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Journal of Virology, December 2007, p. 13456-13468, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01619-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Dynamics of T-Cell Responses and Memory T Cells during Primary Simian Immunodeficiency Virus Infection in Cynomolgus Macaques{triangledown}

Ingrid Karlsson,1 Benoît Malleret,1 Patricia Brochard,1 Benoît Delache,1 Julien Calvo,1 Roger Le Grand,1,2 and Bruno Vaslin1,2*

CEA, Service d'Immuno-Virologie, DSV/iMETI, IPSC, Fontenay-aux-Roses, France,1 Université Paris XI, UMRE01, Orsay, France2

Received 25 July 2007/ Accepted 26 September 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular immune responses make an important contribution to both the control of human immunodeficiency virus (HIV) replication and disease progression. We used a pathogenic model of SIVmac251 infection of cynomolgus macaques to longitudinally evaluate cellular immune responses in association with various rates of disease progression. We found an inverse relationship between plasma viral load and the simian immunodeficiency virus (SIV)-specific T cells responses in peripheral blood and lymph nodes. SIV-specific T-cell responses in peripheral blood were transient during primary infection, with the highest responses detected around 3 months after infection. There was also a transient increase of central memory CD8+ T cells in peripheral blood during primary infection, and effector memory T-cell counts in peripheral lymph nodes were increased. This study emphasizes the importance of the early virus-specific immune responses in the outcome of HIV/SIV disease and provides details about the changes of virus-specific immune responses over time.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cellular immune response is an important element in the control of viral infections (30), including human immunodeficiency virus (HIV) infections (39); indeed, both CD4+ and CD8+ T-cell responses contribute to the control of HIV infection (53). During acute HIV or simian immunodeficiency virus (SIV) infection, the CD8+ T-cell response increases following the increase in viral load, and there is an inverse relationship between the CD8+ T-cell response and viral load during primary infection (9, 31, 44, 50, 67, 68). During chronic infection, HIV-specific CD8+ T-cell proliferation (42) and polyfunctional HIV-specific CD8+ T cells (5) are maintained in nonprogressors. In vivo depletion of CD8+ T cells in SIV-infected macaques implicates CD8+ T cells in the control of viral replication during both acute (60) and chronic (26, 60) infection. Efficient CD8+ T-cell responses are dependent on CD4+ T-cell help (28). Indeed, high CD4+ proliferative responses have also been associated with slow progression in HIV-infected patients (8, 58). In addition to helper functions, CD4+ T cells may have a cytolytic function that contributes to the control of HIV infection (1, 70).

Despite virus-specific cellular responses during primary HIV infection, the host fails to prevent establishment of chronic infection. HIV infection and depletion of CD4+ T cells impairs both CD4+ and CD8+ T-cell responses. SIV models have demonstrated an early massive depletion of memory CD4+ T cells at sites of viral replication (33, 37). Moreover, HIV preferentially infects HIV-specific CD4+ T cells (16). In addition to infection and depletion of HIV-specific immune cells, the chronic viral replication causes chronic immune activation and exhaustion of the immune system. Impairment of both CD4+ and CD8+ memory T-cell function has been evidenced by weakened cytokine and proliferative responses and by the upregulation of inhibitory T-cell surface markers in HIV-related, as well as SIV-related, disease (13, 19, 54, 55, 61, 63).

Although HIV/SIV-specific cellular immune responses have been extensively studied, we are unaware of any studies that have longitudinally monitored antigen-specific CD4+ and CD8+ T-cell cytokine and antigen-specific lymphocyte proliferative responses during primary infection in relation to disease progression. The early frequency of HIV-specific CD8+ T cells and cytolytic function are important for the control of viral load (9, 31, 44, 50, 67, 68). Therefore, it would be valuable to investigate the changes of virus-specific T-cell cytokine and proliferative responses during primary infection and the corresponding viral replication; this may help in the evaluation and design of HIV vaccines. We report here the changes in the numbers of naive and memory T cells and virus-specific cellular immune responses during primary infection in relation to long-term disease progression. We used a model of SIVmac251 infection of cynomolgus macaques: this model shows a variety of progression rates and closely resembles HIV infection in humans (15, 46, 57).

Our longitudinal follow-up displayed that of SIV-specific T-cell responses in peripheral blood were transient during primary infection, increased during acute infection and declined during chronic infection. High CD4+ T-cell and CD8+ T-cell gamma interferon (IFN-{gamma}) responses during early infection were associated with better viral control in the long term. Central memory CD8+ T-cell counts in peripheral blood increased with similar kinetics. We also monitored the evolution of naive and memory T cells and SIV-specific immune responses in peripheral lymph nodes. Central and effector memory CD8+ T-cell numbers in peripheral lymph nodes increased during SIV infection. The proliferative SIV-specific response in lymph nodes was lower and increased later than that in blood and was inversely correlated to plasma viral load.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and infections. We studied 22 adult male cynomolgus macaques (Macaca fascicularis), each weighing 3.5 to 4.5 kg, imported from Mauritius and kept according to European guidelines for animal care (Journal Officiel des Communautés Européennes, L358, 18 December 1986). Eighteen macaques were inoculated intravenously with pathogenic SIVmac251 (47) and divided into three groups of six animals. The animals of group 1 received 50 50% animal infectious doses (AID50). Those of group 2 received 50 AID50 and were treated with zidovudine (AZT; 4.5 mg/kg), lamivudine (3TC; 2.5 mg/kg), and indinavir (20 mg/kg), as previously described (3); drugs were given orally, twice per day, from 4 h postinfection until 28 days postinfection. The animals of group 3 received 5,000 AID50 of the virus.

