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The Journal of Immunology, 2001, 166: 506-516.
Copyright © 2001 by The American Association of Immunologists

Relative Resistance in the Development of T Cell Anergy in CD4+ T Cells from Simian Immunodeficiency Virus Disease-Resistant Sooty Mangabeys1

Pavel Bostik2,*, Ann E. Mayne*, Francois Villinger*, Kenneth P. Greenberg*, Jonathan D. Powell{dagger} and Aftab A. Ansari*

* Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322; and {dagger} Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite high viral loads, T cells from sooty mangabey (SM) monkeys that are naturally infected with SIV but remain clinically asymptomatic, proliferate and demonstrate normal Ag-specific memory recall CD4+ T cell responses. In contrast, CD4+ T cells from rhesus macaques (RM) experimentally infected with SIV lose Ag-specific memory recall responses and develop immunological anergy. To elucidate the mechanisms for these distinct outcomes of lentiviral infection, highly enriched alloreactive CD4+ T cells from humans, RM, and SM were anergized by TCR-only stimulation (signal 1 alone) and subsequently challenged with anti-CD3/anti-CD28 Abs (signals 1 + 2). Whereas alloreactive CD4+T cells from humans and RM became anergized, surprisingly, CD4+ T cells from SM showed marked proliferation and IL-2 synthesis after restimulation. This resistance to undergo anergy was not secondary to a global deficiency in anergy induction of CD4+ T cells from SM since incubation of CD4+ T cells with anti-CD3 alone in the presence of rapamycin readily induced anergy in these cells. The resistance to undergo anergy was reasoned to be due to the ability of CD4+ T cells from SM to synthesize IL-2 when incubated with anti-CD3 alone. Analysis of phosphorylated kinases involved in T cell activation showed that the activation of CD4+ T cells by signal 1 in SM elicited a pattern of response that required both signals 1 + 2 in humans and RM. This function of CD4+ T cells from SM may contribute to the resistance of this species to SIV-induced disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sooty mangabey monkeys, a non-human primate species from West Africa, much like a number of other African nonhuman primate species, are naturally infected with the SIV, but remain clinically asymptomatic throughout their lives (1, 2). Select SIV isolates from the sooty mangabey, when experimentally injected into Asian rhesus or pig-tailed macaques, lead to infection and a disease course remarkably similar to human HIV-1 infection and AIDS (3). The lack of disease development in the natural hosts was found to be not simply due to viral load since naturally infected sooty mangabeys raised in captivity appear to have up to 105-107 virus particles/ml of plasma (levels known to induce disease and death in both macaque species and humans) and yet do not demonstrate any signs of disease (2). Furthermore, experimental boosting of the endogenous viral load and/or experimental infection of SIV-seronegative mangabeys with a dose and isolate of SIV that has been documented to cause disease and death in Asian macaques does not lead to any detectable disease, respectively (2, 4, 5). Thus, a paradox is apparent in that if the lack of disease is due to the development of a virus-specific immune response in naturally infected mangabeys, why are there persistent high levels of plasma viremia and why does this not lead to disease in these animals?

Our laboratory has been studying the nature of the immune response in these naturally infected disease-resistant sooty mangabeys and comparing their immune responses with those in SIV-infected disease-susceptible rhesus macaques with the hypothesis that knowledge gained from such studies may provide important information as to the mechanism(s) of disease resistance/susceptibility in these two species (4, 6, 7, 8, 9, 10, 11, 12, 13). During the course of these studies, it was noted that unfractionated PBMCs from sooty mangabeys consistently showed high spontaneous proliferation, and a high frequency of cells remain viable without external stimuli irregardless of the media used to culture these cells. Furthermore, whereas SIV-infected rhesus macaques much like HIV-1-infected humans (14, 15, 16, 17) demonstrate an accelerated loss of their Ag-specific CD4+ T cell responses (see Fig. 1Go), sooty mangabeys failed to show such loss in memory T cell responses. The decreased responses of SIV-infected rhesus macaques were not secondary to loss of CD4+ T cells and could not be ascribed to dysfunction of the APCs. It was reasoned that one mechanism that could account for such distinct outcomes of Ag-specific immune responses was likely due to perturbations of the intracellular chain of events following TCR ligation induced in CD4+ T cells in rhesus macaques but not sooty mangabeys. As an initial step toward addressing these mechanisms, studies were performed to determine the requirements and mechanisms by which CD4+ T cells, one of the major cell lineages involved in AIDS pathogenesis, become activated in the disease-resistant sooty mangabeys as compared with disease-susceptible rhesus macaques and, for purposes of control, human CD4+ T cells.



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  		FIGURE 1. Kinetics of the TT-specific CD4+ T cell proliferative response of SIV-infected rhesus macaques and sooty mangabeys. Six adult rhesus macaques (RM), three SIV-seronegative (SM-), and three SIV-seropositive (SM+) mangabeys were hyperimmunized with TT as described in Materials and Methods. Three of the six rhesus macaques (RM inf.) were then infected with 100 tissue culture infective dose 50% of SIVmac239 (at 2 wk, indicated by arrow). PBMC samples from all 12 monkeys obtained before (preimmunization) and at varying times after immunization were cocultured with autologous herpes papio-transformed cell lines that had been pulsed overnight with 10 µg/ml OVA (control) or 10 µg/ml of TT, washed, and irradiated (8000 rad). The cocultures were incubated for 48 h and each culture was pulsed with [3H]thymidine and harvested 16 h later. Cultures were performed in triplicate and the mean cpm of triplicate cocultures with TT-pulsed APCs was divided by the mean cpm of the triplicate cocultures with OVA-pulsed APCs to derive stimulation indices. All PBMC samples from a single monkey were performed in a single assay. The percent SD of triplicate samples was always <10%. Data shown represent values obtained on serial samples from the same monkey and are representative of all three monkeys per group. The arrow denotes the time when the monkeys were infected with SIV.

