Global Dysfunction of CD4 T-Lymphocyte Cytokine Expression in Simian-Human Immunodeficiency Virus/SIV-Infected Monkeys Is Prevented by Vaccination

JOURNAL OF VIROLOGY, Apr. 2003, p. 4695 4702 Vol. 77, No. 8 0022-538X/03/$08.00 0 DOI: 10.1128/JVI.77.8.4695 4702.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Global Dysfunction of CD4 T-Lymphocyte Cytokine Expression in Simian-Human Immunodeficiency Virus/SIV-Infected Monkeys Is Prevented by Vaccination Paul F. McKay, 1 Dan H. Barouch, 1 Jörn E. Schmitz, 1 Ronald S. Veazey, 2 Darci A. Gorgone, 1 Michelle A. Lifton, 1 Kenneth C. Williams, 1 and Norman L. Letvin 1 * Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, 1 and Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana 70433 2 Received 7 October 2002/Accepted 23 January 2003 Human immunodeficiency virus type 1 infection results in a dysfunction of CD4 T lymphocytes. The intracellular events contributing to that CD4 T-lymphocyte dysfunction remain incompletely elucidated, and it is unclear whether aspects of that dysfunction can be prevented. The present studies were pursued in a rhesus monkey model of AIDS to explore these issues. Loss of the capacity of peripheral blood CD4 T lymphocytes to express cytokines was first detected in simian immunodeficiency virus-infected monkeys during the peak of viral replication during primary infection and persisted thereafter. Moreover, infected monkeys with progressive disease had peripheral blood CD4 T lymphocytes that expressed significantly less cytokine than infected monkeys that had undetectable viral loads and intact CD4 T-lymphocyte counts. Importantly, CD4 T lymphocytes from vaccinated monkeys that effectively controlled the replication of a highly pathogenic immunodeficiency virus isolate following a challenge had a preserved functional capacity. These observations suggest that an intact cytokine expression capacity of CD4 T lymphocytes is associated with stable clinical status and that effective vaccines can mitigate against CD4 T-lymphocyte dysfunction following an AIDS virus infection. * Corresponding author. Mailing address: Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215. Phone: (617) 667-2766. Fax: (617) 667-8210. E-mail: nletvin@bidmc.harvard.edu. CD4 T lymphocytes are critical for a functional immune system, providing the signals required for the development of antigen-specific responses by both B and CD8 T lymphocytes. CD4 T lymphocytes provide the cytokine environment and second signals necessary for the development of antibody responses and are also required for the generation and maintenance of effective cytotoxic T lymphocytes (CTL) (3, 6, 7, 12, 29). In fact, loss of CD4 help results in a reduced capacity of CTL to protect against new viral infections and results in a failure of immune control of the replication of viruses, such as Epstein-Barr virus and cytomegalovirus, that cause clinical infection (5, 13, 14). Abnormalities in CD4 T lymphocytes have been described in human immunodeficiency virus (HIV)-infected individuals that profoundly affect the global competency of their immune function. A reduction in the proliferative capacity of CD4 T lymphocytes in response to HIV antigens has been demonstrated (9, 15, 18, 19, 23, 24). HIV type 1 (HIV-1) replication also inhibits antigen-specific CD4 T-lymphocyte progression through the cell division cycle (26). Finally, a loss of the CD4 T lymphocytes ability to secrete cytokines in response to viral antigen stimulation has been described in infected individuals (9, 17). Since activated CD4 T cells are infected and destroyed by HIV-1, these sequelae of infection are not surprising (8, 27). However, we know little about the early status and temporal evolution of CD4 T-lymphocyte abnormalities during the course of an immunodeficiency virus infection. Cohorts of HIV-infected individuals have been described who do not develop progressive clinical disease, maintaining effective HIV-specific CTL, as well as CD4 T lymphocytes that demonstrate robust proliferation and cytokine secretion in response to viral antigens (10, 23). Moreover, we and others have demonstrated that prior vaccination can alter the clinical outcome of an immunodeficiency virus infection, protecting against the evolution of CD4 T-lymphocyte loss (2, 25). Although difficulties have been encountered in eliciting immunity with prototype vaccines that is likely to prevent HIV infection following exposure to the virus, these types of observations suggested the feasibility of prior vaccination delaying progressive clinical AIDS in individuals who become infected. The biologic mechanisms underlying the protection afforded by vaccine-elicited immune responses remain unknown. In the present studies, we have assessed the functional capacity of CD4 T lymphocytes in rhesus monkeys both prospectively during the course of a simian immunodeficiency virus (SIV) infection and in a cohort of SIV/SHIV-infected animals with nonprogressive disease. We have also evaluated the protection against functional CD4 T-lymphocyte dysfunction seen in monkeys who are vaccinated prior to an immunodeficiency virus infection. These studies suggest that preserved CD4 T-lymphocyte function may be central to the control of disease progression following immunodeficiency virus infection. 4695

4696 MCKAY ET AL. J. VIROL. MATERIALS AND METHODS Animals and viruses. Heparinized blood samples were obtained from rhesus monkeys (Macaca mulatta). All animals were maintained in accordance with the guidelines of the Committee on Animals for the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (11a). The viruses used in this study were SHIV-89.6P, SHIV-89.6, SIVmac251, and SIVsmE543. SHIV-89.6 and SHIV-89.6P are chimeric viruses consisting of the SIVmac239 backbone and the HIV-1 89.6 envelope gene, which was cloned from a primary patient R5/X4 dualtropic HIV-1 isolate (21). CD4 counts and viral loads. Peripheral blood CD4 T-lymphocyte counts were determined by multiplying the total lymphocyte count by the percentage of CD3 CD4 T cells assessed by flow cytometry. Plasma viral RNA levels were measured either by using a branched-chain DNA assay with a detection limit of 500 copies/ml as previously described (11) or by using a quantitative real-time reverse transcriptase PCR (RT-PCR) technique as previously described (20). The analytical sensitivity of this assay is typically 300 copy equivalents of the SIV gag sequence per reaction. Plasmid DNA vaccination protocol. The vaccination protocol used has been previously described in detail (1). Briefly, double-cscl-banded maxipreparations of HIV-1 89.6P env (KB9) and SIVmac239 gag plasmid DNAs were injected into both quadriceps muscles of rhesus monkeys. Animals treated with interleukin- 2/immunoglobulin (IL-2/Ig) protein were inoculated twice daily with 0.5 mg/day for 14 days after DNA vaccine administration. Other animals received 5 mg of IL-2/Ig plasmid on day 2 after DNA vaccination. The vaccination regimen consisted of inoculation at week 0, followed by boost immunizations at weeks 4, 8, and 40. IL-2 treatments were included only at weeks 0 and 4. Animals were challenged at week 46 with 100 50% monkey-infective doses of SHIV-89.6P by the intravenous route. Antibodies. All of the antibodies used in this study were directly coupled to fluorescein isothiocyanate, phycoerythrin, phycoerythrin-texas red, or allophycocyanin. The following monoclonal antibodies (MAbs) were used: anti-cd3 fluorescein isothiocyanate (SP34; BD Pharmingen, San Diego, Calif.), anti-cd8 phycoerythrin Texas Red (7PT; kindly provided by S. F. Schlossman, Dana- Farber Cancer Institute, Boston, Mass.), anti-gamma interferon (anti-ifn- ) allophycocyanin (B27; BD Pharmingen), anti-tumor necrosis factor alpha (anti- TNF- ) allophycocyanin (MAb11; BD Pharmingen), and anti-il-2 allophycocyanin (MQ1-17H12; BD Pharmingen). PBMC stimulation and intracellular cytokine staining. Unfractionated fresh peripheral blood lymphocytes (PBMC) were cultured at 37 C ina5%co 2 environment for 6 h in the presence of RPMI medium alone (unstimulated) or 100 ng of phorbol myristate acetate (PMA) per ml plus 1 g of ionomycin per