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CD8⁺ T Cell Dynamics during Primary Simian Immunodeficiency Virus Infection in Macaques: Relationship of Effector Cell Differentiation with the Extent of Viral Replication¹

Valérie Monceaux^*, Laurence Viollet^2,*, Frédéric Petit^2,*, Raphaël Ho Tsong Fang^*, Marie-Christine Cumont^*, John Zaunders, Bruno Hurtrel^* and Jérôme Estaquier^3,*

^* Unité de Physiopathologie des Infections Lentivirales, Institut Pasteur, Paris, France; and Centre for Immunology, St Vincent’s Hospital, Darlinghurst, New South Wales, Australia

	Abstract

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Immunological and virological events that occur during the earliest stages of HIV-1 infection are now considered to have a major impact on subsequent disease progression. We observed changes in the frequencies of CD8^bright T cells expressing different chemokine receptors in the peripheral blood and lymph nodes of rhesus macaques during the acute phase of the pathogenic SIVmac251 infection; the frequency of CD8^bright T cells expressing CXCR4 decreased, while the frequency of those expressing CCR5 increased. These reciprocal changes in chemokine receptor expression were associated with changes in the proportion of cycling (Ki67⁺) CD8^bright T cells, and with the pattern of CD8^bright T cell differentiation as defined by expression of CCR7 and CD45RA. In contrast, during the primary phase of the attenuated SIVmac251nef infection, no major change was observed. Whereas during the acute phase of the infection with pathogenic SIV (2 wk postinfection) no correlate of disease protection was identified, once the viral load set points were established (2 mo postinfection), we found that the levels of cycling and of CCR5- and CXCR4-positive CD8^bright T cells were correlated with the extent of viral replication and therefore with SIV-infection outcome. Our data reveal that, during primary SIV infection, despite intense CD8 T cell activation and an increase in CCR5 expression, which are considered as essential for optimal effector function of CD8⁺ T cells, these changes are associated with a poor prognosis for disease progression to AIDS.

	Introduction

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Chemokines and chemokine receptors play a critical role in T cell homeostasis and in the migration of various cell types including memory and effector T cells (1, 2, 3, 4). Two important subdivisions of chemokine receptors are those which control recirculation of resting naive and memory cells through T cell areas of secondary lymphoid tissue, CXCR4 and CCR7, and those which direct activated and effector T cells under inflammatory conditions, CXCR3 and CCR5 (5, 6, 7, 8, 9). In antiviral immunity, this localization of effector CD8⁺ T cells plays an extremely important role in promoting host recovery and virus clearance (10, 11, 12). The primary acute phase of HIV type-1 (HIV-1) and SIV infections is characterized by an early burst of viral replication, an exponential increase in plasma viral load, the dissemination and seeding of the virus in all peripheral lymphoid organs, and the induction of the host immune response against the virus (13, 14, 15, 16, 17, 18). The plasma viral load reaches a steady-state at the end of this primary phase, 2–6 mo after infection that is predictive of progression toward AIDS (19, 20, 21, 22, 23).

CD8⁺ T cells are thought to play an important role in controlling HIV and SIV viral replication and an HIV-specific CTL response is rapidly induced at the same time as the decline in viral titers (18, 24, 25, 26, 27). However, HIV-infected individuals usually fail to control virus replication efficiently. Several reports have shown an apparent nonresponsiveness of circulating CD8⁺ T cells that may reflect one mechanism by which immunodeficiency viruses escape the immune response (28, 29, 30, 31, 32).

However, an optimal antiviral defense requires efficient mechanisms for targeting activated T cells to sites of infection. Insight into the regulation of CD8 T cell trafficking is therefore essential to understanding how viral infections are controlled. CCR5 expression seems to be required for optimal CD8 effector T cell function in vivo and CCR5 may be involved in the recruitment and positioning of virus-specific T cells (33, 34, 35). In HIV-1 infection in particular, where CCR5⁺CD4⁺ T cells are the main targets in early infection, colocalization of CCR5⁺ effector CD8⁺ T cells would be predicted to be especially important. In contrast, CCR7 functions as a homing receptor in migration of CD8⁺ T cells to the lymph nodes (LNs), ⁴ but is down-regulated after stimulation with Ag (36, 37). The following differentiation lineage for CD8⁺ T cells has therefore been proposed: CD45RA⁺CCR7⁺ (naive) CD45RA-CCR7⁺ (central memory, TCM) CD45RA-CCR7^– (effector memory, TEM) CD45RA⁺CCR7^– (terminally differentiated T cells, TDT) (32). Moreover, the CCR7^– subset of CD8⁺ T cells express perforin-containing granules (a feature of CTLs) (38, 39). It is unclear how the expression of chemokine receptors on CD8⁺ T cells is affected by the physiological response during primary HIV infection.

Circulating lymphocytes represent only 2% of all T lymphocytes, whereas LN lymphocytes account for 30% of the lymphocyte pool (40) and are considered as the main sites of both ongoing T cell activation and viral replication. The SIV-infection model is especially well-suited for analyzing T cell dynamics and changes in chemokine receptor expression on CD8⁺ T cells during primary infection, because it is possible to control precisely the inoculum used, the timing and route of inoculation, and to obtain LNs early in infection. In contrast, study of acute HIV-1 infection is extremely limited by availability of both peripheral blood and lymphoid tissue, and in nearly all cases involves highly variable cross-sectional data rather than longitudinal data from different individuals with different outcomes. In this study rhesus macaques were either infected with the pathogenic SIVmac251 strain or the attenuated SIVmac251nef molecular clone. We looked at the relationships between changes in chemokine receptor expression on CD8⁺ T cells, in both peripheral blood and LNs, and the rate of viral replication and progression to AIDS.

	Materials and Methods

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Animals, virus infection, and LN collection

Rhesus macaques (Macaca mulatta) that had been housed and cared for in accordance with European Union guidelines were inoculated i.v. (n = 10) with 10 50% animal-infectious doses of the pathogenic SIVmac 251 strain. Four animals were inoculated with the SIVmac251nef molecular clone provided by A. M. Aubertin (Institut National de la Santé et de la Recherche Médicale Unité 74, Strasbourg, France). SIVmac251nef was derived from the BK28 clone (41). Axillary and inguinal LNs were collected at selected times after infection and placed in RPMI 1640, before being cut and gently teased through a fine sterile mesh. Cells were counted under a light microscope. The total number of lymphocytes per LN was determined as function of the initial weight. The numbers of each lymphoid subset within LNs were calculated by multiplying the percentage of lymphoid cells, as assessed by flow cytometry using specific mAbs, by the numbers of cells retrieved from one LN, as previously described (23).