 
It has been known for some time now that although the TCR exhibits remarkable specificity in the peptide-MHC complex that it can interact with, the outcome of this interaction (signal 1) is not the function of the peptide, but the context in which it is presented to the T cell (18, 19). Thus, such interaction may either result in a tolerogenic or an immunogenic signal based on the absence or presence of interaction between sets of molecules expressed by T cells and their natural ligands on the APCs termed as costimulatory molecules and their ligands, respectively (signal 2). Although the engagement of the TCR alone may lead to apoptosis and is one method for the induction of tolerance, there is now a large body of data showing that delivery of signal 1 in the absence of the costimulatory signal may not lead to cell death but a prolonged period of unresponsiveness termed "anergy" (20, 21, 22). Although a number of sets of cell surface molecules on T cells and APCs have been shown to function as receptors and their respective ligands providing costimulatory signals (signal 2), the interaction between CD28 on the T cell surface and CD80/CD86 on the APCs have been the most extensively studied (23, 24, 25, 26, 27). Although the precise biochemical pathways involved by which CD28 ligation promotes CD4+ T cell costimulation remain to be defined, it is clear that signals provided by such costimulation promote proliferation by inducing IL-2 expression both at the transcriptional and posttranscriptional levels (28).

Thus, the knowledge that full immune activation of CD4+ T cells requires not only interaction between the TCR and its cognate peptide bearing MHC class II molecules (signal 1) but also requires interaction between costimulatory molecules such as CD28 and their natural ligands CD80/86 (signal 2) (29, 30, 31) was exploited to define whether differences exist in the activation requirements of CD4+ T cells from sooty mangabeys as compared with rhesus macaques and humans. Results of these studies constitute the basis of this report.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Source of samples

The blood samples were obtained from normal, healthy adult rhesus macaques (Macacca mulatta), asymptomatic (following achievement of viral load set point 3–6 mo after infection) rhesus macaques experimentally infected with 100 tissue culture infective doses 50% of SIVmac239 (grown in PHA blasts from rhesus macaques), and adult healthy SIV-seronegative and -seropositive sooty mangabeys (Cercocebus atys) housed at the Yerkes Regional Primate Research Center of Emory University. All animals were maintained according to the guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, and the Health and Human Services guidelines "Guide for the Care and Use of Laboratory Animals." Human blood samples were obtained from adult healthy laboratory volunteers.

Immunization of monkeys

Each monkey was injected s.c. with 100 µg of tetanus toxoid (TT; Pasteur Mérieux Connaught, Ontario, Canada) incorporated in IFA followed by two booster doses of TT (10 µg), each administered i.v. in sterile PBS at 2-wk intervals. A similar dose and schedule was then administered for keyhole limpet hemocyanin (KLH)3 (Pierce, Rockford, IL).

Measurement of Ag-specific proliferative responses

PBMC were obtained by Ficoll-Hypaque density centrifugation and cryopreserved. An aliquot of these PBMCs before initiation of the immunizations was incubated with supernatant fluid containing herpes papio virus and resulting transformed cells were cryopreserved to be used as autologous APCs. The PBMC samples obtained from one monkey before and at varying times after immunization were assayed in a single assay. The cryopreserved transformed cells were thawed, washed, and incubated with either 10 µg/ml chicken OVA or 10 µg/ml of TT overnight at 37°C in a 7% CO2 humidified atmosphere. Aliquots of these cells were phenotyped by FACS analysis and found to express CD14, CD20, and low-density CD56 but not CD3, CD4, and CD8, consistent with the view that these cells represented B and/or monocyte but not T cell lineage. It should be noted that non-human primate monocytes express CD56 (32). The Ag-pulsed cells were irradiated (8000 rad), washed, and adjusted to 1 x 105 cells/ml and 0.1 ml was dispensed into individual wells of a 96-well microtiter plate. The PBMCs were adjusted to 1 x 106/ml and cocultured with TT-pulsed APCs or OVA-pulsed APCs in a volume of 0.1 ml. All cultures were performed in triplicate. Controls also included cultures of PBMCs alone and APCs alone in media. Cultures were incubated for 5 days with the addition of 2 U/ml IL-2 on day 3. On day 5, the cultures were pulsed with 1 µCi of [3H]thymidine (sp. act., 2 Ci/mM; NEN, Boston, MA) in a volume of 0.02 ml of media, harvested after a 14-h incubation, and the mean uptake of [3H]TdT was determined. A stimulation index was calculated by dividing the mean cpm of the TT-specific response by the mean cpm of the OVA-nonspecific response.

Preparation of CD4+ T cells

The CD4+ cells were separated from freshly isolated PBMCs using Dynabeads M450 CD4 and detached with Detachabead (both from Dynal, Lake Success, NY). The purity of the cell population isolated was always >99.0% as determined by FACS analysis. The fact that optimal proliferation of CD4+ T cells requires the presence of monocytes was used to check for the degree of purity of the CD4+ T cells. An aliquot of isolated CD4+ T cells was thus cultured with a predetermined optimum concentration of PHA-P (0.1%) in the presence (positive control) and absence of 10% autologous non-CD4+ T cells. Cultures of rhesus macaque and human CD4+ T cells alone failed to proliferate in the presence of PHA-P, denoting the absence of adherent cells. In contrast, highly purified CD4+ T cells from TT-immunized mangabeys when cultured alone proliferated in the presence of PHA-P but not in the presence of TT unless autologous APCs were added. In select experiments, the CD4+ highly enriched population was further fractionated into CD45 RA+ and RA- populations with the addition of the appropriate mAb reagent (clone L48; BD Biosciences, San Jose, CA) followed by the separation using anti-mouse Ig immunobeads (Dynal).

Media

Media at all times refers to RPMI 1640 supplemented with 1% penicillin-streptomycin, 2 mM L-glutamine (all from Life Technologies, Long Island, NY), and 10% of a preselected lot of FCS (HyClone, Logan, UT).

Preparation of allogeneic primed CD4+ T cells and T cell lines

The purified CD4+ T cells were cultured in a 30-ml tissue culture flask at 2 x 106/ml with 2 x 106/ml of irradiated (previously identified as allogeneic) unfractionated PBMCs from the same species. The cultures were incubated at 37°C in a 7% CO2 humidified atmosphere for 7 days and then fed with 10 U/ml recombinant human IL-2 (courtesy of Hoffmann-LaRoche, Nutley, NJ) and incubated for an additional 3 days. These cultures were boosted with the same source of allogeneic irradiated cells (2 x 106/ml) on day 10 followed by the addition of an additional 10 U/ml recombinant human IL-2 on day 13. and the cultures were then harvested on days 15 and 16. An aliquot of these cells was subjected to FACS analysis and routinely found to be >99.9% CD4+. For select experiments, such alloprimed CD4+ T cell lines were subjected to limited dilution cloning (0.3–1 cell/well with 5 x 104 irradiated allogeneic cells) and the positively growing wells were gradually expanded and maintained on the same allogeneic irradiated stimulator cells until 3–4 x 108 cells were obtained. Aliquots of 107 cells were then cryopreserved and used in later studies.