Determination of plasma SIV RNA load. Viral RNA was prepared from 200 µl of cell-free plasma, using the HighPure viral RNA kit (Roche Diagnostics, Meylan, France), according to the manufacturer's instructions. RNA was eluted in 50 µl of nuclease-free water and frozen immediately at –80°C until analysis. SIVmac251 virus titrated by the branched-chain DNA assay and diluted in and EDTA-treated plasma samples from macaques not infected with SIV was used to generate a standard curve (serial 10-fold dilutions). Three titrated SIVmac251-infected EDTA-treated plasma samples and two EDTA-treated plasma samples from SIV-negative macaques were used as positive and negative reverse transcription-PCR controls, respectively.

Standards, controls, and viral RNA samples were extracted and tested in parallel under the same conditions. The SIVmac251 gag cDNA sequence, ligated into the plasmid pCR4-TOPO (Invitrogen) and purified with a HiSpeed Maxiprep kit (Invitrogen), was used as a positive control for PCR.

One-tube reverse transcription-PCR was performed in an iCycler real-time thermocycler (Bio-Rad) under the following conditions: 25 µl of reaction mixture containing 50 mM KCl, 20 mM Tris-HCl (pH 8.3), 0.8 mM deoxynucleoside triphosphates, 3 mM MgCl2, 20 U of RNasin (Promega), 25 U of murine leukemia virus reverse transcriptase (Applied Biosystems), 0.625 U of Hot-Start iTaq DNA polymerase (Bio-Rad), 450 nM concentrations of each primer, 250 nM fluorogenic probe, and 10-µl RNA elution samples. The probe and primers, as described by Hofmann-Lehmann et al. (24), were designed according to the sequence within the conserved SIV gag region. The sequence of the primer for reverse transcription was 5'-CAATTTTACCCAGGCATTTAATGTT-3' (25 bp). PCR was carried out using the same primer and with the sense primer 5'-GCAGAGGAGGAAATTACCCAGTAC-3' (24 bp). The TaqMan probe sequence was 5'-TGTCCACCTGCCATTAAGCCCGA-3' (23 bp). This probe had a fluorescent reporter dye, FAM (6-carboxyfluorescein), attached to its 5' end and the quencher dye TAMRA (6-carboxytetramethyl-rhodamine) attached to its 3' end. Samples were heated for 30 min at 46°C and 4 min at 95°C, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min.

All amplifications were performed in duplicate, with the standard RNA template dilution, over 7 orders of magnitude. A correlation coefficient of up to 97% was obtained, with a sensitivity of 100 copies (or fewer) per ml.

CD4+ T-cell, CD8+ T-cell, and B-cell counts. CD4+ and CD8+ T-cell counts were determined in 30-µl aliquots of whole blood by immunostaining with anti-CD3-fluorescein isothiocyanate (FITC; clone FN18; CliniSciences, Montrouge, France), anti-CD4 PE (clone L200; BD Biosciences, Grenoble, France), and anti-CD8 PC5 (clone B9.11; Beckman Coulter, France) antibodies. For B cells, anti-CD20 FITC (clone B9.E9; Beckman Coulter) was used. Blood samples were incubated with antibodies for 15 min at room temperature, and the red blood cells were lysed with 600 µl of fluorescence-activated cell sorting (FACS) lysing solution (BD Biosciences). Stained cells were washed in phosphate-buffered saline (PBS) and fixed (Cell-Fix; BD Biosciences). The data were acquired on a FACScan instrument using CellQuest software (BD Biosciences). The proportions of CD3+ CD4+ and CD3+ CD8+ cells were determined in the lymphocyte gate, defined in terms of light-scattering properties, using CellQuest software. Absolute counts were calculated from the absolute blood count of lymphocytes obtained by automated cell counting (Coulter MDII; Coultronics, Margency, France).

Cell isolation. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood, either by Ficoll gradient centrifugation or by using CPT tubes (BD Biosciences), according to the manufacturer's recommendations. Residual red blood cells were lysed by hypotonic shock for 5 min, followed by a wash in PBS. Whole axillary or inguinal lymph nodes were mechanically disrupted and passed through a strainer with 40-µm pores (BD Biosciences), and cells were washed in PBS.

Quantification and phenotyping of naive, central memory, and effector memory T cells. For quantification of naive and memory T cells, PBMC were immunostained with anti-CD3 FITC (clone FN18; CliniSciences), anti-CD4 PerCP (clone L200; BD Biosciences), anti-CD28 PE (clone 28.2; BD Biosciences), and anti-CD95 APC (clone DX2; BD Biosciences). Naive, central memory, and effector memory T cells were defined as CD28+ CD95–, CD28+ CD95+, and CD28– CD95+, respectively (56). Stained cells were washed in PBS and fixed (Cell-Fix). Stained and fixed cells were acquired on an LSRI instrument using CellQuest software (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).