Determination of serum viral load and quantitative analysis of productively infected cells

Determination of serum viral load. Viral load was determined by quantification of serum SIV RNA level by real-time quantitative RT-PCR. SIV RNA copy number was determined by reference to a standard curve prepared by RT-PCR amplification of serial dilutions of an in vitro-transcribed SIV gag RNA. RNA dilutions were divided into aliquots and frozen immediately at –70°C. RNA from sera of SIV-infected macaques was purified using the TRI REAGENT BD kit (Molecular Research Center). Primers specific to the SIV RNA sequence were designed (5'-GCA GAG GAG GAA ATT ACC CAG TAC-3' and 5'-CAA TTT TAC CCA GGC ATT TAA TGT T-3'). A probe, also specific to the SIV RNA sequence, was tagged with a fluorescent reporter dye (FAM) at the 5' end and a quencher dye at the 3' end (5'-TGT CCA CCT GCC ATT AAG CCC GA-3'). A fluorescence signal was detected with an ABI Prism 7700 sequence detector (Applied Biosystems). Data were captured and analyzed using the Sequence Detector Software (SDS).

Productively infected cells. We quantified the viral load in frozen LNs by in situ hybridization using a ³⁵S-labeled RNA probe derived from pBluescript encoding the SIVmac nef gene. Infected cells, detected as spots, were counted in the paracortical zone on a minimum of three sections using a Nikon-FXA microscope. The numbers of LN-positive cells (spots) counted were then divided by the surface of the entire LN section, and results were expressed as positive cell numbers/2 mm² section as previously described (17, 23, 42). The mean count obtained for three slides is indicated.

Lymphocyte immunophenotyping and flow cytometry

Surface staining. Cells were quantified by flow cytometry with the following fluorochrome-labeled mAbs: anti-rhesus monkey CD3 conjugated with FITC (clone FN18; BioSource International), anti-human CD4 conjugated with allophycocyanin (clone M-T477; BD Biosciences), anti-human CD8 conjugated with PE, PerCP or allophycocyanin (clone Leu2a; BD Biosciences), anti-human CD16-PE (clone 3G8), anti-human CD20-PE (clone 2H7), anti-human CD45RA-FITC and CD45RA-PE (clone 2H4; Coulter). Abs were added to 100 µl of whole blood collected on EDTA or to 2 x 10⁵ LN cells. Cells were incubated for 15 min at room temperature. Erythrocytes were lysed using 2 ml of diluted IOTest 3 lysing solution (Beckman Coulter). The cells were washed once in PBA buffer (PBS-1% BSA-10 mM NaN₃), and resuspended in PBA containing 1% paraformaldehyde (PBA-PF). Anti-human-CD45RO Abs could not be used as they do not cross-react with the homologous monkey Ag. For anti-human CCR7 staining (clone 2H4; BD Biosciences) a multistep procedure was used. Briefly, cells were first incubated with purified mouse anti-human CCR7 Ab (20 min at 4°C) and then washed twice in PBA buffer. The cells were then incubated with a biotinylated rat anti-mouse IgM (clone R6-60.2; BD Biosciences) (20 min at 4°C). After two washes in PBA buffer, cells were incubated with streptavidin-allophycocyanin (BD Biosciences) for 20 min at 4°C. After extensive washing, Abs against CD45RA, and CD8 were added. Erythrocytes were then lysed and the cells were washed and fixed before flow cytometric analysis. Other Abs were used to analyze the activation state of T cells subsets. These Abs included anti-HLADR-PE and anti-CD28-PE (BD Biosciences/BD Pharmingen). To quantify chemokine receptor expression, anti-human CXCR4-PE (BD Biosciences/BD Pharmingen) and unlabeled purified mouse anti-human CCR5 were used. Briefly, 50 µl of whole blood or 2 x 10⁵ LN cells were incubated with either Abs (10 µg/ml) or matched isotype controls (Beckman Coulter) for 1 h at 4°C. After washing, cells were then incubated with a 1/50 dilution of the FITC-conjugated F(ab')₂ goat anti-mouse IgG (DAKO) for CCR5 (45 min at 4°C). After washing, cells were incubated with a saturating concentration of mouse Ig or rabbit Ig. After 30 min at 4°C, labeled anti-CD8 mAb was added. Finally, erythrocytes were lysed and the cells were fixed in 1% paraformaldehyde.

Intracellular TIA-1 and Ki67 stainings. For intracellular stainings, 100 µl of whole blood or 2 x 10⁵ LN cells were incubated with CD8-PerCP (clone SK1; BD Biosciences) for 15 min at room temperature. Erythrocytes were lysed and washed with PBS. Cells were then fixed and permeabilized with 2 ml of the Permeafix reagent (BD Pharmingen/BD Biosciences) (20 min at room temperature). After washings with PBA containing 0.05% saponin (PBA-Sap), the cells were incubated for 45 min with either Ki67-FITC mAb (clone Ki67; DAKO) or TIA-1-FITC mAb (clone GMP-17; Beckman Coulter). After two washes in PBA-Sap, the cells were resuspended in PBA-PF. For intracellular staining, an isotypic control incubated with a mouse Ig G1 (IgG1)-FITC Ab instead of Ki67-FITC was processed in parallel for each sample and was used to set the gate for Ki67⁺ cells. All analyses were performed with a FACSCalibur flow cytometer with CellQuest software (BD Biosciences).

Chemotaxis

Quantitative chemotaxis transmigration assays were done using a Transwell system (Corning). Cells isolated from LNs 2 mo after infection (10⁵ cells/insert in 200 µl) were placed in the upper chamber of each well (6.5 mm in diameter, 8 µm pore size with polycarbonate membrane), whereas chemokines were placed in the lower chamber. Inserts were removed after 4 h at 37°C in a 5% CO₂ atmosphere and 500 µl of the sample in the lower compartment was collected. These cells were labeled with anti-CD4 mAb and then counted by flow cytometry (FACScan; BD Biosciences). For cell counts, an elapsed time of 3 min was used with a high fluid speed. Experiments were done in duplicate. This protocol was only conducted on LN cells and not peripheral blood cells, as Ficoll-Hypaque purification of PBMCs can acutely affect chemokine receptor expression (data not shown; Ref. 43).