ml. All cultures contained brefeldin A (GolgiPlug; BD Pharmingen) at 10 g/ml to disrupt Golgi apparatus transport, thereby causing intracellular cytokine accumulation. The cultured cells were stained with MAbs specific for cell surface molecules prior to red blood cell lysis and fixation (Immunoprep reagent system and a Q-prep workstation; Beckman Coulter, Fullerton, Calif.). The PBMC were washed once with phosphate-buffered saline (PBS) 2% fetal calf serum and then permeabilized with Cell Fix/Perm solution (BD Pharmingen) in accordance with the permeabilization protocol. Cells were washed twice with 2.5 volumes of 1 Perm/Wash buffer and then stained with 1 g of anticytokine MAb per 10 6 cells. Anticytokine MAb titers for optimal staining were determined in preliminary experiments. Cells were washed twice with 2 ml of 1 Perm/Wash buffer and once with PBS and then fixed in 1.5% formaldehyde PBS. Samples were analyzed on a FACScalibur instrument with CellQuest software. Data analysis was performed with CellQuest software, and graphs were prepared with Microsoft PowerPoint 98 (Microsoft, Redmond, Wash.). Statistical analyses. The mean cytokine expression percentages of each group were compared by using an analysis of variance with Bonferroni s multiplecomparison test. A P value of 0.05 was considered significant. RESULTS CD4 T-lymphocyte cytokine expression during the course of SIVmac251 infection. To assess the evolution of functional CD4 T-lymphocyte abnormalities following AIDS virus infection, a pilot study was done to evaluate IFN- expression by CD4 T lymphocytes in two rhesus monkeys during the period of a primary SIVmac251 infection. PBMC from these monkeys were stimulated in vitro with PMA and ionomycin, stained with a MAb specific for IFN-, and evaluated by flow cytometric FIG. 1. The percentage of CD4 T lymphocytes that can produce IFN- decreases rapidly following SIVmac251 infection. PBMC were stimulated with optimal concentrations of PMA and ionomycin, stained, and analyzed for IFN- expression on gated CD4 T cells. analysis, gating on the CD3 CD8 / cell populations. The PBMC were cultured in the presence of brefeldin A to disrupt the Golgi network and allow cytokine protein accumulation. Control PBMC, cells that received no stimulation, showed little accumulation of cytokines under these culture conditions. In cells stimulated with PMA and ionomycin, significant intracellular cytokine accumulation was observed in the CD4 T lymphocytes (data from a representative monkey are shown in Fig. 1, day 0 panel). Interestingly, the capacity of this population of cells to express IFN- was reduced to 35% of the preinfection level by day 10 following SIVmac251 infection and to 21% of the preinfection level at the time of peak viremia on day 14 (Fig. 1). The specificity of this functional T-lymphocyte abnormality was then evaluated by determining the IFN- expression in the CD8 T-lymphocyte population in these same PBMC samples. In contrast to the CD4 T-lymphocyte population, the CD8 T lymphocytes did not show a comparable reduction in the ability to produce IFN-. Furthermore, the reduction in the potential of the CD4 T-lymphocyte populations to produce IFN- correlated with viral expansion in these monkeys and was reduced to the lowest levels at the time of peak viral replication (Fig. 2A). The number of peripheral blood CD4 T lymphocytes was not significantly reduced in these animals during the period of study (data not shown). These data suggest that a global and profound defect in CD4 T-lymphocyte function occurs during primary immunodeficiency virus infection. IFN- expression by CD4 T lymphocytes does not recover after immune control of viremia. The PBMC from a further four animals were similarly analyzed during the first days following SIVmac251 infection to determine if this specific re-