PBMC stimulation and intracellular cytokine staining. A total of 2 x 106 PBMC were stimulated with either 2 µg of aldrithiol-2 (AT-2)-inactivated SIVmac239/ml, 0.5 µg of Staphylococcus enterotoxin A (SEA; Sigma-Aldrich, Saint-Quentin Fallavier, France)/ml, and 0.5 µg of Staphylococcus enterotoxin B (SEB; Sigma-Aldrich)/ml or with AT-2-treated SupT1 microvesicles or medium alone (RPMI 1640, 10% fetal calf serum, and antibiotics) as negative controls. AT-2-inactivated SIVmac239 (ARP1018.1) and its negative control (ARP1018.2) were obtained from Jeff Lifson (Frederick, MA) through the EU Program EVA Centralized Facility for AIDS Reagents (NIBSC, Potters Bar, United Kingdom). SIV AT-2 and AT-2 microvesicle stimulations were continued for 24 h with brefeldin A (10 µg/ml; Sigma-Aldrich) during the last 5 h. SEA+SEB and medium-alone stimulations were continued for 6 h with brefeldin A (10 µg/ml) during the last 5 h. Preliminary experiments showed that these stimulation conditions gave the highest IFN-{gamma}, interleukin-2 (IL-2), and tumor necrosis factor (TNF) responses (data not shown). After stimulation, the cultures were treated with EDTA (2 mM; Sigma-Aldrich) to separate any cell aggregations formed during stimulation. Cells were subsequently fixed and permeabilized using FACS lysing and FACS permeabilizing solutions (BD Biosciences) according to the manufacturer's instructions. After permeabilization the cells were immunostained with anti-CD3 Alexa700 (clone SP34-2; BD Biosciences), anti-CD4 PerCP (clone L200; BD Biosciences), anti-CD69 PE (clone FN50; BD Biosciences), anti-IFN-{gamma} FITC (clone 4S.B3; BD Biosciences), and anti-IL-2 APC (clone MQ1-17H12; BD Biosciences) or anti-TNF APC (clone MAB11; BD Biosciences). Stained cells were washed in PBS and fixed by using BD Cell-Fix. Staining of CD3 and CD4 after permeabilization allowed detection of these markers even when downregulated in response to stimulation. Stained cells were acquired on an LSRI instrument using CellQuest software and analyzed with FlowJo software. Approximately 100,000 to 200,000 events in the lymphocyte gate were collected per sample. The level of cytokine expression was defined within activated CD69+ cells. The background level of cytokine staining varied from sample to sample, but was typically <0.01% of CD4+ or CD4– T cells. The only samples considered positive were those in which the percentages of cytokine-stained cells were at least double those of the background and in which there was a distinct population of cytokine-positive cells. Samples not fulfilling these criteria were set to 0.01%. CD8+ T cells were defined as CD3+ CD4– within the lymphocyte gate. Control experiments indicated that CD3+ CD4– and CD3+ CD8+ gave results similar to those of double-positive (CD4+ CD8+) and double-negative (CD4– CD8–) T lymphocytes and made up <5% of the T lymphocytes in PBMC (data not shown).

T-cell proliferation assay. A total of 105 PBMC were seeded in flat-bottom 96-well plates and stimulated with either 2 µg of AT-2-inactivated SIVmac239/ml or AT-2-treated SupT1 microvesicles as negative controls or with 5 µg of concanavalin A (Sigma-Aldrich)/ml as positive controls. Then, 1 µCi of [3H]thymidine (Amersham Biosciences, Orsay, France) was added during the final 8 h of culture on day 3 for concanavalin A and on day 5 for inactivated SIVmac239 activated PBMC. Cells were harvested onto glass fiber filters by using a cell harvester (Skatron, Lier, Norway). [3H]thymidine incorporation was measured in a Microbeta 1 450 liquid scintillation counter (EG&G Wallac, Turku, Finland). The results are expressed as stimulation indexes calculated by dividing the mean counts per minute (cpm) for triplicate antigen- or mitogen-activated wells by the mean cpm for the triplicate negative control wells. The background proliferation differed between samples but was generally <1,000 cpm.

Statistical analysis. The nonparametric Spearman correlation test was used to test for correlation between different T-cell populations and viral load, cytokine response, or proliferative response. The Mann-Whitney test was used to compare the T-cell responses, the proportions of naive and memory T cells, and the viral loads of different groups of macaques. The Wilcoxon test was used to compare the numbers of naive and memory T cells at different time points or in different tissues. In some cases, we used the area under the curve for analysis of the amplitude of variation of a given variable throughout primary infection. Statview software (SAS Institute, Inc., Cary, NC) was used for statistical analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evolution of plasma viral load and CD4+ T cells during SIVmac251 infection of cynomolgus macaques. Eighteen macaques were infected intravenously with SIVmac251. One group (n = 6) was inoculated with a high viral dose (the 5,000 AID50 group), and another group (n = 6) was inoculated with a lower dose (the 50 AID50 group). The third group (n = 6) received the same lower dose (50 AID50) and was given antiretroviral therapy (ART) from 4 h postinfection until 28 days postinfection (the 50 AID50+ART group); the treatment given has been demonstrated to lower the viral load during primary infection and improve control of the viremia in the long term (3). We monitored the evolution of the viral load in the three groups for 226 days (Fig. 1). The viral load in the plasma of macaques infected with the high viral dose increased earlier than that in plasma in macaques infected with the lower dose, but the peaks were not higher, and viremia was not significantly different at the set point (128 days postinfection). ART delayed and lowered the plasma viral load peak. During chronic infection (128 to 274 days postinfection), the viral load as a function of time, measured as the area under the curve, was significantly lower (P = 0.0453) in the treated than in the untreated macaques (Fig. 1), confirming previous observations (3).