Preparation of anergic cells

The allogeneic primed CD4+ T cells were adjusted to 2 x 106/ml and then cultured in 30-ml flasks with media alone (control) or in flasks precoated overnight with anti-CD3 mAbs (clone FN-18; BioSource International, Camarillo, CA, for nonhuman primates and clone UCHT-1; BD Biosciences, for human cells). The cultures were incubated at 37°C in a 7% CO2 humidified atmosphere for 3 days. The cells were then washed and placed in media alone for 24–48 h, harvested, and subjected to testing for potential anergy as described below. The CD4+ T cells incubated in media or following culture with immobilized anti-CD3 and the rest period expressed CD2, CD3, CD4, CD25, CD69, CD71, and TCR {alpha}{beta} and low levels of CD28 which increased following activation.

Anergy analysis

The cells were resuspended in media at 1 x 106/ml and 0.1 ml of the population to be tested (anti-CD3 stimulated) and the control cells (incubated in media alone) were dispensed into individual wells of a 96-well microtiter plate. To triplicate cultures was added either 0.1.ml of media (background control), 0.1 ml of media containing 1 x 106/ml irradiated allogeneic sensitizing cells (Ag-specific control), anti-CD3/anti-CD28-conjugated immunobeads (3 beads/target cell) prepared as described elsewhere (33) or 10 U/ml recombinant human IL-2 (positive control). The concentration of anti-CD3/anti-CD28 and IL-2 that gives maximal proliferation and synthesis of IL-2 was previously determined. The microculture plates were then incubated for 72 h at 37°C in a 7% CO2 humidified atmosphere, pulsed with [3H]TdT, and assayed as described above. The mean uptake of [3H]thymidine by triplicate cultures was calculated and the SD derived. In some experiments, supernatant fluids from the challenge cultures were removed after the microtiter plates were centrifuged and 0.1 ml of this supernatant fluid was added to individual wells of a 96-well microtiter plate previously seeded with the IL-2-starved (105 cells/well) HT-2 cell line (HT-2; American Type Culture Collection, Manassas, VA). The cultures were pulsed with [3H]thymidine and the mean cpm ± SD was calculated. Addition of 1 µg/well anti-IL-4 to either the supernatant fluid from the anti-CD3-pretreated or media-treated sooty mangabey CD4+ T cells did not appreciably alter the results, denoting that the proliferation of the HT-2 cell line was not likely due to the presence of IL-4.

Use of cyclosporin A (CSA), rapamaycin, and hydroxyurea

In efforts to determine the stage at which anergy is induced, use was made of previously defined reagents that have been utilized for similar studies with murine CD4+ T cells (34). This included the use of CSA (Calbiochem, San Diego, CA), rapamycin (Calbiochem), and hydroxyurea (Sigma, St. Louis, MO). The CSA, rapamycin, and hydroxyurea were dissolved in ethanol to prepare stock solutions. Appropriate concentrations were added to the cultures at the time of anergy induction with the use of anti-CD3 mAb alone. CSA and rapamycin were added to these cultures at a concentration of 100 nM and hydroxyurea at 4 µM; the cultures were then washed and subjected to anergy analysis as outlined above. The concentrations of CSA, rapamycin, and hydroxyurea were predetermined for each species by a protocol as described elsewhere (34).

Analysis of cytokines by anti-CD3 or anti-CD3/anti-CD28-activated CD4+ T cells

Aliquots of alloactivated CD4+ T cells from three normal rhesus macaques, three seronegative mangabeys, and three adult human volunteers were cultured at 4 x 106 cells/ml with either media alone, anti-CD3, or anti-CD3/anti-CD28-conjugated immunobeads. Supernatant fluids from these cultures were collected at 24, 48, and 72 h and subjected to quantitation of the levels of IL-2, IFN-{gamma}, IL-3, and TNF-{alpha} using a standard enzyme immunoassay as described elsewhere (7, 8). Standard curves utilizing our laboratory-prepared nonhuman primate and commercially available human recombinant cytokines were used in parallel to derive the data on the quantitative levels of the cytokines.

Analysis of mitogen-activated protein kinase (MAPK) activity

A critical threshold of anti-CD3 Ab concentration was shown to be required for the induction of phosphorylated extracellular signal-related kinase (ERK) in highly enriched alloactivated CD4+ T cells from humans and rhesus macaques. In efforts to demonstrate differences in the threshold of anti-CD3 required to induce phosphorylation of ERK in CD4+ T cells from mangabeys, macaques, and humans, aliquots of such alloactivated highly enriched (>99%) CD4+ T cells from all three species were stimulated with a previously defined suboptimal dose of anti-CD3 alone and for purposes of control an optimal dose of anti-CD3/CD28 in vitro in 24-well plates for 10 min at 37°C. The cells were then immediately lysed in 150 µl of SDS sample buffer that was supplemented with 1 mM sodium vanadate. In addition, aliquots of the same alloactivated CD4+ T cells from rhesus macaques and mangabeys were subjected to anergy induction by incubation of the cells at 1 x 106/ml with anti-CD3 Abs for 3 days as described above. After the rest period, the cells were adjusted to 4 x 106/ml and challenged with anti-CD3/anti-CD28 in 24-well plates for 10 min at 37°C and lysed. Aliquots of the lysates corresponding to the equal number of cells from each specimen were then subjected to PAGE using a 10% gel and subjected to Western blot analysis using 1 µg/ml anti-phospho-ERK or anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were incubated with an alkaline phosphatase-conjugated goat anti-mouse Ig Ab (Southern Biotechnology Associates, Birmingham, AL) diluted 1:5000 and developed. Integrated densities (ID) of the bands were obtained using NIH Image Analysis software 1.61 and p-ERK indexes were calculated as follows: p-ERK index = (IDp-erk/IDerk) x 100.