FIG. 2. Loss of functional CD4 T lymphocytes but preservation of functional CD8 T lymphocytes following SIVmac251 infection. (A) The number of CD4 T lymphocytes that secrete IFN- in response to PMA plus ionomycin decreases during the initial phase of intense viral replication during primary infection. In contrast, the number of CD8 T lymphocytes that are capable of secreting IFN- is maintained. (B) Four rhesus monkeys were infected with SIVmac251 on day 0, and their CD4 and CD8 T lymphocytes were assessed for IFN- production following in vitro stimulation with PMA and ionomycin at various times during the course of infection. The CD4 T-cell potential for IFN- production decreased concurrent with the rise in viremia during primary infection. Upon partial viral containment, the potential of these monkeys CD4 T lymphocytes to produce IFN- did not return to normal levels. 4697

4698 MCKAY ET AL. J. VIROL. TABLE 1. Viral loads and absolute CD4 T-cell numbers per milliliter of peripheral blood of infected monkeys that were either typical progressors or had nonprogressive disease Group and monkey Viral challenge Viral load CD4 T-cell count/ml Typical progressors H627 SIVsmE543 4,714,100 718 H628 SIVsmE543 349,198 317 H629 SIVsmE543 3,492,250 677 H632 SIVsmE543 27,106,900 1,373 H633 SIVsmE543 55,422 772 H634 SIVsmE543 1,299,541 282 H636 SIVsmE543 11,329,900 566 T798 SIVmac239 5,803 130 Nonprogressors 253 96 SIVmac251 500 961 403 91 SIVmac251 (J5) 500 859 206 93 SHIV-89.6 500 977 556 92 SHIV-89.6 500 1,032 287 94 SHIV-89.6 500 799 duction in the IFN- expression of CD4 T lymphocytes was universally observed. In fact, this reduction in IFN- expression by CD4 T lymphocytes following infection with SIVmac251 was observed in the PBMC of all of the members of this second cohort of animals (percentages ranged from 13 to 32% of preinfection levels) (Fig. 2B). As observed in the PBMC from the first two animals that were studied, the loss of IFN- expression by the CD4 PBMC was also coincident with the rise in viremia in these four animals (Fig. 2B). Importantly, the capacity of these CD4 T lymphocytes to produce IFN- after in vitro stimulation never recovered following control of viral replication. Moreover, the CD8 T lymphocytes in the same PBMC samples did not evidence a similar reduction in IFN- expression (Fig. 2B). These data suggest that the functional defects of CD4 T lymphocytes failed to improve even after primary viremia was controlled. Cytokine expression by CD4 T lymphocytes in SIV/SHIVinfected monkeys with various clinical sequelae of infection. We then sought to determine whether the cytokine expression profile of CD4 T lymphocytes from chronically SIV/SHIVinfected rhesus monkeys correlated with the clinical status of the monkeys. PBMC from both uninfected control monkeys and SIV/SHIV-infected experimental monkeys were evaluated. The infected monkeys were categorized as clinical nonprogressor or progressor animals on the basis of the viral RNA levels in their plasma, peripheral blood CD4 T-lymphocyte counts, and clinical status. The nonprogressor monkeys had been infected for more than 3 years, had maintained low plasma viral RNA levels, had relatively normal peripheral blood CD4 T-lymphocyte counts, and showed no clinical signs of disease progression (Table 1). The progressor monkeys had been infected for 6 months or longer, had high and varied levels of plasma viral RNA, had peripheral blood CD4 T-lymphocyte counts that were equal to or less than 50% of the preinfection levels, and showed signs of clinical disease (Table 1). PBMC of the monkeys were stimulated with PMA and ionomycin, and expression of IFN-, TNF-, and IL-2 by CD4 T lymphocytes was assessed. Upon stimulation, a large percentage of CD4 peripheral blood T lymphocytes from FIG. 3. Cytokine expression by CD4 T lymphocytes in healthy, uninfected monkeys (A), chronically infected monkeys (B), and monkeys that demonstrated signs of progressive disease (C). Three cohorts of monkeys were selected on the basis of the nature of their disease progression and assessed for the capacity of their CD4 T lymphocytes to produce the cytokine IFN-, TNF-, or IL-2. Panel A shows the expression profiles of CD4 T lymphocytes from animals that were uninfected (n 8), demonstrating the typical cytokine profile of healthy rhesus monkeys. Panel