Figure 1
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  		FIG. 1. Plasma viral RNA levels during SIVmac251 infection of cynomolgus macaques. (A) Plasma viral loads of individual macaques are shown. Top panel: open symbols, six cynomolgus macaques inoculated intravenously with 5,000 AID50. Middle panel: solid symbols, six cynomolgus macaques inoculated intravenously with 50 AID50. Bottom panel: gray symbols, six cynomolgus macaques inoculated intravenously with 50 AID50 and given antiretroviral treatment (AZT, 3TC, and indinavir) starting 4 h postinfection until 28 days postinfection. (B) Plasma viral load during chronic infection 123 to 273 days after infection, expressed as the area under the curve (AUC), for the three different groups: 5,000 AID50, 50 AID50, and 50 AID50+ART. Horizontal lines represent the median in each group. The treated macaques have a significantly improved control of the viremia (P = 0.0453 [Mann-Whitney test]).

 
Plasma viral load correlated with the decrease of central memory CD4+ T cells in peripheral blood. The evolution of CD4+ T cells during primary infection and during early and asymptomatic chronic infection (until 226 days postinfection) was monitored as a proportion of values on day 0 (Fig. 2). On day 0, the numbers of peripheral total CD4+ T cells in blood were between 337 and 1,659 cells/µl, with a median of 962 ± 363 cells/µl (n = 18). During acute infection, there was a rapid decline of CD4+ T cells in peripheral blood, followed by a rebound to a level just below baseline and then by a gradual diminution after primary infection. The decline in CD4+ T-cell counts during acute infection was, as for plasma viral load, earlier in the macaques infected with a high dose than in macaques infected with a low dose, with nadir values at days 9 and 11 after infection, respectively. ART delayed and lowered the acute CD4+ T-cell decline: the lowest values in the treated group were observed 21 days after infection. The CD4+ T-cell nadir values for the treated macaques during acute infection were significantly higher than those for the untreated macaques (P = 0.0104), whereas there was no difference (P = 0.8728) between the macaques infected with the high dose of virus and the macaques infected with the lower dose.


Figure 2
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  		FIG. 2. Changes in the numbers of total, naive, central memory, and effector memory CD4+ T cells in peripheral blood during SIVmac251 infection of cynomolgus macaques and correlations with plasma viral load. (A) Changes in the absolute counts of total, naive, central memory, and effector memory CD4+ T cells in peripheral blood, presented as percentages of the values on day 0. For visual reasons, one time point for effector memory cells is above (341%), the maximum value of the y axis. Left panels: open symbols, six cynomolgus macaques inoculated intravenously with 5,000 AID50. Middle panels: solid symbols, six cynomolgus macaques inoculated intravenously with 50 AID50. Right panels: gray symbols, six cynomolgus macaques inoculated intravenously with 50 AID50 and given antiretroviral treatment (AZT, 3TC, and indinavir) starting 4 h postinfection until 28 days postinfection. (B) Correlations between the losses of total (Spearman correlation, P = 0.2033), naive (Spearman correlation, P = 0.1973), central memory (Spearman correlation, P = 0.0297), or effector memory (Spearman correlation, P = 0.6007) CD4+ T cells in peripheral blood on day 226 postinfection and plasma viral load on day 226 postinfection. Values for the 50 AID50+ART group are indicated in gray.

 
We found no significant correlation (P = 0.2033) between the loss of total CD4+ T cells and plasma viral load on day 226 postinfection. However, there was a highly significant negative correlation between the CD4+ T-cell count and plasma viral load at all time points (P < 0.0001) (data not shown). There was a significant correlation (P = 0.0297) between the loss of central memory CD4+ T cells and plasma viral load on day 226 postinfection. The evolution of the numbers of naive, central memory, and effector memory CD4+ T cells during the course of SIV infection is shown in Fig. 2. On day 0, the median proportions of naive, central memory, and effector memory CD4+ T cells in peripheral blood were 64% ± 14%, 27% ± 11%, and 4% ± 9%, respectively (n = 18). The loss of central memory CD4+ T cells correlated with viral load, but the loss of naive and effector memory CD4+ T cells did not. However, in two of the three macaques with a viral load in plasma above 105 copies per ml during chronic infection the total population of CD4+ T cells, including naive CD4+ T cells, was very low. In the third macaque with a high plasma viral load, the total CD4+ T-cell count remained relatively stable, although the central memory CD4+ T-cell counts did decrease detectably.

Increase in peripheral blood central memory CD8+ T cells during primary SIV infection. As for CD4+ T cells, there was a diminution of CD8+ T cells in peripheral blood during acute infection, followed by a rebound (Fig. 3). On day 0, the number of CD8+ T cells was between 247 and 1,331 cells/µl of blood, with a median of 869 ± 294 cells/µl of blood (n = 18). The median proportions of naive, central memory, and effector memory CD8+ T cells on day 0 were 34% ± 17%, 15% ± 5%, and 48% ± 16%, respectively (n = 18). Central memory CD8+ T-cell counts increased during primary infection (Fig. 3). This was most evident around 100 days after infection when the numbers of central memory CD8+ T cells were significantly higher than on day 0 (P = 0.0123). However, this increase was transient, and during early and asymptomatic chronic infection the numbers of central memory CD8+ T cells were not significantly different (day 226 postinfection, P = 0.9826) from the baseline value. In some macaques, the numbers of naive CD8+ T cells increased during acute infection. In two of the three macaques with a plasma viral load of more than 105 copies per ml during chronic infection, the total population of CD8+ T cells, and especially naive CD8+ T cells, was very low. The CD8+ T-cell counts and the counts of different subsets of CD8+ T cells did not correlate with the level of viral load; also, we found no evidence of any significant effects of ART (Fig. 3).