Cloning of the IL-2 promoter sequence

DNA from highly enriched populations of CD4+ T cells from five rhesus macaques and five sooty mangabeys was isolated using the Genomic DNA purification kit (Promega, Madison, WI) and subjected to PCR amplification using the following primers: 5'-CTGGCAATAAGGGCTGAGTG-3' and 5'-GAGGTTACTGTGAGTA GTGAT-3'. The resulting ~1.4-kb PCR product was purified in Sea-Plaque low-melting agarose (FMC, Rockland, ME), cloned into the pGEM-T vector (Promega), and sequenced. Sequences were analyzed using the GCG Wisconsin Package analysis software (GCG, Madison, WI).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of the Ag-specific CD4+ T cell response in SIV-infected rhesus macaques and sooty mangabeys

It has been previously shown that HIV-1-infected humans demonstrate an accelerated loss of their Ag-specific CD4+ T cell responses (14, 15, 16, 17). To characterize such CD4+ T cell recall responses in SIV-infected nonhuman primates, the proliferative responses of CD4+ T cells from three uninfected rhesus macaques, three SIV-infected rhesus macaques, three SIV-seronegative mangabeys, and three SIV-seropositive mangabeys were measured. All 12 monkeys were immunized with KLH and TT as described in Materials and Methods and since the trend of responses to KLH was identical to that of TT, only the responses to TT are presented. Blood samples from the SIV-noninfected and -infected rhesus macaques and mangabeys were analyzed for their T cell response at approximately similar time periods in efforts to validate comparative analysis of data. As seen in Fig. 1Go, PBMC samples from all 12 monkeys showed significant TT-specific proliferative responses within 2–3 wk following the final booster dose. The proliferative response was primarily mediated by MHC class II-restricted CD4+ T cells since depletion of CD4+ T cells and pretreatment of Ag-pulsed APCs with 10 µg/ml anti-MHC class II monomorphic mAb (but not Ig isotype control Ab) before assay led to >85% decrease in the responses (data not shown). The PBMC samples from the rhesus macaques following SIV infection showed a marked reduction in the TT-specific proliferative response over time as compared with noninfected rhesus macaques. Interestingly, the kinetics of the TT-specific proliferative response of PBMC samples from SIV-seronegative and -seropositive sooty mangabeys in contrast was similar. It is important to note that the magnitude of the Ag-specific response of mangabeys immunized while SIV seropositive was similar to that seen in SIV-seronegative mangabeys. The decreased responses of SIV-infected rhesus macaques were not secondary to loss of CD4+ T cells, since significant (>75% of the baseline) numbers of CD4+ T cells were still present in samples that were analyzed. In addition, this decrease could not be ascribed to dysfunction of the APCs since the same autologous herpes-transformed cells were used for the assay. Thus, although SIV infection clearly leads to immunological suppression (unresponsiveness) in rhesus macaques, SIV infection does not appear to affect either the generation or retention of Ag-specific memory T cell responses in sooty mangabeys. The mechanisms for such distinct outcomes in SIV-infected rhesus macaques and sooty mangabeys were reasoned to be either due to depletion of Ag-specific memory T cells or due to dysfunctional intracellular signaling events following TCR ligation in rhesus macaques but not mangabeys.

Relative resistance of mangabey CD4+ T cells to undergo anergy

One mechanism for the marked reduction of Ag-specific responses in HIV-1-infected humans and as described above in SIV-infected rhesus macaques was reasoned to be due to the induction of Ag-specific unresponsiveness or anergy (35, 36, 37, 38). To formally address this issue, highly enriched populations of alloprimed CD4+ T cells from three SIV-seronegative sooty mangabeys, three normal rhesus macaques, and three healthy adult human volunteers were either incubated in media (control) or subjected to induction of anergy by incubation with immobilized anti-CD3 mAb alone as described in Materials and Methods. These cultures were then subjected to analysis of anergy by challenge with the sensitizing allogeneic cells, anti-CD3/anti-CD28-conjugated immunobeads, media alone (background control), or IL-2. Representative data of 6 identical experiments (which thus represents 18 samples from each species) are presented in Fig. 2Go. As seen, while CD4+ T cells from rhesus macaques and humans preincubated with media (induction phase) proliferate when challenged by coculture with the allosensitizing cells, anti-CD3/anti-CD28 immunobeads, and IL-2, aliquots of the same CD4+T cells preincubated with immobilized anti-CD3 (induction phase) showed a marked diminution in their response to challenge with the sensitizing cells or anti-CD3/anti-CD28 (Fig. 2Go, A and B). This failure of the anti-CD3-incubated cells to proliferate was not secondary to the lack of cell viability since they did proliferate when cultured with IL-2. In contrast to the results obtained with CD4+ T cells from rhesus macaques and humans, similarly prepared CD4+ T cells from SIV-seronegative sooty mangabeys when cultured with media alone or anti-CD3 mAb alone gave a relatively similar amount of proliferation when challenged with the sensitizing cells, anti-CD3/anti-CD28, or IL-2 (Fig. 2GoC). These data were highly reproducible and provided evidence that in fact CD4+ T cells from SIV-seronegative sooty mangabeys consist of cells that are relatively resistant to the induction of anergy, at least as defined with the use of anti-CD3 (signal 1 only) for the induction phase. T cell anergy is characterized by a decrease in IL-2 production upon rechallenge. In efforts to demonstrate that the decrease in proliferation was indeed secondary to a decrease in IL-2 synthesis, an identical experiment was conducted, except instead of measuring cell proliferation, supernatant fluids were removed and added to the IL-2-dependent cell line which was pulsed with [3H]thymidine, and the mean uptake was determined. As seen in Fig. 3Go, the results were virtually similar to those seen with the proliferation assay. Thus, CD4+ T cells from rhesus macaques and humans when pretreated with anti-CD3 alone failed to synthesize IL-2 upon rechallenge with the sensitizing allogeneic cells or anti-CD3/anti-CD28 beads, but CD4+ T cells from sooty mangabeys similarly pretreated with anti-CD3 alone synthesized readily detectable levels of IL-2 upon challenge with the sensitizing allogeneic cells or anti-CD3/anti-CD28. To exclude the possibility that the proliferation of the HT-2 cells treated with supernatant fluid from the sooty mangabey CD4+ T cells were not false positive due to the presence of IL-4, in one parallel experiment the same protocol was followed except anti-IL-4 (1 µg/well) or control mouse IgG1 (1 µg/ml) was added to the supernatant fluid from cultures of the sooty mangabey cells before its addition to the HT-2 cell line. No detectable difference in the uptake of [3H]thymidine was noted between the HT-2 cell line that was cultured with anti-IL-4-treated, normal IgG1-treated, or untreated supernatant fluids (data not shown).