B shows the expression profile of the CD4 T lymphocytes of monkeys that had been infected with SIV/ SHIV for 3 years and yet demonstrated no signs of progressive disease (n 5). Panel C shows the cytokine expression profile of CD4 T lymphocytes of animals with progressive disease, low CD4 T-cell counts, and heavy viral loads (n 8). uninfected animals expressed both TNF- and IL-2 and a smaller percentage expressed IFN- (Fig. 3). Interestingly, this cytokine expression profile of the CD4 T lymphocytes from uninfected monkeys showed little variation between animals (20% IFN- [range, 14 to 29%], 72% TNF- [range, 64 to 82%], 65% IL-2 [range, 60 to 72%]; n 8). However, there was a small difference between the cytokine expression profiles of the peripheral blood CD4 T lymphocytes from these normal monkeys and those from SIV/SHIV-infected clinical nonprogressor monkeys. The animals that were clinical nonprogressors had a lower percentage of peripheral blood CD4 T lymphocytes expressing TNF- and IL-2, with no reduction in the percentage expressing IFN-, following in vitro stimulation (Fig. 3). The peripheral blood CD4 T lymphocytes from this group of clinical nonprogressor monkeys (n 5) also had remarkably consistent profiles of cytokine expression: 21%

VOL. 77, 2003 CD4 T-CELL DYSFUNCTION IN AIDS 4699 TABLE 2. Viral loads and absolute CD4 T-cell numbers per milliliter of peripheral blood of vaccinated and infected monkeys 150 days after a challenge with highly pathogenic SHIV-89.6P Treatment and monkey Viral challenge Viral load CD4 T-cell count/ml Sham vaccination KPE SHIV-89.6P 5,475 840 KPB SHIV-89.6P 24,667 12 DNA vaccines 820 SHIV-89.6P 1,021 567 811 SHIV-89.6P 177,019 272 702 SHIV-89.6P 500 754 DNA vaccines IL-2/Ig protein 798 SHIV-89.6P 1,323 888 772 SHIV-89.6P 1,166 667 839 SHIV-89.6P 500 720 712 SHIV-89.6P 500 1,085 DNA vaccines IL-2/Ig plasmid 893 SHIV-89.6P 651 1,276 833 SHIV-89.6P 500 858 483 SHIV-89.6P 500 1,554 728 SHIV-89.6P 719 968 IFN- (range, 13 to 31%), 50% TNF- (range, 37 to 62%), and 47% IL-2 (range, 30 to 59%). Finally, dramatic defects in the CD4 T-lymphocyte cytokine expression profiles were evident in the blood of the cohort of animals that had typical disease progression after infection, with high viral RNA levels and reduced peripheral blood CD4 T-lymphocyte counts. The percentage of CD4 T lymphocytes from these animals (n 8) that expressed IFN- was reduced by 50%, to 9% (range, 2.5 to 23%), and the percentage that expressed TNF- or IL-2 after stimulation was reduced to minimum levels (TNF-, 24% [range, 14 to 36%]; IL-2, 12% [range, 6 to 20%]) (Fig. 3). Thus, the functional capacity of CD4 T lymphocytes to express cytokines is associated with clinical status in SIV/SHIV-infected monkeys. We also measured the cytokine expression profiles by the total CD8 T-lymphocyte population and found the cytokine expression capacity of these cells to be within normal limits until the end stage of disease progression. We observed a small but statistically significant difference between the TNF- and IL-2 cytokine expression capacities of CD8 T lymphocytes from normal animals and those from progressors. This slight cytokine dysfunction was only detectable in latestage disease and likely reflects the severe immune system failure in those animals (data not shown). Plasmid DNA vaccination of monkeys protects their CD4 T lymphocytes from dysfunctional cytokine expression after a challenge with pathogenic SHIV-89.6P. We also assessed the cytokine expression profiles of peripheral blood CD4 T lymphocytes from a group of monkeys that were vaccinated and then subsequently challenged with SHIV-89.6P. Four groups of animals were evaluated in this study: one received sham vaccines, one received plasmid DNA constructs expressing HIV-1 89.6P Env (KB9) and SIVmac239 Gag, one received these same plasmid DNA constructs and IL-2/Ig protein, and a final group received these plasmid DNA constructs and an IL-2/Ig-expressing plasmid (Table 2). Following a challenge with SHIV-89.6P, all of the vaccinated monkeys maintained FIG. 4. Effective vaccination