Figure 3
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  		FIG. 3. Changes in the numbers of total, naive, central memory, and effector memory CD8+ T cells in peripheral blood during SIVmac251 infection of cynomolgus macaques and correlations with plasma viral load. (A) Changes in the absolute counts of total, naive, central memory, and effector memory CD8+ T cells in peripheral blood, presented as percentages of the values on day 0. Left panels: open symbols, six cynomolgus macaques inoculated intravenously with 5,000 AID50. Middle panels: solid symbols, six cynomolgus macaques inoculated intravenously with 50 AID50. Right panels: gray symbols, six cynomolgus macaques inoculated intravenously with 50 AID50 and given antiretroviral treatment (AZT, 3TC, and indinavir) starting 4 h postinfection until 28 days postinfection. (B) Correlations between the losses of total (Spearman correlation, P = 0.3536), naive (Spearman correlation, P = 0.2419), central memory (Spearman correlation, P = 0.7304), or effector memory (Spearman correlation, P = 0.0996) CD8+ T cells in peripheral blood on day 226 postinfection and plasma viral load on day 226 postinfection. The 50 AID50+ART group is indicated in gray.

 
Decrease in CD4+ T cells and increase in CD8+ T cells and B cells in peripheral lymph nodes and correlation with viral load in plasma. Peripheral lymph node biopsies were performed before infection and at 15, 38, and 245 days postinfection. Before infection, the main cell populations among lymphocytes of peripheral lymph nodes were CD4+ T cells, CD8+ T cells, and B cells (CD20+), with medians of 53% ± 7%, 26% ± 5%, and 9% ± 6%, respectively (n = 18). After SIV infection there was a gradual decrease in the proportion of CD4+ T cells in the total lymphocyte population (Fig. 4). By 245 days postinfection this decrease significantly correlated (P = 0.0058) with plasma viral load. There was a concomitant increase of the proportion of both CD8+ T cells and B cells correlating with the viral load (P = 0.0371 and P = 0.0488, respectively) (Fig. 4).


Figure 4
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  		FIG. 4. Changes in the proportions of CD4+ T cells, CD8+ T cells, and B cells in peripheral lymph nodes during SIVmac251 infection of cynomolgus macaques and correlations with plasma viral load. (A) Changes in the proportions of CD4+ T cells, CD8+ T cells, and B cells in peripheral lymph nodes, presented as percentages of the values on day 0. Left panels: open symbols, six cynomolgus macaques inoculated intravenously with 5,000 AID50. Middle panels: solid symbols, six cynomolgus macaques inoculated intravenously with 50 AID50. Right panels: gray symbols, six cynomolgus macaques inoculated intravenously with 50 AID50 and given antiretroviral treatment (AZT, 3TC, and indinavir) starting 4 h postinfection until 28 days postinfection. (B) Correlations between the losses of CD4+ T cells (Spearman correlation, P = 0.0058), CD8+ T cells (Spearman correlation, P = 0.0371), or B cells (Spearman correlation, P = 0.0488) CD8+ T cells in peripheral lymph nodes on day 245 postinfection and plasma viral load on day 226 postinfection (pi). The 50 AID50+ART group is indicated in gray.

 
Naive and memory T cells in peripheral blood and peripheral lymph nodes. We evaluated the proportion of naive, central memory, and effector memory cells among the CD4+ and CD8+ T-cell populations in peripheral lymphoid tissue, as we had already been done for peripheral blood. Due to technical problems, values for the proportions of naive and memory T cells in peripheral lymphoid tissues before infection of the 18 macaques were not available. We therefore compared the lymphoid tissues of 4 uninfected macaques and the peripheral blood of 18 other uninfected macaques (data not shown). There was a significantly lower (P = 0.0022) proportion of effector memory CD8+ T cells in peripheral lymph node samples than in peripheral blood. The proportion of naive CD8+ T cells was significantly higher (P = 0.0022) among cells from peripheral lymph node than from peripheral blood, whereas the proportions of central memory CD8+ T cells were similar. The median proportion of naive, central memory, and effector memory CD8+ T cells in peripheral lymph nodes were 80% ± 6%, 16% ± 2%, and 2% ± 1%, respectively, in uninfected macaques (n = 4). The median proportions of naive, central memory, and effector memory CD4+ T cells in peripheral lymph nodes were 68% ± 6%, 31% ± 6%, and 0.08% ± 0.08%, respectively, in uninfected macaques (n = 4), and these values were not significantly different from those for peripheral blood.

Increased proportion of memory CD8+ T cells in the peripheral lymphoid tissues of SIV-infected macaques. We compared the changes in the proportions of naive, central memory, and effector memory cells in CD4+ and CD8+ T-cell populations in peripheral lymphoid tissues after SIV infection in 18 macaques and used 4 uninfected macaques as a reference (Table 1) . The proportion of central memory CD4+ T cells was significantly lower, and the proportion of naive CD4+ T cells was significantly higher in peripheral lymphoid tissue in untreated SIV+ macaques than in SIV– macaques. In SIV+ macaques receiving postexposure ART, the proportions of memory CD4+ T cells were not significantly different from those in SIV– macaques, except for a decrease at the latest time point (P = 0.0550). In the ART group, the mean proportion of naive CD4+ T cells tended to increase but was not significantly different from that in the SIV– group (P = 0.1356 on day 38 and after 245 days). The CD4+ effector memory cell counts were similar in SIV– and SIV+ macaques, except that there was an increase 245 days after infection in the treated macaques.