Statistical analyses

A Mann-Whitney U test was used to determine whether differences in means were significant. Differences were considered to be significant if p < 0.05. Spearman Rank correlations were calculated to evaluate correlations. Best-fit lines are shown.

	Results

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Dynamics of viral replication and cell counts in acute SIVmac251 infection

Ten rhesus macaques (seven females and three males) were infected with 10 animal-infectious doses of the pathogenic SIVmac251 isolate i.v. Animals were 8.50 ± 0.97 years old and had an initial CD4 T cell count in the blood of 875/mm³± 518. Infection of rhesus macaques with the SIVmac251 resulted in a peak of viremia (mean: 1.3 x 10⁷ SIV RNA copies/ml, range: 2.6 x 10⁵ to 3.6 x 10⁷) by day 14 (Fig. 1). This peak was followed by a decrease in viremia that reached a steady state at 2 mo at different levels in different individuals. The steady states were 2 logs lower in five SIVmac251-infected macaques (nos. 0191, 0300, 0341, 0312, 94870; mean: 2.7 x 10³ SIV RNA copies/ml, range: 10² to 10⁴) than in the other five monkeys (nos. 94746, 94852, 94856, R97087, 94860; mean: 1.2 x 10⁵ SIV RNA copies/ml, range: 2 x 10⁴ to 3.4 x 10⁵; p = 0.009). These set points are predictive of further progression to AIDS, identifying slow and moderate progressors, respectively (23, 42). Three animals (nos. 94746, 94852, and R97087), followed for clinical progression toward disease, developed AIDS (a wasting syndrome with cachexia and opportunistic infections) 1–2 years after infection (Table I). Two others (nos. 94856 and 94860) were asymptomatic during the year follow-up period of the study but they lost around 50% of their initial CD4⁺ T cell counts and displayed viral loads higher than 10⁴ copies/ml. The five other primates remained asymptomatic during the study having lost only around 25% of the CD4⁺ T cells and with viral loads remaining <10⁴ copies/ml (Table I). In all infected primates, anti-SIV Abs were detectable in blood 4 wk postinfection. The concentration of these Abs increased thereafter (data not shown). According to our recent results (23, 42), none of this new group of infected primates were rapid progressors, i.e., they did not develop AIDS (wasting syndrome with cachexia, opportunistic infections, and an absence of SIV Ab response) within 6 mo. To determine the extent of the early viral seeding in peripheral lymphoid organs, we also analyzed peripheral LNs. These LNs were sequentially retrieved from each of these animals 0, 7, 14 days and 2 mo after infection. The numbers of SIV productively infected cells peaked on day 14 postinfection (Fig. 1) and decreased by the end of this early phase. The set point levels at 2 mo postinfection were significantly lower in slow progressors (0.24 ± 0.24/2 mm²) than in moderate progressors (2.78 ± 2.84/2 mm²; p = 0.009). The set point level of SIV⁺ RNA-expressing cells at 2 mo postinfection was correlated with the plasma viral load at this time (r = 0.85, p = 0.006). Within the first week of infection, global lymphopenia (B and T cells), around the peak of replication, was observed in peripheral blood (Table II). This observation is in agreement with a recent report on SIV-infected macaques (44). Two months after infection, CD8⁺ T cells and B cells were restored to their preinfection levels or even higher, whereas CD4⁺ T cells remained at a lower level than before infection. The extent of further CD4 T cell decline, as mentioned before, is predictive of further disease evolution toward AIDS (Table I). In contrast to that observed in the blood, LN cell counts revealed that the size of the lymphocyte pools, 2 wk postinfection, is maintained or even higher whereas the enlargement of LNs at 2 mo postinfection was associated with a 2- to 3-fold increase in the pool of CD8⁺ and CD20⁺ cells (Table II). We do not know whether the early lymphopenia was due to cell death, cell redistribution from the blood into tissue sites of SIV replication, or both.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Kinetic analysis of viremia in SIV-infected macaques. A, Viral loads (n = 10) and (B) number of replicating virus cells within LNs (n = 8) in the individual macaques during the acute phase of infection.

Because CCR5 expression seems to be required for optimal CD8 effector T cell function and CCR5 may be involved in the recruitment and positioning of virus-specific T cells (33, 34, 35), we next studied the dynamics and profiles of chemokine receptor expression on CD8^bright T cells from peripheral blood and LNs. Flow cytometric analysis of peripheral blood from healthy macaques showed that most CD8^bright T cells expressed CXCR4 and that a lower proportion expressed CCR5 (67.84 ± 18.61% and 20.8 ± 15.14%, respectively). We also found that CD8^bright T cells from peripheral LNs displayed similar chemokine receptor expression profiles as those from peripheral blood; most of the CD8 ^bright T cells expressed CXCR4 (89.44 ± 10.61%) whereas only 13.84± 7.28% expressed CCR5 (Fig. 2A). Costaining of CD8^bright T cells showed reciprocal expression of CXCR4 and CCR5 (Fig. 2B). It has been reported that expression of CCR5 in human CD8⁺ T cells is restricted to the CD45RA negative subset (45, 46). Here, we similarly found in healthy macaques that CCR5 was mainly expressed on the CD45RA^–CD8^bright T cell subset (Fig. 2, C and D).

