated SIV-specific T-cell responses in these monkeys at regular intervals following infection. PBMCs were exposed in vitro to a pool of Gag peptides, and the CD4⁺ or CD8⁺ T lymphocytes were assessed for IFN- and IL-2 production. Very few IFN--producing CD4⁺ and CD8⁺ T lymphocytes (Fig. 4), and no IL-2-producing CD4⁺ and CD8⁺ T lymphocytes, were detected in these Mamu-A*01^– monkeys (data not shown). This result is consistent with previous studies from our laboratory (9, 10). SIV-specific antibody responses in these monkeys were also evaluated. At day 84, anti-Env antibody responses were detected for monkeys with low to moderate set point viral RNA levels, and no responses were detected for animals with high set point viral RNA levels (data not shown).

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FIG. 4. Low frequency of Gag-specific T-cell responses following SIVmac251 infection. Six rhesus monkeys were infected with SIVmac251 by the intravenous route on day 0, and their CD4+ (A) or CD8+ (B) T lymphocytes were assessed at various times after infection for IFN- production following in vitro exposure to a Gag peptide pool.

Loss of functional CD4⁺ T lymphocytes but preservation of functional CD8⁺ T lymphocytes following SIVmac251 infection. We then sought to evaluate the ability of the CD4⁺ and CD8⁺ T-lymphocyte populations of these monkeys to produce cytokines upon SEB stimulation. The percentage of CD4⁺ T lymphocytes that secreted IFN- (Fig. 5A) or IL-2 (Fig. 5B) was maintained in animals with low set point viral loads. However, the capacity of these populations of cells to secrete IFN- (Fig. 5A) or IL-2 (Fig. 5B) was reduced dramatically by day 14, at the time of peak viremia, in animals that developed moderate to high set point viral loads. Furthermore, the reduction in the potential of the CD4⁺ T-lymphocyte populations to produce IFN- (Fig. 5C) or IL-2 (Fig. 5D) persisted and correlated with set point plasma SIV RNA levels in these monkeys. In contrast to the CD4⁺ T-lymphocyte populations, the CD8⁺ T lymphocytes did not demonstrate a comparable reduction in their ability to produce cytokines following SEB stimulation (Fig. 5A and B), and no correlations were detected between reductions in the potential of the CD8⁺ T-lymphocyte populations to produce cytokines and the set point viral loads in these monkeys (Fig. 5C and D).

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FIG. 5. Loss of functional CD4+ T lymphocytes but preservation of functional CD8+ T lymphocytes following SIVmac251 infection. Six rhesus monkeys were infected with SIVmac251, and their CD4+ and CD8+ T lymphocytes were assessed at various times after infection for IFN- (A) or IL-2 (B) production following in vitro stimulation with SEB. Results for individual monkeys are shown from left to right in increasing order of the animals' set point plasma viral RNA levels. A positive correlation was observed for these animals between the loss of the ability of their CD4+ T lymphocytes to produce IFN- (C) or IL-2 (D), defined as (Day 0 – Day 140)/Day 0, and their set point plasma viral RNA levels.

Loss of function of memory CD4⁺ T lymphocytes remaining after SIVmac251 infection. Finally, we sought to explore the mechanism underlying this loss of cytokine production capacity by all CD4⁺ T lymphocytes during primary SIVmac251 infection. Cytokines are produced predominantly by memory CD4⁺ T lymphocytes; however, memory CD4⁺ T cells can be selectively destroyed in monkeys during the first 14 days following SIVmac251 infection. The global loss of the capacity to produce cytokines in these monkeys may therefore be due to a selective depletion of activated memory CD4⁺ T lymphocytes or be a consequence of memory CD4⁺ T lymphocytes having lost their capacity to produce cytokines.

To evaluate the relative contributions of each of these mechanisms to the loss of CD4⁺ T-lymphocyte function during the period of primary infection, we characterized the functional capacity of the residual memory CD4⁺ T lymphocytes in these monkeys. The fraction of memory CD4⁺ T lymphocytes (CD95⁺) that produced IFN- or IL-2 in response to SEB was assessed by intracellular staining at regular intervals following infection. Production of Ki-67, perforin, and granzyme B in unstimulated memory CD4⁺ T lymphocytes was also monitored (Fig. 6A). The ability of memory CD4⁺ T lymphocytes to secrete IFN- and IL-2 was maintained in animals with low set point viral loads. In contrast, the capacity of this subpopulation of cells to express these cytokines was dramatically reduced by the time of peak viremia on day 14 in animals with moderate to high set point viral loads. Furthermore, this reduction in the potential of the memory CD4⁺ T-lymphocyte populations to produce IFN- persisted and correlated with set point plasma SIV RNA levels in these monkeys (Fig. 6B). Monkeys with low to moderate set point viral loads had a high turnover of memory CD4⁺ T lymphocytes as determined by expression of Ki-67, while this cell population in monkeys with high viral loads had a poor proliferative capacity (Fig. 6A). Memory CD4⁺ T lymphocytes of animals with high viral loads also demonstrated a transient increase in perforin (Fig. 6A) and granzyme B (data not shown) expression at the time of peak viremia on day 14. These data therefore indicate that the loss of functional CD4⁺ T lymphocytes during primary SIVmac251 infection is associated with both a selective depletion of memory CD4⁺ T lymphocytes and a loss of the functional capacity of the remaining memory CD4⁺ T lymphocytes.