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  		TABLE 1. Median proportion of naive and memory T cells in peripheral lymph nodes in uninfected and SIVmac251-infected cynomolgus macaques

 
The proportion of CD8+ effector memory cells was higher at all time points in the SIV+ untreated macaques and after 245 days in macaques receiving postexposure ART than in SIV– macaques. At 38 days after infection, there was a higher proportion of central memory CD8+ T cells and a lower proportion of naive CD8+ T cells in peripheral lymphoid tissue in SIV+ untreated macaques than in SIV– macaques; there was only a tendency for this pattern in SIV+ macaques receiving postexposure ART.

SIV-specific proliferative response in peripheral lymph nodes inversely correlates with viral load during chronic infection. The SIV-specific proliferative response in PBMC and lymph node mononuclear cells (LNMC) was monitored during SIVmac251 infection by determining 3[H]thymidine incorporation (Fig. 5). In PBMC, the SIV-specific proliferative response showed some fluctuations over time but was detected at one or several time points in 17 of the 18 macaques studied. In general, responses increased until around day 100 postinfection and gradually decreased thereafter. The macaque without a detectable SIV-specific proliferative response had the highest plasma viral load during chronic infection (>105 copies/ml). The area under the curve of the proliferative response between day 0 and day 226 postinfection was significantly and inversely correlated with the viral load at 226 days after infection (P = 0.0085). The SIV-specific proliferative response was not different in macaques receiving postexposure ART during early infection from that in untreated macaques infected with the same dose of virus (P = 0.5887).


Figure 5
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  		FIG. 5. Changes in the SIV-specific proliferative responses in PBMC and LNMC during SIVmac251 infection of cynomolgus macaques and correlations to plasma viral load. (A) Changes in the SIV-specific proliferative responses in PBMC (top) and LNMC (bottom) during SIV infection. Left panels: open symbols, six cynomolgus macaques inoculated intravenously with 5,000 AID50. Middle panels: black symbols, six cynomolgus macaques inoculated intravenously with 50 AID50. Right panels: gray symbols, six cynomolgus macaques inoculated intravenously with 50 AID50 and given antiretroviral treatment (AZT, 3TC, and indinavir) starting 4 h postinfection until 28 days postinfection. (B) Top panel: inverse correlations between the total SIV antigen-specific proliferative response in PBMC until day 226, expressed as the area under the curve, and plasma viral load on day 226 postinfection (pi) (Spearman correlation, P = 0.3150). Bottom panel: inverse correlations between the SIV antigen-specific proliferative response in LNMC on day 245 postinfection and plasma viral load on day 226 postinfection (pi) (Spearman correlation, P = 0.0042). The 50 AID50+ART group is indicated in gray.

 
The SIV-specific proliferative response in LNMC was also analyzed by testing before infection and at 15, 38, and 245 days after infection. The proliferative response was most evident at the last available time point: 245 days postinfection. On day 245 postinfection, the SIV-specific proliferative response was inversely correlated with the plasma viral load (P = 0.0042). The response tended to be higher in the macaques receiving postexposure ART during early infection than in untreated macaques infected with the same dose of virus (P = 0.0618).

SIV-specific IFN-{gamma} and TNF response in CD4+ and CD8+ T cells correlated with plasma viral load during chronic infection. We monitored the SIV-specific cytokine responses in CD4+ and CD8+ T cells of peripheral blood by intracellular cytokine staining and flow cytometry (Fig. 6). The IFN-{gamma} responses in CD4+ and CD8+ T cells were similar in terms of strength and kinetics. There were some fluctuations, but in general and as for proliferative responses, the responses increased until around day 100 postinfection and gradually decreased thereafter. In some macaques, there was a IFN-{gamma} response peak in early infection, i.e., around day 28. The IFN-{gamma} response, as a function of time between day 0 and day 226 postinfection, was compared to the viral load during chronic infection (day 226 postinfection): there was a trend toward an inverse correlation for CD4+ T cells (Fig. 7, P = 0.1225) and CD8+ T cells (Fig. 7, P = 0.1693). Excluding one atypical macaque showing an early and sustained control of viral replication, the inverse correlations between viral load and the SIV-specific IFN-{gamma} response of CD4+ T cells and CD8+ T cells was significant (P = 0.0274 and P = 0.0455, respectively). Moreover, a strong IFN-{gamma} response in CD4+ T cells correlated with a strong IFN-{gamma} response in CD8+ T cells (P = 0.0020). No difference in the IFN-{gamma} responses between treated and untreated macaques was found. The kinetics of the IFN-{gamma} response in CD8+ T cells were similar to the kinetics of central memory CD8+ T cells, although the two variables were not significantly correlated.


Figure 6
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  		FIG. 6. Changes in the SIV-specific IFN-{gamma} and IL-2 responses in CD4+ and CD8+ T cells during SIVmac251 infection of cynomolgus macaques. Changes in the SIV-specific IFN-{gamma} (A) and IL-2 (B) responses in CD4+ T cells (top) and CD8+ T cells (bottom) during SIV infection. Left panels: open symbols, six cynomolgus macaques inoculated intravenously with 5,000 AID50. Middle panels: solid symbols, six cynomolgus macaques inoculated intravenously with 50 AID50. Right panels: gray symbols, six cynomolgus macaques inoculated intravenously with 50 AID50 and given antiretroviral treatment (AZT, 3TC, and indinavir) starting 4 h postinfection until 28 days postinfection.