prevents loss of functional CD4 T lymphocytes. CD4 T lymphocytes from monkeys that had been vaccinated and then challenged with highly pathogenic SHIV-89.6P were assessed for the potential to produce IFN- (top panel), TNF- (center panel), and IL-2 (lower panel) 150 days following a challenge. Vaccination with plasmid DNA constructs plus IL-2/Ig plasmid prevented a significant loss of the CD4 T cells ability to produce all three cytokines. lower plasma viral RNA levels and higher peripheral blood CD4 T-lymphocyte counts than did the sham-vaccinated monkeys. Moreover, the degree of clinical control evidenced by the experimentally vaccinated monkeys correlated with the potency of vaccine-elicited CTL responses, with the IL-2/Ig protein-augmented plasmid DNA-vaccinated monkeys having better control of the virus than the monkeys receiving vaccines containing plasmid DNA alone and those receiving the IL-2/Ig plasmid-augmented plasmid DNA vaccines having the best control of the virus (2). Peripheral blood CD4 T-lymphocyte expression profiles were assessed in all of these monkeys on day 150 following a viral challenge (Fig. 4). PBMC of these monkeys were stimulated with PMA and ionomycin, and the percentage of CD4 T lymphocytes that expressed cytokines was assessed. The CD4 T lymphocytes of the vaccinated monkeys had a preserved capacity to produce IFN-, TNF-, and IL-2, with the better-protected monkeys having a better-preserved capacity to produce the cytokines. In fact, the cytokine expression profiles of the CD4 T lymphocytes of the animals that had been

4700 MCKAY ET AL. J. VIROL. TABLE 3. Statistical analysis of the cytokine profiles of CD4 T lymphocytes from various cohorts of rhesus monkeys. a Cytokine Mean no. of cytokine-positive cells/10 6 cells SEM Normal LTNP DNA IL-2/Ig Progressor IFN- 202.4 12.2 214.3 33.5 252.6 28.7 91.2 21.3 b TNF- 723.8 24.4 497.0 49.1 625.3 38.9 238.0 24.7 c IL-2 649.9 16.2 469.4 60.2 399.1 72.9 117.6 18.3 d a A four-way comparison, using an analysis of variance with Bonferronis multiple-comparison test, of the cytokine production potential of CD4 T lymphocytes from healthy, long-term nonprogressing (LTNP), optimally vaccinated, and progressor animals demonstrates that the CD4 T lymphocytes from vaccinated animals are protected from statistically significant dysregulation of cytokine production. The IFN-, TNF-, and IL-2 cytokine secretion potentials of CD4 T lymphocytes from normal, LTNP, and vaccinated animals are highly significantly different from those of lymphocytes from animals that are normal progressors with clinical disease. b Normal and DNA IL-2/Ig versus progressor, P 0.001; LTNP versus progressor P 0.01. c Normal LTNP, and DNA IL-2/Ig versus progressor, P 0.001. d Normal, LTNP and DNA IL-2/Ig versus progressor, P 0.001. optimally vaccinated most closely resembled the phenotype of clinically nonprogressing animals. A statistical comparison of the cytokine secretion potentials of CD4 T lymphocytes from the uninfected healthy monkeys, clinical nonprogressor monkeys, vaccinated and then challenged monkeys, and monkeys with progressive clinical disease demonstrated highly significant differences (Table 3). CD4 T lymphocytes from animals that had clinical signs of progressive disease had a significantly lower potential to secrete IFN- than did CD4 T lymphocytes from the other groups of animals (P 0.001, healthy normal versus clinical progressor; P 0.01, long-term nonprogressor versus clinical progressor; P 0.001, DNA plus IL-2/Ig versus clinical progressor). Dysregulation was also observed in the TNF- and IL-2 cytokine secretion potential of CD4 T lymphocytes from animals that had progressive clinical disease (P 0.001, healthy normal, long-term nonprogressor, and DNA plus IL-2/Ig versus clinical progressor). The functional integrity of the peripheral blood CD4 T lymphocytes of these monkeys was also assessed in a viral antigen-stimulated T-lymphocyte proliferation assay. The TABLE 4. CD4 T-lymphocyte IFN- ELISPOT responses in PBMC of surviving vaccinated monkeys 1.5 years following a challenge a