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FIG. 6. Loss of function in the memory CD4+ T lymphocytes following SIVmac251 infection. The fraction of total memory CD4+ T lymphocytes (CD95+) that produced IFN- or IL-2 in response to SEB, or the fraction of total memory CD4+ T lymphocytes expressing Ki-67 or perforin at various times following infection, was assessed by MAb staining and flow cytometric analysis (A). A positive correlation was observed for these animals between the loss of the ability of their memory CD4+ T lymphocytes to produce IFN-, defined as (Day 0 – Day 140)/Day 0, and their set point plasma viral RNA levels (B).

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Although the peripheral blood CD4⁺ T-cell counts of the rhesus monkeys in the present study remained relatively stable during the period of acute SIVmac251 infection, central memory CD4⁺ T lymphocytes were selectively depleted during this early phase of intensive viral replication. In fact, the dramatic loss of central memory CD4⁺ T cells was apparent when the expression of a number of different combinations of cell surface molecules—CD28/CD95, CD45RA/CD95, CCR7/CD28/CD95 and CCR7/CD45RA—was used to define this cell subpopulation. Moreover, the loss of the CD4⁺ T-lymphocyte subset defined by each of these antibody combinations had a comparable positive association with set point viral load. This finding supports the argument that these different combinations of surface molecules do indeed define the same subpopulation of memory CD4⁺ T lymphocytes.

In a previous study in which we assessed peripheral blood memory CD4⁺ T-lymphocyte subpopulations in a group of vaccinated and SIVmac251-challenged monkeys, we observed no correlation between the preservation of effector memory CD4⁺ T cells and survival (10). However, in the present study, we observed a positive correlation between the loss of effector memory CD4⁺ T lymphocytes and set point viral loads in six unvaccinated SIVmac251-infected animals. The apparent inconsistency of the findings in these two studies is likely a consequence of differences in the rates of disease progression and plasma viral RNA levels in the cohorts of monkeys. In the earlier study, the set point plasma viral RNA levels of the infected animals that were previously vaccinated were maintained at 10³ to 10⁶ copies per ml of plasma. However, in the present study, two unvaccinated rapid-progressor monkeys had set point plasma viral RNA levels of 10⁸ copies per ml, and these two animals died by day 150 following infection. During primary SIVmac251 infection, the magnitude of the effector memory CD4⁺ T-cell subset is controlled by two factors, the destruction of the CCR5⁺CD4⁺ effector memory cells by the virus and the proliferation and regeneration of this cell population from central memory precursors. We and others have shown that in rapid-progressor animals, the proliferation of memory CD4⁺ T cells, as determined by Ki-67 expression, is impaired (16). However, in normal-progressor or slow-progressor animals, the depletion of effector memory CD4⁺ T cells can be partly or completely masked by newly generated cells from the rapidly proliferative memory pool (Fig. 6A). Therefore, although loss of effector memory CD4⁺ T cells occurs in vaccinated and infected animals, this pool of cells is also being regenerated. This equilibrium masks an association between the loss of effector memory CD4⁺ T cells and plasma viral loads. However, the memory CD4⁺ T cells failed to replenish the effector memory CD4⁺ T cells in the rapid-progressor monkeys. Thus, a positive correlation was found between the loss of effector memory CD4⁺ T cells and plasma viral loads in these animals.

While CD4⁺ T lymphocytes mediate helper immune function in HIV/SIV-infected individuals (7), we observed a subset of memory CD4⁺ T cells with cytolytic capacities in the present study. A population of effector memory CD4⁺ T lymphocytes was present in the peripheral blood of the monkeys at a low frequency before SIV infection and was markedly expanded at the time of peak viremia on day 14 following infection. This population of memory CD4⁺ T lymphocytes also had a transient increase in perforin and granzyme B expression on day 14. These results suggest that effector memory CD4⁺ T cells may play a role in containing viral infections, perhaps through cytolytic activity.

CD4⁺ T lymphocytes are dysfunctional in HIV/SIV-infected individuals (2-5, 8, 14, 15, 17, 22, 24, 28, 29). While a reduction in the expression of both IL-2 and IFN- by these CD4⁺ T lymphocytes has been demonstrated, the kinetics of the emergence of these defects and the mechanisms underlying this dysfunction have not been fully elucidated. In the present study, we show that deficiencies in cytokine production by CD4⁺ T lymphocytes are global, apparent even after exposure of the cells to the polyclonal T-cell superantigen SEB, and loss of CD4⁺ T-lymphocyte function directly correlates with set point plasma SIV RNA levels. We also demonstrate that loss of functional CD4⁺ T lymphocytes during primary SIVmac251 infection is explained by both a selective depletion of central and effector memory CD4⁺ T cells and a loss of the functional repertoire of the remaining memory CD4⁺ T lymphocytes.

The findings in the present study indicate that clinical measurement of peripheral blood total CD4⁺ T-lymphocyte counts during acute HIV infection may not have predictive value. Rather, the quantitation of memory CD4⁺ T-lymphocyte loss and the assessment of CD4⁺ T-lymphocyte function during the first several weeks after infection may provide important immune correlates of clinical outcome.

 
We are grateful to Mark Cayabyab and Victoria Love for helpful conversations.

This work was supported in part with funds from the intramural research program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH; Center for HIV-AIDS Vaccine Immunology (CHAVI) grant AI067854; and Harvard University Center for AIDS Research (CFAR) program P30 AI060354.

 
* Corresponding author. Mailing address: Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, RE113, P.O. Box 15732, Boston, MA 02215. Phone: (617) 667-2766. Fax: (617) 667-8210. E-mail: nletvin{at}bidmc.harvard.edu

Published ahead of print on 23 May 2007.

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Journal of Virology, August 2007, p. 8009-8015, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.00482-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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