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Chemokine receptor expression on CD8bright cells of healthy macaques. A, Percentages of CD8+ T cells expressing CXCR4 and CCR5 in whole blood (left panel) and LNs (right panel) are shown. Each symbol represents one healthy macaque (n = 14). Bars represent the geometric means. The proportions were calculated as follow: (CCR5+CD8+/CCR5+CD8+ + CCR5–CD8+) x 100. B, Proportions of CD8+ T cells expressing CXCR4 and/or CCR5 in whole blood (left panel) and LNs (right panel) are shown. Results are the mean ± SD of three healthy macaques. C, Representative dot plots of coexpression of CD45RA with CXCR4 (left panel) and CCR5 (right panel) in whole blood gating on CD8+ T cells are shown. D, Proportions of CD45RA+ and CD45RA–CD8+ T cells expressing CXCR4 (left panel) and CCR5 (right panel). Results are the mean ± SD of three healthy macaques. The proportions were calculated for each subsets as follow: (CXCR4+CD45RA+/CXCR4+CD45RA+ + CXCR4–CD45RA+) x 100.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Dynamics of chemokine receptor expression on CD8bright cells during primary SIV infection. The proportions of CD8bright cells expressing (A) CXCR4 and (B) CCR5 in whole blood (left panel) and LNs (right panel) are shown. Each symbol represents one individual macaque. C, Chemotactic response of CD8+ T cells. The responses of CD8+ T cells from LNs of macaques before (SIV–) and 2 mo after SIV infection (SIV+) are shown. The effect of adding different concentrations of RANTES and MIP-1 (100 ng, 10 ng, and 2 ng/ml) was assessed. Results are expressed as chemotactic index. Data are the mean ± SD of four individual macaques.

Changes in CCR5 and CXCR4 expressions are associated with changes in T cell activation during the acute phase of SIVmac251 infection

Several reports have found that CCR5 and CXCR4 are differentially modulated following in vitro cell stimulation (45, 46, 49, 50). We first assessed whether stimulation of CD8⁺ T cells of healthy macaques either with Con A in the presence of IL-2 or following engagement of the TCR/CD3 complex using a specific CD3 mAb modulated the expression of chemokine receptors. We found a down-regulation of CXCR4 expression and up-regulation of CCR5 expression after stimulation (data not shown). This reciprocal change in chemokine receptor expression on CD8⁺ T cells has been previously reported for CD4⁺ T cells (45, 46, 50).

We next evaluated in vivo T cell activation by measuring the expression of Ki67, a nuclear Ag expressed in the G₁, G₂, S, and M phases but not the G₀ phase of the cell cycle (42, 51). We found that the number of cycling CD8^bright T cells in the blood greatly increased at day 14 (28.7 ± 13.7%) compared with preinfection levels (4.54 ± 1.20%, p = 0.0015) (Fig. 4A). This higher proportion of cycling CD8^bright T cells was mainly limited to the CD45RA^– cell subset (Fig. 4B). The proportion of cycling CD8^bright T cells in the blood was 2-fold higher 2 mo after infection (7.34 ± 2.59%, p = 0.004) than before infection (Fig. 4A).

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Dynamics of cycling CD8bright cells during primary SIVmac251 infection. The percentage of CD8bright cells expressing Ki67 was assessed by flow cytometry in whole blood (A and B) and LNs (C and D) of SIV-infected macaques. B and D, The fractions of cycling CD45RA+ and CD45RA– T cells. Each symbol represents one individual macaque.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. T cell activation in SIVmac251-infected macaques. The expression of HLA-DR (A) and CD28 (B) on CD8bright cells from peripheral blood (left) and LNs (right) was assessed in individual macaques before and 2 mo after infection. Results are the mean ± SD of eight macaques.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Changes during primary SIVmac251nef infection. A, Kinetic analysis of viremia in SIVmac251nef-infected macaques. Viral loads of four individual macaques (256, ; 94854, ; 970112, ; 97035, ) during the acute phase of infection. B, The percentage of CD8bright cells expressing Ki67 was assessed by flow cytometry in whole blood and in the fractions of CD45RA+ and CD45RA– T cells. C, The proportions of CD8bright cells expressing CXCR4 (left panel) and CCR5 (right panel) in whole blood are shown. Each symbol represents one individual macaque.

These results reveal significant differences between individual SIV-infected macaques with respect to viral replication, chemokine receptor expression, and extent of T cell activation. The plasma viral load levels as well as the numbers of SIV productively infected cells were not statistically significant between moderate and slow progressors at day 14 postinfection (Fig. 7, A and B). At the end of this acute phase, both plasma viremia and the numbers of SIV productively infected cells showed a significant correlation with progression toward disease. Together these findings suggest that the initial extent of viral seeding and replication in the LNs (day 14) is not a predictive factor of progression toward disease. However, once the initial seeding has occurred, the capacity of the host to rapidly control the level of viral replication may represent a crucial component in determining the subsequent viral set point that will predict the evolution toward AIDS. These data are reminiscent of our recent reports showing an association between T cell activation and apoptosis and disease progression (23, 42, 52, 53).

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Correlation between viral replication and changes in immunological parameters at 2 wk postinfection. The level of viral replication, the percentage of cycling cells (Ki67), and the changes in CCR5 and CXCR4 expressions (percent of either CXCR4 or CCR5 at 2 wk minus the percentage of either CXCR4 or CCR5 at day 0) in whole blood (A) and in LNs (B) of SIVmac251nef- (nef, n = 3, ), and of SIVmac251-infected macaques of either slow (slow, n = 5, ) or moderate progressors (moderate, n = 5, ). A Mann-Whitney U test was used to determine whether differences in means were significant (NS, not statistically different). C, Regression analysis between the extent of viral replication in the LNs at 2 mo postinfection with 1) the percentage of cycling cells in the LNs, 2) the changes in CCR5 expression in the LNs, and 3) and the changes in CXCR4 expression in the LNs at 2 wk postinfection. The Spearman rank test was calculated to evaluate correlations. Best-fit lines are shown.

View larger version (37K): [in this window] [in a new window]   FIGURE 8. Correlation between viral replication and changes in immunological parameters at 2 mo postinfection. The level of viral replication, the percentage of cycling cells (Ki67), and the changes in CCR5 and CXCR4 expressions (the percentage of either CXCR4 or CCR5 at 2 mo minus the percentage of either CXCR4 or CCR5 at day 0) in whole blood (A) and in LNs (B) of SIVmac251nef- (nef, n = 3, ), and of SIVmac251-infected macaques of either slow (slow, n = 5, ) or moderate progressors (moderate, n = 5, ). A Mann-Whitney U test was used to determine whether differences in means were significant (NS, not statistically different). C, Regression analysis between the extent of viral replication in the LNs at 2 mo postinfection with 1) the percentage of cycling cells in the LNs, 2) the changes in CCR5 expression in the LNs, and 3) and the changes in CXCR4 expression in the LNs at 2 mo postinfection. The Spearman rank test was calculated to evaluate correlations. Best-fit lines are shown.