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  		FIGURE 2. Induction of anergy in allogeneic primed CD4+ T cells from normal non-infected rhesus macaques, seronegative sooty mangabeys, and normal healthy adult humans. Highly purified CD4+ T cells from rhesus macaques (A), humans (B), and sooty mangabeys (C) were primed against allogeneic stimulator cells as described in Materials and Methods and then either incubated in media alone (control) or with immobilized anti-CD3 Abs for 48 h, washed, and then rested in media for 24–48 h. Aliquots of these CD4+ T cells were then challenged with the same allogeneic cells, anti-CD3/anti-CD28, and IL-2 and for purposes of control media alone. Cultures were always performed minimally in triplicate and after incubation for 48 h pulsed with [3H]thymidine, harvested 16–18 h later, and the mean cpm ± SD calculated. The percent SD of triplicate samples was always <10%. Results shown are representative of six experiments performed similarly and each experiment included use of CD4+ T cells from three rhesus macaques, three sooty mangabeys, and three human volunteers.

 


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  		FIGURE 3. Induction of anergy in allogeneic primed CD4+ T cells from normal non-infected rhesus macaques, seronegative sooty mangabeys, and normal healthy adult humans as measured by IL-2 production. The protocol was identical to that described in the legend to Fig. 2Go, except at the time of pulsing the cultures with [3H]thymidine, the microtiter plate was centrifuged and the supernatant fluids were collected and added to the IL-2-dependent HT-2 cell line (105 cells/well). The cell line was pulsed with [3H]thymidine (1 µCi/well), harvested 16–18 h later, and proliferation of HT-2 cells was measured by the incorporation of thymidine.

 
In efforts to determine whether the difference in the induction of anergy in CD4+ T cells from rhesus macaques, humans, and mangabeys was not due to a difference in the kinetics of anergy induction in the CD4+ T cells from mangabeys, alloprimed CD4+ T cells from three mangabeys were precultured with anti-CD3-immobilized mAb for 24, 48, 72, 96, and 120 h. Aliquots of the same alloprimed CD4+ T cells untreated (for purposes of control) and the anti-CD3-pretreated cells were then assayed for theirresponse to the allosensitizing cells, anti-CD3, or anti-CD3/anti-CD28 beads. Representative data in Fig. 4Go show that although the response in general does decrease with anti-CD3 exposure past 48 h, it is clear that these cells do proliferate and the stimulation index remains high. Thus, the difference in the ability to undergo anergy by CD4+ T cells from the mangabeys was reasoned not to be due to a difference in the kinetics of anergy induction. It is important to note that optimal induction of anergy in alloprimed CD4+ T cells from rhesus macaques occurred at 48–72 h following culture with immobilized anti-CD3, after which there was a rapid occurrence of cell death.



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  		FIGURE 4. Kinetic parameters of the relative resistance of mangabey CD4+ T cells to undergo anergy. A large batch of alloprimed CD4+ T cells from three sooty mangabeys were prepared and cryopreserved. An aliquot was thawed and incubated for 24, 48, 72, 96, and 120 h with immobilized anti-CD3 mAb (clone FN-18) in a reverse order with respect to time so that they could all be harvested at the same time. The cells were then washed and challenged with the initial irradiated allogeneic cells, anti-CD3, anti-CD3/anti-CD28 and with media for purposes of background control. The response of an aliquot of the same alloprimed CD4+ T cells was compared with that of these anti-CD3-pretreated CD4+ T cells. Cultures were incubated for 2 days; pulsed for 16–18 h with [3H]thymidine (1 µCi/well), harvested, and the mean uptake ± SD was calculated. The percent SD of triplicate samples was always <10%.

 
Next, a series of experiments was conducted to determine whether this functional resistance to the induction of anergy in sooty mangabeys was retained following SIV seroconversion. Thus, highly purified alloprimed CD4+ T cells from a set of three SIV-seropositive healthy mangabeys and for comparison three SIV-seronegative healthy mangabeys were either cultured in media alone (control) or with immobilized anti-CD3 for 3 days and then challenged with the sensitizing allogeneic cells, anti-CD3 alone, anti-CD3/anti-CD28-conjugated immunobeads, or media (control), and proliferation of representative samples is shown in Fig. 5Go. CD4+ T cells from both SIV seronegative and seropositive mangabeys failed to demonstrate anergy upon preculture with anti-CD3 and subsequent challenge with either anti-CD3 or anti-CD3/ anti-CD28. These data suggest that this functional difference among rhesus macaques, humans, and mangabeys is present in the mangabeys before seroconversion and retained after seroconversion and suggests that this function is not secondary to the naturally acquired virus infection.



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  		FIGURE 5. Effect of SIV status on the anergy induction in CD4+ T cells from sooty mangabeys. Aliquots of cryopreserved alloprimed CD4+ T cells from three SIV-seronegative and three SIV-seropositive sooty mangabeys were cultured with immobilized anti-CD3 (clone FN-18) or media for 3 days. Such media or anti-CD3-pretreated cells were then challenged with either anti-CD3 or anti-CD3/anti-CD28. Representative results from a single-seropositive and a single-seronegative sooty mangabey are shown.

 
Effect of CSA, rapamycin, and hydroxyurea on the induction of anergy

To gain further insights into the mechanisms by which CD4+ T cells from sooty mangabeys resist the induction of anergy, the immunosuppressive agents such as CSA, rapamaycin, and hydroxyurea (34) have been used to dissect the stage and/or pathways at which anergy induction is mediated. Highly enriched alloprimed CD4+ T cells from three mangabeys, three rhesus macaques, and three humans were cultured in vitro for 3 days with anti-CD3 in the absence or presence of CSA, rapamycin, and hydroxyurea. As seen previously, alloprimed CD4+T cells from rhesus macaques and humans pretreated with anti-CD3 (signal 1 only) led to a marked diminution in their subsequent response to challenge with the allosensitizing cells or anti-CD3/anti-CD28-conjugated immunobeads (Fig. 6Go, A and B), whereas the alloprimed CD4+ T cells from mangabeys did not demonstrate the induction of anergy (Fig. 6GoC). This lack of response in human and rhesus macaques was not due to viability issues since an aliquot of the same cells did proliferate in the presence of IL-2. Although the addition of CSA inhibited the induction of anti-CD3-induced anergy, the addition of rapamycin augmented the induction of anti-CD3-induced anergy and the addition of hydroxyurea did not affect the induction of anergy in cultures from rhesus macaques and humans. Interestingly, although the addition of both CSA and hydoxyurea had no effect (did not facilitate) on the induction of anergy in mangabey cells, the addition of rapamycin clearly did facilitate the induction of anergy in cultures from mangabeys. Once again, this failure to respond to challenge with the sensitizing allogeneic cells or anti-CD3/anti-CD28 following culture with anti-CD3 in the presence of rapamycin was not due to lack of cell viability, as the response of these cells from all three species to the exogenous addition of IL-2 was retained (Fig. 6Go). It should be noted that the drugs used were present only during the induction phase and the cells were washed several times before rechallenge and thus not present during rechallenge. These data provide evidence to support the notion that the pathway of anergy induction in rhesus macaques is similar to that in mice and humans (21, 22, 34, 39), but the uniqueness lies in the ability of sooty mangabey CD4+ T cells to synthesize IL-2 in response to signal 1 alone.