Treatment and monkey CD4 T-lymphocyte response (no. of SFC/10 6 PBMC) Gag1 Gag2 Env1 Env2 Sham vaccination KPE 120 110 15 0 KPB 0 0 0 0 DNA vaccines 820 135 375 10 0 811 0 0 0 0 702 115 35 10 0 DNA vaccines IL-2/Ig protein 772 380 330 55 5 839 240 20 25 20 712 580 170 185 0 DNA vaccines IL-2/Ig plasmid 893 890 1,305 310 135 833 600 170 60 5 483 420 375 200 20 728 340 90 30 5 a ELISPOT responses of PBMC depleted of CD8 T lymphocytes and stimulated with the Gag1, Gag2, Env1, and Env2 peptide pools. ELISPOT responses are reported as numbers of spot-forming cells (SFC) per 10 6 PBMC. Backgrounds were consistently 15 SFC/10 6 PBMC and were subtracted from the observed responses. CD4 T lymphocytes from the vaccinated animals that controlled viremia following a challenge proliferated normally in response to SIV Gag protein, while the CD4 T lymphocytes of those animals that controlled virus replication less well evidenced decreased proliferation responses. In fact, a strong inverse correlation was demonstrated between the mean SIV Gag-stimulated proliferation of total PBMC and the mean number of viral RNA copies per milliliter in these monkeys (Fig. 5). The generation of neutralizing antibodies (NAbs) was also determined in these animals. We found that the vaccinated animals developed high NAb titers while the control animals did not (1) and that these differences in NAb titers persisted at all study time points (data not shown). Furthermore, the specific response of the peripheral blood CD4 T lymphocytes to SIV Gag or HIV-1 Env protein, as assessed by ELISPOT assays, was also preserved in the optimally vaccinated but not in the control sham-vaccinated animals (Table 4). These data indicate that monkeys evidencing vaccine-related clinical protection against progressive disease following AIDS virus infection demonstrate preserved virus-specific CD4 T-lymphocyte function. FIG. 5. An inverse correlation was observed between the ability of CD4 T lymphocytes of SHIV-89.6P-infected rhesus monkeys to proliferate and the viral burden in the animals. Animals are indicated by individual triangles (progressive disease) or by circles (healthy nonprogressor). The correlation was shown to be highly significant (P 0.0001). DISCUSSION While infections of rhesus monkeys with a number of distinct immunodeficiency virus strains have been characterized, no single lentivirus isolate has been shown to cause a disease in these monkeys that perfectly models human AIDS. The consequences of these immunodeficiency virus infections in monkeys range from nonpathogenic to highly pathogenic. Although the uncloned SIVmac251 and the cloned SIVmac239 viruses

VOL. 77, 2003 CD4 T-CELL DYSFUNCTION IN AIDS 4701 cause a disease in monkeys that in many ways resembles HIV- 1-induced disease in humans, set point viral loads in animals infected with these viruses are greater than HIV-1 loads in humans and the disease in these animals is more rapidly progressive than human AIDS. Some of the sooty mangabey SIV isolates (SIVsm) are even more highly pathogenic in rhesus monkeys than the SIVmac isolates. For example, SIVsmE543 typically causes a disease in rhesus monkeys that progresses to death within 2 years of infection. The chimeric SHIVs include some constructs that are nonpathogenic in monkeys (e.g., SHIV-89.6) and others that have been passaged in monkeys to render them highly pathogenic (e.g., SHIV-89.6P). By studying monkeys infected with a number of different SIV and SHIV isolates, we are able to model the immunopathogenesis of varied manifestations of HIV-1 infection in humans. Accruing data have suggested that CD4 T lymphocytes are dysfunctional in HIV-1-infected individuals with evidence of illness. Although PBMC proliferative responses to HIV-1 antigens have been shown to be maintained in clinically nonprogressing HIV-1-infected individuals, they are weak or absent in a majority of people who have progressive disease (18, 22, 23). A decrease in the expression of both IL-2 and IFN- by CD4 T lymphocytes from HIV-1-infected people has also been described (4, 16, 28). The findings