 

Figure 7
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  		FIG. 7. Correlations between the SIV-specific IFN-{gamma}, IL-2, and TNF responses in CD4+ and CD8+ T cells and plasma viral load during chronic SIVmac251 infection of cynomolgus macaques. Correlations are shown between responses for SIV-specific IFN-{gamma} for days 21 to 226 after infection (A), IL-2 for days 21 to 177 after infection (B), and TNF on day 226 after infection (C) in CD4+ T cells (top) and CD8+ T cells (bottom) and plasma viral load on day 226 postinfection (pi). AUC, area under the curve. The 50 AID50+ART group is indicated in gray.

 
The SIV-specific IL-2 responses in CD4+ and CD8+ T cells in peripheral blood displayed kinetics similar to those of the IFN-{gamma} responses, increasing until around day 100 postinfection and then decreasing. Unlike the IFN-{gamma} responses, IL-2 responses in CD4+ and CD8+ T cells of peripheral blood did not correlate with plasma viral load, and the difference in the IL-2 responses between treated and untreated macaques was not significant. Only a small proportion of the responding cells were double positive for IFN-{gamma} and IL-2, and because the IL-2 responses were already low, there were too few events for meaningful analysis. However, a high IL-2 response correlated with a high IFN-{gamma} response (data not shown).

A previous study by Gauduin et al. (21) showed that the SIV-specific TNF response by CD4+ T cells was stronger than the IFN-{gamma} and IL-2 responses during chronic SIV infection. Therefore, and since there were very low or undetectable IL-2 responses at 150 days postinfection, we studied the TNF responses on day 226. SIV-specific TNF responses were detected in CD4+ and CD8+ T cells from most macaques. There was a significant inverse correlation (P = 0.0373) between the TNF response in CD8+ and the plasma viral load on any given day, but there was no correlation between the TNF response in CD4+ T cells and plasma viral load on the same day (Fig. 7). A high TNF response correlated with a high IFN-{gamma} response (data not shown).

We attempted to evaluate the IFN-{gamma} and IL-2 responses in CD4+ and CD8+ T cells from peripheral lymph nodes before infection and at 15 and 38 days postinfection. However, using the protocol applied to PBMC we were not able to detect any cytokine responses in LNMC.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cellular immune responses are an important part of the control of HIV infection (39, 53). We report here the changes in naive and memory cells, and the virus-specific responses of CD4+ and CD8+ T cells, during primary infection using the model of SIVmac251 infection of cynomolgus macaques. In a cohort of 18 cynomolgus macaques infected with the same pathogenic viral strain, we describe diverse rates of progression, closely resembling HIV infections in humans. On our study, 3 of 12 untreated macaques demonstrated difficulties in controlling viremia with a set point of >105 viral RNA copies/ml (128 days postinfection); this is consistent with a recent analysis in humans in which 2 of 23 patients had a plasma viral load above 105 viral RNA copies/ml after 1 year of infection (59). One of the twelve untreated macaques had an undetectable plasma viral load at set point; in a human cohort of 426 patients, 6.7% had a plasma viral load of <500 copies/ml 5 years after seroconversion (36). The remaining macaques displayed a broad range of plasma viral load in between these extreme values. Thus, this model allowed longitudinal follow-up of the cellular immune responses and changes in naive and memory T-cell populations during primary infection; the findings could then be compared to the various rates of disease progression.

We used plasma viral load as a marker of disease progression, since it has been shown to be a good predictive marker of survival of SIV infection (35, 64) and the set point levels correlating to disease progression in HIV (41). Another marker of HIV disease progression is the loss of CD4+ T cells (40). We found a better correlation between plasma viral load and the losses of peripheral blood central memory CD4+ T cells, compared to the total CD4+ T cells, as similarly described by others during SIV infection in Indian rhesus macaques (61), which generally show a more rapid disease progression (57). Memory CD4+ T cells express CCR5 (6), the main coreceptor for SIV (11), which makes these cells susceptible to infection. In two of the three macaques with a high plasma viral load (>105 viral RNA copies/ml) at the set point, there was also a depletion of naive CD4+ and CD8+ T cells, as described in SIV infection of rhesus macaques (38, 48). Depletion of naive T cells may reflect changes in T-cell homeostasis and regeneration during progressive disease, as described in humans (14). In peripheral lymph nodes, the primary sites of HIV replication (52), we found declining total CD4+ T cells correlating with plasma viral load. Extensive apoptosis in T-cell areas of peripheral lymph nodes during primary SIV infection has been correlated with rapid disease progression in rhesus macaques (43). The proportion of central memory CD4+ T cells was lower in peripheral lymph nodes of SIV+ macaques than at baseline, whereas the proportions of central memory and effector memory CD8+ T cells were higher. A transient increase in central memory CD8+ T cells was also observed in peripheral blood during primary infection, as previously described in rhesus macaques (38).

The kinetics of cellular immune responses appeared to be similar to the kinetics of memory CD8+ T cells. In peripheral blood, the highest SIV-specific proliferative IFN-{gamma} and IL-2 responses were during primary infection coinciding with an increase of CD8+ central memory T cells. In peripheral lymph nodes, the highest proliferative responses, as well as the highest CD8+ effector memory T-cell proportions, were in most cases during early chronic infection. However, the limited possibilities of lymph node biopsies did not allow fine kinetic analysis at these sites, and an even higher response around 3 months after infection, as for peripheral blood, cannot be excluded. Memory T cells are the best cytokine responders upon antigen stimulation (65), and although the kinetics of CD8+ memory T cells followed the kinetics of SIV-specific responses, we were not able to demonstrate a direct correlation between the two variables.