CCR7 is another chemokine receptor that plays a major role in the homing of CD8⁺ T cells to the T cell areas of LNs. We analyzed the changes in the expression of the CD45RA and CCR7 Ags using the following interpretation of T cell differentiation pattern as previously described (32, 54, 55): CD45RA⁺CCR7⁺ (naive) CD45RA^–CCR7⁺ (central memory, TCM) CD45RA^–CCR7^– (effector memory, TEM) CD45RA⁺CCR7^– (terminally differentiated T cells). Five animals, two slow and three moderate progressors, were followed up.

While the percentages of naive and central memory cells rapidly declined in the blood following SIV infection, the proportions of effector memory increased at day 14 (Fig. 9) but no differences were observed between slow and moderate progressors. However, as noted for cycling cells and changes in CCR5 and CXCR4 expression, the difference in CCR7 expression between slow and moderate progressors became apparent after 2 mo of infection. Thus, the percentage of effector memory T cells was higher in moderate progressors (MPs: 61 ± 23%) compared with slow progressors (SPs: 22 ± 1%, Fig. 9). The lower proportion of effector memory cells in slow progressors was associated with an increased proportion of terminally differentiated T cells. Thus, a full T cell lineage commitment was observed in the two slow progressors compared with the three moderate progressors in which the proportions of TDT remain lower at 2 mo (MPs: 31 ± 14%; SPs: 57 ± 1%).

View larger version (32K): [in this window] [in a new window]   FIGURE 9. Pattern of T cell differentiation during primary SIVmac251 infection. Cells from blood (left panel) and LNs (right panel) were analyzed by flow cytometry gating on CD8bright cells. The percentages of naive (RA+R7+), central memory (TCM, RA–R7+), effector memory (TEM, RA–R7–), and terminal differentiated T cells (TDT, RA+R7–) of two slow (no. 0191, and no. 0300, ) and three moderate progressors (no. R97087, ; no. 94852, ; and no. 94856, ) are shown.

CTL lysis of infected cells occurs primarily through the granule exocytosis pathway and the expression of perforin seems to be more restricted than serine proteases (38, 56). The expression of perforin seems to define differentiated effector CD8⁺ T cells (38, 39) and several reports have now suggested that, during the asymptomatic phase the cell lysis, capability of CD8⁺ T cells is reduced due to low perforin expression (39, 57, 58, 59). However, there is little data during primary infection, and we investigated TIA-1 expression, a polyadenylate-binding protein localized to the granules of cytolytic T cells and functionally related to perforin. Here, we found that the level of TIA-1-expressing CD8^bright T cells in the LNs is greater in SIV-infected macaques at 2 mo postinfection than in uninfected macaques (Fig. 10A). The proportion of TIA-1-CD8^bright T cells in the LNs was higher in moderate progressors than in slow progressors. In addition, gating on the different CD8^bright T cell subsets, we observed that at 2 mo postinfection most of the TIA-1-expressing cells are effector memory T cells (CD45RA^–CCR7^–) and to a lesser extent, also fully terminal differentiated T cells (CD45RA⁺CCR7^–). Low levels of TIA-1 were expressed in naive CD8^bright T cell subsets (CD45RA⁺CCR7⁺) (Fig. 10B). These findings show that an increase in cells with a CTL phenotype among CD8^bright T cells was associated with cell proliferation (Ki67), T cell differentiation, and the magnitude of viral replication. Thus, at 2 mo, the CD8^bright T cell subset in moderate progressors expanded and expressed more TIA-1 in response to viral replication. In contrast, CD8^bright T cells with lower TIA-1 expression were expanded at 2 mo in slow progressors.

View larger version (27K): [in this window] [in a new window]   FIGURE 10. TIA-1 expression in CD8+ T cells during primary SIVmac251 infection. A, Representative staining for TIA-1 in CD8+ T cells from the same monkey at day 0 and at 2 mo postinfection. The percentage is shown. B, Data from uninfected (SIV–, n = 4), and SIV-infected macaques (slow progressors, n = 3 and moderate progressors, n = 3) are shown. The p value was obtained using the nonparametric Mann-Whitney U test. C, Perforin expression in CD8+ T cells with each CCR7/CD45RA phenotype at day 0 and 2 mo postinfection. Representative results from three individual macaques are shown.

	Discussion

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CCR5 is considered as crucial in the recruitment and positioning of virus-specific T cells (33, 34, 35). Here, we found after in vitro stimulation that activated CD8^bright T cells express high levels of CCR5 but lower CXCR4. In mice, it has been also reported that T cytotoxic type 1 effector CD8⁺ T cells, which produce type-1 cytokines, predominantly express CCR5 while T cytotoxic type 2 effector CD8⁺ T cells, which produce type-2 cytokines, express high levels of CXCR4 but not CCR5 (60). Our results on the dynamics of chemokine receptor expression upon pathogenic SIVmac251 infection may be at least in part the consequence of in vivo CD8 T cell activation as indicated by Ki67 staining and changes in the lineage commitment of T cell subpopulations. In nonpathogenic SIVmac251nef, despite intense viral replication at day 14, we did not observe major changes in the dynamics of CD8^bright T cells, especially in the expression of CCR5. These observations suggest that during the acute phase, a certain threshold of SIV Ags is required to activate CD8⁺ T cells and trigger the expression of CCR5. However, the differences in CD8 phenotype (CXCR4 increase) in the SIVmac251nef could still suggest an indirect effect of nef in pathogenic SIV-infected macaques. Recently, we have observed that, during primary HIV infection, CCR5 expression and proliferation of CCR5⁺CD8⁺ T cells were also higher in HIV-infected individuals than in healthy donors (61). There is also evidence that viral infections induced, on virus epitope-specific tetramer-positive CD8⁺ T cells, cell surface expression of CCR5 (62, 63). Similar to humans (47, 50, 64), CCR5 is mainly expressed on the CD45RA^low T cell subset in macaques and CCR5 expression has been reported to be restricted to memory and effector CD8⁺ T cells but not naive CD8⁺ T cells (62). Although we have not directly shown the level of chemokine receptor on the different CD8⁺ T cell subsets, due to the limited panel of labeled Abs that cross-react in macaques, we found an increase in the proportion of effector memory (CD45RA^–CCR7^–) CD8^bright T cells in moderate progressors concomitant with CCR5 increase. Moreover, our data showing that RANTES and MIP-1 induce a greater CD8 T cell migration in SIV-infected macaques than in healthy macaques confirm that CCR5 expressed on CD8⁺ T cells physiologically functions as a receptor for chemokines.