in the present nonhuman primate studies are consistent with these observations. The loss of IFN-, TNF-, and IL-2 secretion capacity by a CD4 T-lymphocyte population has significant ramifications for the function of these cells. IFN-, an important Th1-type cytokine that is associated with the activation of CD8 T lymphocytes and the upregulation of major histocompatibility complex class I on antigen-presenting cells, mediates both antiviral and antitumor activities. TNF- is a multifunctional inflammatory cytokine that also has antiviral properties. IL-2 is essential for expansion and survival of T lymphocytes. Abnormalities in the secretion of these cytokines likely contribute to the immune defects associated with AIDS virus infections. In the present analysis of SIV- and SHIV-infected rhesus monkeys, the ability of the monkeys total CD4 T-lymphocyte population to produce IFN- upon polyclonal stimulation was shown to be dramatically reduced during the first days following infection, and this reduction coincided temporally with the peak of viral replication in the animals (Fig. 1 and 2). Since this loss of CD4 T-lymphocyte function occurred before the detection of a virus-specific CD8 CTL response, the expansion and maturation of effector CTL took place in a suboptimal helper cytokine environment. Moreover, although all of the animals in the prospective study went on to control viral replication and at least transiently maintained normal numbers of peripheral blood CD4 T lymphocytes following infection, the ability of these CD4 T lymphocytes to produce IFN- did not return to normal (Fig. 2). These findings build on previous observations made in studies of CD4 T lymphocytes from HIV-1-infected humans. However, most of the studies of human cells have assessed defects in cytokine production only in antigen-specific CD4 T-lymphocyte subpopulations. The present studies demonstrate that these defects are present globally in all CD4 T lymphocytes. Moreover, CD4 T lymphocytes from HIV-1- infected humans have, for the most part, been evaluated at single time points and only in selected individuals. We have evaluated this cell population and its functional repertoire prospectively through the period of primary infection and into the establishment of chronic infection. Our selected cohort of long-term nonprogressor animals retained highly functional peripheral blood CD4 T lymphocytes, whereas animals with progressive disease exhibited dysfunctional CD4 T-lymphocyte cytokine expression. These data support the notion that the ability of an immunodeficiency virus-infected individual s CD4 T lymphocytes to express cytokines is reflective of their functional capacity and correlates with disease status. Finally, we and others had previously shown that monkeys with high-frequency vaccine-elicited CTL that were challenged with a pathogenic AIDS virus were protected from many of the clinical sequelae of that infection. Such animals had low viral loads, preserved CD4 T lymphocytes, and no evidence of clinical disease. However, the mechanisms accounting for their persistent clinical protection were not clear. In the present study, we have shown a significant correlation between the ability of the PBMC from these animals to proliferate in response to the SIV Gag protein and the mean number of viral RNA copies per milliliter of plasma. Moreover, the CD4 T lymphocytes from these vaccinated and then infected animals had a preserved capacity to express cytokines, comparable to what was seen in animals that were infected with nonpathogenic viruses. Therefore, the present observations demonstrate that an immune correlate of this persisting clinical protection in vaccinated and then challenged monkeys is a preserved capacity of the peripheral blood CD4 T lymphocytes to proliferate in response to viral antigen and express important immunomodulatory cytokines. ACKNOWLEDGMENTS We thank Nicole Siciliano, Michael Piatak, Jr., Jeffrey D. Lifson, and Vanessa M. Hirsch for generously providing assistance and reagents. 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