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  		FIGURE 6. Effect of CSA, rapamycin, and hydroxyurea on the induction of anergy by anti-CD3 (signal 1 alone) treatment of alloprimed CD4+ T cells from rhesus macaques, sooty mangabeys, and humans. Aliquots of the alloprimed CD4+ T cells from three rhesus macaques (A), three human adult volunteers (B), and three sooty mangabeys (C), were cultured for 3 days with immobilized anti-CD3 mAb in the absence (control) and presence of CSA (100 nM), rapamycin (100 nM), and hydroxyurea (4 µM). The cells were then washed three times, rested for 24 h in media, and then challenged with IL-2, the sensitizing irradiated allogeneic cells or anti-CD3/anti-CD28 mAb. Results are representative of three similar experiments.

 
Analysis of ERK in CD4+ T cells from macaques and mangabeys

Data from previous studies show that the p21ras/MAPK/ERK signaling pathway plays a crucial role in TCR-mediated signaling leading to ERK phosphorylation and IL-2 synthesis (40, 41). The full activation and synthesis of IL-2 by T cells require costimulation (42, 43, 44). Anergic cells have been shown to exhibit defects in multiple signaling pathways, including the p21ras/MAPK/ERK pathway (45, 46, 47, 48, 49). It was thus of interest to determine whether the CD4+ T cells from the two monkey species differ in the ERK activation pattern induced by TCR stimulation. Purified CD4+ T cells from three rhesus macaques, three sooty mangabeys (two seronegative and one seropositive), and two humans (as a control) were stimulated with anti-CD3 Ab (signal 1) alone or anti-CD3/anti-CD28 Abs (signal 1 and 2) as described in Materials and Methods and analyzed for the content and phosphorylation status of ERK by Western blot (Fig. 7GoA). All of the samples showed a similar content of ERK1/ERK2 proteins as expected and the relative amounts of phosphorylated ERK were also comparable in extracts from the anti-CD3/anti-CD28-stimulated cells from all three species. Interestingly, under the stimulating conditions employed, while rhesus macaque- and human-derived CD4+ T cells showed very low phosphorylation of ERK after anti-CD3 (signal 1) stimulation alone, CD4+ T cells from sooty mangabey exhibited a substantial increase in phosphorylated ERK. Furthermore, when purified CD4+ T cells were preincubated with anti-CD3 alone (signal 1) and challenged with anti-CD3/anti-CD28 (signals 1 + 2) only the sooty mangabey cells showed substantial ERK phosphorylation (Fig. 7GoB). These data provide further support to the view that there are indeed differences in the TCR signaling of CD4+ T cells from sooty mangabey as exemplified by the readily detectable activation of the MAPK/ERK pathway by signal 1 alone.



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  		FIGURE 7. Western blot analysis for the presence of ERK and phosphorylated ERK in rhesus macaque, sooty mangabey, and human CD4+ T cells. A, Aliquots of freshly isolated CD4+ T cells from two humans, three rhesus macaques, and three sooty mangabeys were stimulated by anti-CD3 alone ({alpha}CD3) or anti-CD3/anti-CD28 ({alpha}CD3/28)-coated beads as described in Materials and Methods, lysed, and Western blots performed on the lysates for the presence of ERK ({alpha}-ERK) and phospho-ERK ({alpha}-pERK). B, Aliquots of freshly isolated CD4+ T cells from two rhesus macaques and two sooty mangabeys were stimulated by anti-CD3, challenged with anti-CD3/CD28, and Western blots performed as above. p-ERK index was calculated from ID of ERK1 bands as follows: p-ERK index = (IDp-erk/IDerk) x 100.

 
Cytokine analysis of anti-CD3-treated CD4+ T cells from macaques, mangabeys, and humans

It has been shown previously that anergy induction leads to a marked decrease in the synthesis of IL-2 by CD4+ T cells (21). In addition, such cells, despite a marked shutdown in IL-2 synthesis, nonetheless remain capable of synthesizing a number of other cytokines such as IFN-{gamma}, IL-3, and TNF-{alpha}, to name a few. In efforts to determine whether the differences noted in second signal requirements by CD4+ T cells from mangabeys vs macaques and humans is reflected in differences in the cytokine levels synthesized, alloprimed CD4+T cells from rhesus macaques, mangabeys, and humans were cultured with media alone (control constitutive expression), with immobilized anti-CD3 alone (signal 1), or the anti-CD3/anti-CD28 (signals 1 + 2)-conjugated immunobeads, and then the supernatant fluids were examined for levels of IL-2, IL-3, IFN-{gamma}, and TNF-{alpha} as described in Materials and Methods. There was relatively little if any detectable level of the cytokines synthesized by CD4+ T cells cultured in media alone (constitutive expression) from either macaques, mangabeys, or humans (data not shown). As seen in Table IGo, anti-CD3/anti-CD28-induced stimulation clearly augments the synthesis of all of the cytokines measured in all three species. There was a hierarchy in the degree of increase with IL-2 followed in decreasing order by IFN-{gamma}, IL-3, and TNF-{alpha} synthesized by the activated CD4+ T cells from all three species. However, contrary to the rhesus macaques and humans, mangabey CD4+ T cells synthesize significant levels of IL-2 when activated by anti-CD3 alone (0.6 ± 0.4 and 7.4 ± 0.4 vs 37.4 ± 10.2 ng/ml, respectively). These differences in the levels of IL-2 synthesized by differentially stimulated (signal 1 vs signals 1 + 2) CD4+ T cells from sooty mangabey vs macaques and humans is also reflected in the ratio values (0.23 vs 0.008 and 0.03, respectively). On the other hand, similar ratios of IFN-{gamma}, IL-3, and TNF-{alpha} among the species tested indicate that the differences in IL-2 production by mangabeys are unique for this cytokine.