CCR5 is widely expressed on T lymphocytes in many different organs particularly at sites of inflammation (65, 66, 67, 68). The injection of RANTES into a chimeric SCID mouse model has been shown to recruit CD8⁺ T cells to the sites of injection (69), while another report demonstrated the localization of infused CCR5 HIV-specific T cells to inflamed tissue (70). Our results during primary SIV infection thus support the idea that these CCR5⁺CD8⁺ T cells, activated within the LNs, may re-enter the circulation, and be capable of directional migration to the inflammatory sites to control viral replication (33, 34, 35). Therefore, because CCR5 expression has been proposed to be essential for optimal CD8⁺ effector T cell function and exert its effector potential at the site of virus infection (33, 34, 35), our data raises a major issue. Indeed, despite higher activation states, higher numbers of cycling CD8⁺ T cells, and higher proportions of CCR5 expressing CD8⁺ T cells, the levels of plasma viremia set point and SIV⁺ replicating cells within LNs remained higher in moderate progressors than in slow progressors. These data support the idea that despite intense CD8⁺ T cell activation and proliferation within LNs, these cells are inefficient in controlling the virus replication during primary infection.

During the asymptomatic phase, it has been proposed that the CD8 T cell response fails to control HIV and SIV infections because CD8⁺ T cells are defective in perforin expression, but not in cytokine production, which could render them less efficient at killing virus-infected cells (59, 71). Here, we found that the proportion of TIA-1-expressing CD8^bright T cells in the LNs is greater in moderate progressors than in slow progressors at 2 mo postinfection. TIA-1 expressing CD8^bright T cells were, at 2 mo, in fact characterized mostly by effector memory phenotype (CD45RA^–CCR7^–).

A skewed maturation of HIV-specific memory CD8⁺ T cells has been previously reported in inducing the accumulation of a preterminally differentiated subset of memory T cells during the asymptomatic phase (32). Although only five animals were analyzed, we found in moderate progressors the accumulation of effector memory CD8⁺ T cells. The absence of a fully differentiated profile might be due to a deregulation of the cell cycle making CD8⁺ T cells more anergic and prone to undergo apoptosis as observed during the asymptomatic phase (30, 31, 53, 72, 73, 74). In this sense, it has been reported that HIV-specific CTL clones rapidly disappear (29), and activated CCR5⁺CD8⁺ T cells are more prone to undergo apoptosis in vitro during primary HIV infection (61). We and other laboratories have previously reported that CD45RA^–CD8⁺ T cells are more prone to undergo apoptosis than CD45RA⁺ during the asymptomatic phase (31, 53, 75). Therefore, whether the abortive differentiation pattern in moderate progressors was the result of an increased death of either effector memory and/or terminal differentiated T cells, or both, during primary infection remains an open question.

Increasing evidence suggests an association between high levels of immune activation during the asymptomatic phase and poor outcome in HIV-infected individuals (76, 77) and in SIV-infected macaques (42). A recent report suggest that the CD8⁺ T cell activation "set point" is also an early predictive marker of further disease evolution toward AIDS in HIV-infected individuals (78) which is in agreement with our data. Moreover, introduction of antiretroviral treatment resulted in rapid decreases in the level of CD8 T cell activation (79, 80) supporting the idea that the threshold of viral replication determines the extent of CD8 T cell activation. In such chronic infection, viral immune evasion could be due to persistence of activated CD8⁺ T cells as proposed by Zajac et al. (81) following an examination of the regulation of virus-specific CD8⁺ T cells during chronic lymphocytic choriomeningitis infection of mice. This unresponsiveness was higher under conditions of CD4 T cell deficiency. Thus, the lack of CD4 T cell help may render these CD8⁺ T cells non-fully differentiated during primary SIV infection and warrants further exploration (82, 83, 84).

Because the duration of antigenic stimulation of CD8⁺ T cells can determine whether subsequent in vivo clonal expansion will be abortive or extensive (85) and that sustained stimulus may induce an extensive proliferation and production of cells potentially capable of inducing destruction of peripheral target tissues, it cannot be excluded that, although provocative, the CD8 T cell response may have a detrimental effect during SIV infection rather than a protective effect. Other reports have shown an active participation of activated CCR5⁺CD8⁺ T cells in the pathogenesis of graft-vs-host-disease (66).

Despite the intense T cell activation and changes in chemokine receptor expression that occur during primary infection, related to the extent of viral replication and further disease progression to AIDS, our results highlight the inability to control viremia that may be, at least in part, due to the lack of maturation of CD8^bright T cells during the acute phase. Better understanding of the mechanisms involved should improve our knowledge of the early steps in the inability of the immune response to control HIV and SIV replication and prevent further progression to AIDS.

	Acknowledgments

 
We acknowledge M. Muller-Trutwin and A. Brussel for helpful discussions and critical reading of our manuscript.

	Disclosures

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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 funded by a grant from the Agence Nationale de Recherche sur le Sida (ANRS; to J.E.). F.P. was supported by a postdoctoral fellowship from Ensemble Contre le Sida and L.V. was supported by a doctoral fellowship from ANRS.

² L.V. and F.P. were equal contributors.

³ Address correspondence and reprint requests to Dr. Jérôme Estaquier, Unité de Physiopathologie des Infections Lentivirales, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France. E-mail address: jestaqui{at}pasteur.fr

⁴ Abbreviations used in this paper: LN, lymph node.

Received for publication May 14, 2004. Accepted for publication March 11, 2005.