In our study, a high SIV-specific peripheral lymphocyte proliferative response in lymph nodes correlated with a lower viral load. High IFN-{gamma} responses in peripheral blood CD4+ and in CD8+ T cells during primary infection also tended to correlate with plasma viral load during early chronic infection. In peripheral lymph nodes, T-cell cytokine responses were undetectable during primary infection, and this may have been a consequence of the numerous naive T cells in these tissues. Previous studies have similarly described low levels of T-cell cytokine responses in lymph nodes (21, 62).

There have been many investigations of cellular immune responses in the context of HIV/SIV infection. Several studies have shown a relationship between high CD4+ or CD8+ T-cell responses and low viral load and/or slow progression (5, 8, 9, 21, 31, 42, 44, 50, 58, 68). Most of these studies have been cross-sectional and/or during chronic infection. One recent longitudinal study of SIV-infected Indian rhesus macaques during primary infection demonstrated a correlation between the loss of antigen nonspecific CD4+ T-cell responses (response to SEB) and plasma viral load (61). However, these researchers could not find evidence for a correlation between SIV-specific cytokine responses and plasma viral load, possibly because the rates of progression and early T-cell dysfunction in Indian rhesus macaques are very much faster than those in cynomolgus macaques. Few studies have reported on both CD4+ and CD8+ T responses, although both are important and they are interrelated (20, 27, 53). We demonstrate here a correlation between a high CD4+ T-cell response and a high CD8+ T-cell response.

Recent studies have emphasized the importance of an efficient IL-2 response by memory T cells for the control of HIV/SIV infection (25, 34, 62, 69). Others have identified the double-positive IFN-{gamma} and IL-2 (7) or the polyfunctional (5, 23) T-cell responses as being important. However, there are also studies a correlating single IFN-{gamma} (8, 32) or TNF (21) T-cell response and plasma viral load. In the present study, we demonstrate an inverse correlation between the viral load and each of the IFN-{gamma} and TNF responses. We could not detect a correlation between IL-2 responses and disease progression. However, it should be noted that in our study we used whole inactivated virus in our cytokine assay, whereas previous studies have used peptides or protein stimulations, and it cannot be excluded that such experimental differences influence the results. The function, not the presence, of HIV-specific T-cell responses inversely correlates with plasma viral load (22, 66). T-cell dysfunction is manifest early in infection (45), and the IL-2 response is lost first (19). Possibly, the low IL-2 responses in our study, consistent with other reports (21, 61), were due to an early T-cell dysfunction. However, some IL-2 responses were detected during primary infection, but these responses declined and were lost before the IFN-{gamma} or TNF responses, agreeing with the reports of the loss of IL-2 responses during HIV/SIV infection (25, 61, 62).

Our cohort of 18 macaques included a group given postexposure ART during acute infection. This treatment lowered and delayed the peak plasma viral load and was associated with better control of viral replication in the long term, as previously shown (3). In these treated macaques, we also found a lower decline of CD4+ T cells in peripheral blood during acute infection, no significant decrease in central memory CD4+ T cells in the peripheral lymph nodes, and a better SIV-specific proliferative response in the peripheral lymph nodes, suggesting preserved T-cell responses during primary infection. Lowering the plasma viral load during acute infection by ART preserves the SIV/HIV-specific cellular immune responses, which in turn may be beneficial for the control of viremia in the long term (49, 51). Similarly, rhesus macaques infected with attenuated SIV have better CD4+ T-cell responses and slower progression than macaques infected by wild-type SIV (21).

Our study demonstrates the importance of SIV-specific cellular immune responses during primary infection for the control of SIV infection. However, there are many other viral and host factors contributing to the efficiency of these responses and to the rate of disease progression. Indeed, disease progression is influenced by factors such as the humoral immune response (18), the HLA subtype (10), the abundance of chemokine receptors (4), cytokine and chemokine production (29), the viral phenotype (2, 12), and viral fitness and replicative capacity (17).

In conclusion, using a model displaying various rates of disease progression we were able to show that high SIV-specific T-cell responses in both peripheral blood and in peripheral lymph nodes during early infection inversely correlated with viral load. There was a transient increase of central memory CD8+ T cells in peripheral blood during primary infection and similarly SIV-specific T-cell responses were detectable during primary infection and then faded in the long term. The present study highlights the importance of the early virus-specific immune responses in the outcome of HIV/SIV disease and provides more extensive details about the changes of virus-specific immune responses over time.


   ACKNOWLEDGMENTS
 
This study was supported by the French national AIDS agency, Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS). B.M. and I.K. were both supported by grants from the ANRS. We also are grateful to the Lennanders Foundation, the Royal Swedish Academy of Sciences, and the Faculty of Medicine of Lund University, Lund, Sweden.

AT-2-inactivated SIVmac239 (ARP1018.1) and its negative control (ARP1018.2) were prepared and donated by Jeff Lifson (Frederick, MA) through the EU Program EVA Centralized Facility for AIDS Reagents (NIBSC, Potters Bar, United Kingdom). We thank Christophe Joubert, Christophe Jouy, Patrick Flament, and Hélène Juin for animal care.


   FOOTNOTES
 
* Corresponding author. Mailing address: CEA, Service d'Immuno-Virologie, DSV/iMETI, 18 Route du Panorama, BP 6, 92 265 Fontenay-aux-Roses, France. Phone: 33 1 46 54 94 78. Fax: 33 1 46 54 77 26. E-mail: bruno.vaslin{at}cea.fr Back

{triangledown} Published ahead of print on 3 October 2007. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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