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  		Table I. Cytokine profile of anti-CD3 as compared with anti-CD3/anti-CD28-conjugated immunobead-activated alloprimed CD4+ T cells from uninfected rhesus macaques, SIV-seronegative mangabeys, and adult human volunteers

 
In addition, the data show that human CD4+ T cells when activated by anti-CD3/anti-CD28-conjugated immunobeads synthesize significantly higher amounts of both IL-2 and IFN-{gamma} than the rhesus macaques and mangabeys. Furthermore, the rhesus macaque CD4+ T cells synthesize a significantly higher amount of IFN-{gamma} than similarly activated CD4+ T cells from mangabeys. These differences were not due to differences in kinetics since the data obtained were essentially similar in trend in samples harvested at other time points (data not shown). Taken together, these data indicate that as opposed to humans and macaques there is a selective induction of IL-2 in sooty mangabey CD4+ T cells stimulated with signal 1 alone and that this phenotype may contribute to the increased proliferative responses seen in mangabey PBMC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The decrease of Ag-specific recall responses in T cells from HIV-1-infected patients is clearly one of the major mechanisms that contributes to generalized immunosuppression, susceptibility to opportunistic infections, and the development of AIDS (14, 15, 16, 17). This decreased responsiveness is not merely secondary to the direct cytolytic effects of the virus and depletion of CD4+ T cells, since such diminished responses are detected very early during the asymptomatic phase of the infection (17) when there are substantial numbers of CD4+ T cells. The decreased responsiveness also cannot be solely attributed to the selective depletion of Ag-specific memory T cell subsets for a number of reasons. First, phenotypic analysis showed no significant differences in the percentage of naive (CD45RA+) CD4+ T cells (there is no reagent currently available to detect monkey CD45RO+ T cells) between SIV-infected asymptomatic rhesus macaques (24.4 ± 6.6, n = 6) and SIV-infected sooty mangabeys (20.1 ± 5.6, n = 8). Although indirect, these data suggest that the remaining CD4+ T cells are likely to be CD45RO+ (unless an unusual subset replaces the potentially depleted CD45RO+ T cells) and that differences in anergy induction are not likely due to frequency issues but due to function. Second, the initiation of highly active antiretroviral therapy has been shown to lead not only to the expansion of existing memory cells (50, 51), but also to the gradual restoration of both Ag-specific and anti-CD3-elicited memory T cell proliferative responses to immune stimuli (52, 53, 54) suggesting that the Ag specific memory cells are present in such patients but are "unresponsive" or "anergic." Further support for this view emanates from the finding that the interruption of highly active antiretroviral therapy in HIV-infected patients rekindles such anergy and leads to the rapid decrease of T cell responses (55). The delineation of the mechanism by which HIV down-modulates these responses would thus be an important step that would help in the design of successful reconstitution therapies. The use of the SIV-infected disease asymptomatic vs the symptomatic nonhuman primate models of SIV infection that show normal vs dysfunctional immune responses, respectively (see Fig. 1Go), provides a powerful means to define the mechanisms that underlie such Ag-specific unresponsiveness. The fact that the restoration or maintenance of Ag-specific T cell proliferative responses is independent of viral load in both HIV infection of humans (56) and in SIV-infected sooty mangabeys (Fig. 1Go) suggests that mechanisms other than viral load are implicated and likely to involve host-mediated intracellular mechanisms. The results of the studies presented herein suggest that the CD4+ T cells from the naturally infected sooty mangabey synthesize IL-2 upon signal 1 activation alone, which prevents anergy induction that is not seen with CD4+ T cells from rhesus macaques and humans. The studies performed always included human cells since conditions, parameters, and previous data on the activation pathway of CD4+ T cells and, in particular, those involving anergy induction have been characterized with rodent and human cells (57, 58) and not rhesus macaques and thus serve as a built in reference control.

Some of the trivial reasons for the relative resistance of CD4+ T cells from sooty mangabeys to undergo anergy in vitro could be the presence of a low but sufficient number of CD4-expressing monocytes; the difference in the kinetics of expression of other cell surface molecules known to regulate cell proliferation such as CTLA-4 and CD40/CD154; or the abnormal expression of CD80/86 by CD4+ T cells from this species that could serve to provide CD4-CD4 autocostimulation. We examined the purity of the CD4+ T cells from both rhesus macaques and mangabeys following allogeneic priming and following culture with immobilized anti-CD3-conjugated immunobeads. These cells from both species were >99% CD4+ and no detectable levels of APC function were noted. Therefore, it is unlikely that the differences noted in the ability to undergo anergy in vitro by CD4+ T cells from mangabeys is due to the presence of small numbers of monocytes/macrophages. Furthermore, observed differences among the species are probably not related to the differences in the level or kinetics of CTLA-4, CD154, and the mitogen-induced CD80/86 expression by PBMCs as determined by flow microfluorometric analysis (Ref. 12 and data not shown). These negative findings were not secondary to the failure of the reagents to react with nonhuman cells since the unfractionated populations of PBMCs and PHA-P-activated PBMCs from these two species analyzed in parallel as positive controls readily expressed these molecules. Thus, we submit that this increased resistance to the induction of anergy by CD4+ T cells from mangabeys is likely to be intrinsic. It is possible, however, that sooty mangabeys evolved to more extensively utilize alternative costimulatory receptors, e.g., TIRC7 (59, 60), ICOS (61, 62, 63, 64), LIGHT (65), or other yet undiscovered receptors and pathways that provide intracellular signaling normally induced by CD28 and CD80/86. Clearly, further studies are needed to identify whether such alternate usage of costimulatory ligands and receptors are the basis of the findings. Even if this was the case, such interactions would have to occur by CD4+/CD4+ T-T cell interactions, which is rarely observed for costimulatory function so far.