	References

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1. Cyster, J. G.. 1999. Chemokines and cell migration in secondary lymphoid organs. Science 286: 2098-2103.[Abstract/Free Full Text]
2. Butcher, E. C., M. Williams, K. Youngman, L. Rott, M. Briskin. 1999. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72: 209-253.[Medline]
3. Kim, C. H., H. E. Broxmeyer. 1999. Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J. Leukocyte Biol. 65: 6-15.[Abstract]
4. Sallusto, F., C. R. Mackay, A. Lanzavecchia. 2000. The role of chemokine receptors in primary, effector, and memory immune responses. Annu. Rev. Immunol. 18: 593-620.[Medline]
5. Mackay, C. R.. 1996. Chemokine receptors and T cell chemotaxis. J. Exp. Med. 184: 799-802.[Free Full Text]
6. Sallusto, F., A. Lanzavecchia, C. R. Mackay. 1998. Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. Today 19: 568-574.[Medline]
7. Premack, B. A., T. J. Schall. 1996. Chemokine receptors: gateways to inflammation and infection. Nat. Med. 2: 1174-1178.[Medline]
8. Wells, T. N., C. A. Power, A. E. Proudfoot. 1998. Definition, function and pathophysiological significance of chemokine receptors. Trends Pharmacol. Sci. 19: 376-380.[Medline]
9. Baggiolini, M.. 1998. Chemokines and leukocyte traffic. Nature 392: 565-568.[Medline]
10. Doherty, P. C., W. Allan, M. Eichelberger, S. R. Carding. 1992. Roles of and T cell subsets in viral immunity. Annu. Rev. Immunol. 10: 123-151.[Medline]
11. Doherty, P. C., D. J. Topham, R. A. Tripp, R. D. Cardin, J. W. Brooks, P. G. Stevenson. 1997. Effector CD4⁺ and CD8⁺ T-cell mechanisms in the control of respiratory virus infections. Immunol. Rev. 159: 105-117.[Medline]
12. Masopust, D., V. Vezys, A. L. Marzo, L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291: 2413-2417.[Abstract/Free Full Text]
13. Little, S. J., A. R. McLean, C. A. Spina, D. D. Richman, D. V. Havlir. 1999. Viral dynamics of acute HIV-1 infection. J. Exp. Med. 190: 841-850.[Abstract/Free Full Text]
14. Soudeyns, H., G. Pantaleo. 1999. The moving target: mechanisms of HIV persistence during primary infection. Immunol. Today 20: 446-450.[Medline]
15. D’Souza, M. P., B. J. Mathieson. 1996. Early phases of HIV type 1 infection. AIDS Res. Hum. Retroviruses 12: 1-9.[Medline]
16. Reimann, K. A., K. Tenner-Racz, P. Racz, D. C. Montefiori, Y. Yasutomi, W. Lin, B. J. Ransil, N. L. Letvin. 1994. Immunopathogenic events in acute infection of rhesus monkeys with simian immunodeficiency virus of macaques. J. Virol. 68: 2362-2370.[Abstract/Free Full Text]
17. Chakrabarti, L., M. C. Cumont, L. Montagnier, B. Hurtrel. 1994. Variable course of primary simian immunodeficiency virus infection in lymph nodes: relation to disease progression. J. Virol. 68: 6634-6643.[Abstract/Free Full Text]
18. Dalod, M., M. Dupuis, J. C. Deschemin, D. Sicard, D. Salmon, J. F. Delfraissy, A. Venet, M. Sinet, J. G. Guillet. 1999. Broad, intense anti-human immunodeficiency virus (HIV) ex vivo CD8⁺ responses in HIV type 1-infected patients: comparison with anti-Epstein-Barr virus responses and changes during antiretroviral therapy. J. Virol. 73: 7108-7116.[Abstract/Free Full Text]
19. Mellors, J. W., L. A. Kingsley, C. R. Rinaldo, Jr, J. A. Todd, B. S. Hoo, R. P. Kokka, P. Gupta. 1995. Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion. Ann. Intern. Med. 122: 573-579.[Abstract/Free Full Text]
20. Watson, A., J. Ranchalis, B. Travis, J. McClure, W. Sutton, P. R. Johnson, S. L. Hu, N. L. Haigwood. 1997. Plasma viremia in macaques infected with simian immunodeficiency virus: plasma viral load early in infection predicts survival. J. Virol. 71: 284-290.[Abstract]
21. Lifson, J. D., M. A. Nowak, S. Goldstein, J. L. Rossio, A. Kinter, G. Vasquez, T. A. Wiltrout, C. Brown, D. Schneider, L. Wahl, et al 1997. The extent of early viral replication is a critical determinant of the natural history of simian immunodeficiency virus infection. J. Virol. 71: 9508-9514.[Abstract]
22. Staprans, S. I., P. J. Dailey, A. Rosenthal, C. Horton, R. M. Grant, N. Lerche, M. B. Feinberg. 1999. Simian immunodeficiency virus disease course is predicted by the extent of virus replication during primary infection. J. Virol. 73: 4829-4839.[Abstract/Free Full Text]
23. Monceaux, V., J. Estaquier, M. Fevrier, M. C. Cumont, Y. Riviere, A. M. Aubertin, J. C. Ameisen, B. Hurtrel. 2003. Extensive apoptosis in lymphoid organs during primary SIV infection predicts rapid progression towards AIDS. AIDS 17: 1585-1596.[Medline]
24. Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, et al 1999. Control of viremia in simian immunodeficiency virus infection by CD8⁺ lymphocytes. Science 283: 857-860.[Abstract/Free Full Text]
25. Ogg, G. S., S. Kostense, M. R. Klein, S. Jurriaans, D. Hamann, A. J. McMichael, F. Miedema. 1999. Longitudinal phenotypic analysis of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes: correlation with disease progression. J. Virol. 73: 9153-9160.[Abstract/Free Full Text]
26. Musey, L., J. Hughes, T. Schacker, T. Shea, L. Corey, M. J. McElrath. 1997. Cytotoxic-T-cell responses, viral load, and disease progression in early human immunodeficiency virus type 1 infection. N. Engl. J. Med. 337: 1267-1274.[Abstract/Free Full Text]
27. Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard, C. E. Irwin, J. T. Safrit, J. Mittler, L. Weinberger, et al 1999. Dramatic rise in plasma viremia after CD8⁺ T cell depletion in simian immunodeficiency virus-infected macaques. J. Exp. Med. 189: 991-998.[Abstract/Free Full Text]
28. Xiong, Y., M. A. Luscher, J. D. Altman, M. Hulsey, H. L. Robinson, M. Ostrowski, B. H. Barber, K. S. MacDonald. 2001. Simian immunodeficiency virus (SIV) infection of a rhesus macaque induces SIV-specific CD8⁺ T cells with a defect in effector function that is reversible on extended interleukin-2 incubation. J. Virol. 75: 3028-3033.[Abstract/Free Full Text]
29. Pantaleo, G., H. Soudeyns, J. F. Demarest, M. Vaccarezza, C. Graziosi, S. Paolucci, M. Daucher, O. J. Cohen, F. Denis, W. E. Biddison, et al 1997. Evidence for rapid disappearance of initially expanded HIV-specific CD8⁺ T cell clones during primary HIV infection. Proc. Natl. Acad. Sci. USA 94: 9848-9853.[Abstract/Free Full Text]
30. Lewis, D. E., D. S. Tang, A. Adu-Oppong, W. Schober, J. R. Rodgers. 1994. Anergy and apoptosis in CD8⁺ T cells from HIV-infected persons. J. Immunol. 153: 412-420.[Abstract]
31. Estaquier, J., M. Tanaka, T. Suda, S. Nagata, P. Golstein, J. C. Ameisen. 1996. Fas-mediated apoptosis of CD4⁺ and CD8⁺ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists. Blood 87: 4959-4966.[Abstract/Free Full Text]
32. Champagne, P., G. S. Ogg, A. S. King, C. Knabenhans, K. Ellefsen, M. Nobile, V. Appay, G. P. Rizzardi, S. Fleury, M. Lipp, et al 2001. Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature 410: 106-111.[Medline]
33. Cerwenka, A., T. M. Morgan, A. G. Harmsen, R. W. Dutton. 1999. Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection. J. Exp. Med. 189: 423-434.[Abstract/Free Full Text]
34. Cook, D. N., M. A. Beck, T. M. Coffman, S. L. Kirby, J. F. Sheridan, I. B. Pragnell, O. Smithies. 1995. Requirement of MIP-1 for an inflammatory response to viral infection. Science 269: 1583-1585.[Abstract/Free Full Text]
35. Nansen, A., O. Marker, C. Bartholdy, A. R. Thomsen. 2000. CCR2⁺ and CCR5⁺ CD8⁺ T cells increase during viral infection and migrate to sites of infection. Eur. J. Immunol. 30: 1797-1806.[Medline]
36. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99: 23-33.[Medline]
37. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J. Exp. Med. 189: 451-460.[Abstract/Free Full Text]
38. Hamann, D., P. A. Baars, M. H. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. R. Klein, R. A. van Lier. 1997. Phenotypic and functional separation of memory and effector human CD8⁺ T cells. J. Exp. Med. 186: 1407-1418.[Abstract/Free Full Text]
39. Zhang, D., P. Shankar, Z. Xu, B. Harnisch, G. Chen, C. Lange, S. J. Lee, H. Valdez, M. M. Lederman, J. Lieberman. 2003. Most antiviral CD8 T cells during chronic viral infection do not express high levels of perforin and are not directly cytotoxic. Blood 101: 226-235.[Abstract/Free Full Text]
40. Westermann, J., R. Pabst. 1990. Lymphocyte subsets in the blood: a diagnostic window on the lymphoid system?. Immunol. Today 11: 406-410.[Medline]
41. Kornfeld, H., N. Riedel, G. A. Viglianti, V. Hirsch, J. I. Mullins. 1987. Cloning of HTLV-4 and its relation to simian and human immunodeficiency viruses. Nature 326: 610-613.[Medline]
42. Monceaux, V., R. H. Fang, M. C. Cumont, B. Hurtrel, J. Estaquier. 2003. Distinct cycling CD4⁺- and CD8⁺-T-cell profiles during the asymptomatic phase of simian immunodeficiency virus SIVmac251 infection in rhesus macaques. J. Virol. 77: 10047-10059.[Abstract/Free Full Text]
43. Sharron, M., S. Pohlmann, K. Price, E. Lolis, M. Tsang, F. Kirchhoff, R. W. Doms, B. Lee. 2000. Expression and coreceptor activity of STRL33/Bonzo on primary peripheral blood lymphocytes. Blood 96: 41-49.[Abstract/Free Full Text]
44. Mattapallil, J. J., N. L. Letvin, M. Roederer. 2004. T-cell dynamics during acute SIV infection. AIDS 18: 13-23.[Medline]
45. Loetscher, P., M. Seitz, M. Baggiolini, B. Moser. 1996. Interleukin-2 regulates CC chemokine receptor expression and chemotactic responsiveness in T lymphocytes. J. Exp. Med. 184: 569-577.[Abstract/Free Full Text]
46. Bleul, C. C., L. Wu, J. A. Hoxie, T. A. Springer, C. R. Mackay. 1997. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc. Natl. Acad. Sci. USA 94: 1925-1930.[Abstract/Free Full Text]
47. Ostrowski, M. A., S. J. Justement, A. Catanzaro, C. A. Hallahan, L. A. Ehler, S. B. Mizell, P. N. Kumar, J. A. Mican, T. W. Chun, A. S. Fauci. 1998. Expression of chemokine receptors CXCR4 and CCR5 in HIV-1-infected and uninfected individuals. J. Immunol. 161: 3195-3201.[Abstract/Free Full Text]
48. de Roda Husman, A. M., H. Blaak, M. Brouwer, H. Schuitemaker. 1999. CC chemokine receptor 5 cell-surface expression in relation to CC chemokine receptor 5 genotype and the clinical course of HIV-1 infection. J. Immunol. 163: 4597-4603.[Abstract/Free Full Text]
49. Signoret, N., M. M. Rosenkilde, P. J. Klasse, T. W. Schwartz, M. H. Malim, J. A. Hoxie, M. Marsh. 1998. Differential regulation of CXCR4 and CCR5 endocytosis. J. Cell Sci. 111: 2819-2830.[Abstract]
50. Unutmaz, D., W. Xiang, M. J. Sunshine, J. Campbell, E. Butcher, D. R. Littman. 2000. The primate lentiviral receptor Bonzo/STRL33 is coordinately regulated with CCR5 and its expression pattern is conserved between human and mouse. J. Immunol. 165: 3284-3292.[Abstract/Free Full Text]