The fact that in vitro TCR stimulation following an anergy induction protocol provides an adequate signal for substantial activation of the MAPK/ERK signaling pathway in CD4+ T cells from sooty mangabeys but not rhesus macaques and humans lends support to the hypothesis that the mechanisms evolved in this species are intrinsic (Fig. 7Go). Clearly, the MAPK/ERK pathway plays an important role in TCR signaling leading to the T cell activation and production of IL-2 (40, 41), and other cytokines, such as IL-3, IL-4, IL-5, and IL-10 (66), and in T cell development and selection (67, 68). It is important to note that the MAPK/ERK pathway receives input not only via TCR stimulation, but also through other receptors, such as CXCR4 (69), CD69 (70), CD38 (71), and pathways that involve calcium-induced signaling (72). At the same time, the TCR-mediated signaling is transduced by more than one pathway (73) and the ultimate full up-regulation of the transcriptional factors NFAT and AP-1 that directly stimulate transcription of IL-2 and other cytokines requires involvement of other stimulatory pathways (74, 75, 76). Also, the fact that although ERK plays a crucial role in Ag-induced proliferation but its targeted inhibition does not induce or block the induction of anergy (77) seems to support the hypothesis that other alternative pathways contribute to the "anergy-resistant" phenotype of sooty mangabey. The substantial increase in ERK phosphorylation in CD4+ T cells from sooty mangabey after stimulation with signal 1 alone (Fig. 7GoB) clearly was not secondary to the variations in the expression of ERK, since similar to previously published data (78) we find comparable amounts of ERK protein in each sample regardless of stimulus. Interestingly, we did not see any difference in ERK stimulation in seropositive vs seronegative sooty mangabeys (Fig. 7Go and data not shown), although it has been reported that ligation of CD4 by HIV-derived gp160 inhibits ERK stimulation (79, 80) and seropositive sooty mangabeys harbor high viral loads (81) and therefore an abundance of SIV-derived gp160. This data would indicate that the potential differences in T cell signaling within CD4+ T cells from sooty mangabey may contribute to the SIV disease-free phenotype of this species, although a more detailed study of the precise pathway by which this results in anergy resistance and/or IL-2 transcription remains to be elucidated.

Our cytokine analysis of anti-CD3-stimulated cells from all three species showed a comparable relative increase in levels of IFN-{gamma}, IL-3, and TNF-{alpha} (Table IGo). However, only the mangabey CD4+ T cells showed a significant induction of IL-2 by ligation of CD3 alone. This indicates that the increase in IL-2 expression is specific for this cytokine among the cytokines examined and is not likely due to global increase in all cytokines synthesized by such cells. It is also important to note that while there are clear differences in the magnitude of IL-2 synthesized by macaques, mangabeys, and humans, as we have described earlier (7, 8, 11), the studies reported herein outline a comparison of the cytokines synthesized by CD4+ T cells treated with anti-CD3 alone vs anti-CD3/anti-CD28-conjugated immunobeads from the same species. The fact that stimulation of mangabey CD4+ T cells with signal 1 alone leads to substantial IL-2 production is an important finding. Whether this difference in IL-2 induction, as well as the resistance to the induction of anergy, is caused solely by differences in signaling pathways or other mechanisms remains to be defined. It has been previously shown that it is not the lack of costimulation that leads to the induction of anergy, but rather anergy is the result of TCR activation in the absence of IL-2 production (82). Several different mechanisms have been shown to regulate IL-2 expression in anergic T cells (83). Down-regulation of IL-2 gene expression in anergic cells was shown to be an active process (84) that involves at least two cis-acting regulatory elements located within the proximal IL-2 promoter. One of the sites, the -180 AP1-like site, was shown to be essential for the down-regulation of IL-2 transcription in anergic cells and anergy induction (85). Interestingly, sequence analysis of the IL-2 promoter region from macaques and mangabeys revealed a single nucleotide substitution within this region only in sooty mangabeys that targets this regulatory site (Fig. 8Go). However, previously published experimental mutations within this site that abolished the down-regulation of the IL-2 promoter in anergic cells were more extensive than the single-base substitution found in the sooty mangabey. Therefore, further studies are needed to elucidate the biological significance of this substitution and its relevance.



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  		FIGURE 8. Comparison of the proximal IL-2 promoter sequences from humans, rhesus macaques, and sooty mangabeys. The proximal IL-2 promoter sequences were cloned from CD4+ T cells from five sooty mangabeys and five rhesus macaques, sequenced, and consensus sequences were aligned with published sequence from humans. Lowercase, bold, and underlined nucleotides indicate variations to the human sequence. Position of TATA boxes and -180 AP-1-like sequence essential for the anergy induction is indicated.

 
The precise biochemical and molecular basis for the induction of anergy continues to be unraveled. It is now becoming increasingly clear that prevention of anergy does not require proliferation (34) since IL-2-induced cell cycle progression from the G1 to S phase, even without proliferation, is sufficient to prevent anergy. This is supported by the studies using the drugs rapamycin and hydroxyurea, which are known to block cell cycle progression from G1 to S and within S phase, respectively. The findings of the studies reported herein suggest that CD4+ T cells from sooty mangabeys while capable of undergoing anergy, as seen with the use of rapamycin, are resistant to anergy induction when provided with signal 1 alone (see Figs. 2Go and 6Go). Thus, biochemical and molecular events other than the pathway by which rapamycin exerts its effect, such as those at which IL-2 is regulated, are likely to be the mechanisms involved by which CD4+ T cells from mangabeys develop relative resistance to anergy induction. Definition of the negative regulatory factors such as corepressors, which regulate IL-2 transcription may lead to the identification of the mechanisms which may have been phylogenetically acquired by CD4+ T cells from sooty mangabeys. Finally, it would be important to determine whether similar resistance to anergy induction by signal 1 alone is also present in other species of nonhuman primates which are chronically and naturally infected with SIV but remain asymptomatic and disease free.


   Acknowledgments
 
We are grateful to the professional and technical staff of the Yerkes Regional Primate Research Center of Emory University for all their help and assistance in the care, maintenance, immunizations, blood collections, and scheduling of the nonhuman primates that were part of the studies reported herein. We are also grateful to the AIDS Reagent and Repository supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, for supplying us some of the reagents that we used during the course of the studies described.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant RO1 AI27057. Back

2 Address correspondence and reprint requests to Dr. Pavel Bostik, Department of Pathology and Laboratory Medicine, Winship Cancer Institute, Emory University School of Medicine, 1365B Clifton Road, Atlanta, GA 30322. Back

3 Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; TT, tetanus toxoid; CSA, cyclosporin A; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; ID, integrated intensity. Back

Received for publication July 20, 2000. Accepted for publication October 5, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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