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Journal of Virology, October 2003, p. 10900-10909, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.10900-10909.2003

Diminished Proliferation of Human Immunodeficiency Virus-Specific CD4+ T Cells Is Associated with Diminished Interleukin-2 (IL-2) Production and Is Recovered by Exogenous IL-2

Christiana Iyasere,1 John C. Tilton,1 Alison J. Johnson,1 Souheil Younes,2 Bader Yassine-Diab,2 Rafick-Pierre Sekaly,2 William W. Kwok,3 Stephen A. Migueles,1 Alisha C. Laborico,1 W. Lesley Shupert,1 Claire W. Hallahan,1 Richard T. Davey Jr.,1 Mark Dybul,1 Susan Vogel,1 Julia Metcalf,1 and Mark Connors1*

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland,1 Laboratory of Immunology, CHUM, University of Montreal, Montreal, Canada,2 Virginia Mason Research Center, Benaroya Research Institute, Seattle, Washington3

Received 12 May 2003/ Accepted 11 July 2003


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ABSTRACT
 
Virus-specific CD4+ T-cell function is thought to play a central role in induction and maintenance of effective CD8+ T-cell responses in experimental animals or humans. However, the reasons that diminished proliferation of human immunodeficiency virus (HIV)-specific CD4+ T cells is observed in the majority of infected patients and the role of these diminished responses in the loss of control of replication during the chronic phase of HIV infection remain incompletely understood. In a cohort of 15 patients that were selected for particularly strong HIV-specific CD4+ T-cell responses, the effects of viremia on these responses were explored. Restriction of HIV replication was not observed during one to eight interruptions of antiretroviral therapy in the majority of patients (12 of 15). In each case, proliferative responses to HIV antigens were rapidly inhibited during viremia. The frequencies of cells that produce IFN-{gamma} in response to Gag, Pol, and Nef peptide pools were maintained during an interruption of therapy. In a subset of patients with elevated frequencies of interleukin-2 (IL-2)-producing cells, IL-2 production in response to HIV antigens was diminished during viremia. Addition of exogenous IL-2 was sufficient to rescue in vitro proliferation of DR0101 class II Gag or Pol tetramer+ or total-Gag-specific CD4+ T cells. These observations suggest that, during viremia, diminished in vitro proliferation of HIV-specific CD4+ T cells is likely related to diminished IL-2 production. These results also suggest that relatively high frequencies of HIV-specific CD4+ T cells persist in the peripheral blood during viremia, are not replicatively senescent, and proliferate when IL-2 is provided exogenously.


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INTRODUCTION
 
In acute or chronic viral infections of humans and experimental animals, virus-specific CD4+ T-cell function is believed to be critical for induction and maintenance of host immunity that mediates effective restriction of viral replication. In numerous viral infections of experimental animals, depletion or disruption of the function of CD4+ T cells results in the impairment of CD8+ T-cell function and a diminished ability to restrict viral replication (8, 28, 29, 33). Many viral infections of humans typically result in induction of CD4+ T-cell responses that can be demonstrated by in vitro proliferation in response to viral antigens long after control of infection due to the persistence of memory cells in the peripheral blood. Unlike most other infections of humans, human immunodeficiency virus (HIV) infection is characterized by the absence of HIV-specific CD4+ T-cell proliferative responses in the vast majority of untreated patients. However, these responses have been detected in some cohorts of patients that restrict HIV replication. Strong HIV-specific CD4+ T-cell responses are found in a relatively rare subgroup of patients, referred to as long-term nonprogressors (LTNP), that maintain low levels of HIV replication without antiretroviral therapy despite prolonged infection (34, 42, 46, 47, 51). In addition, proliferative responses to HIV antigens have been found in patients treated early during acute infection that then have restricted HIV replication when antiretroviral therapy is withdrawn (37, 45, 46). Because HIV infects CD4+ T cells, it was believed that the early loss of HIV-specific proliferative responses may be the result of infection and deletion of HIV-specific cells in the lymphoid tissues when they encounter the virus.

However, several lines of evidence indicate that HIV-specific CD4+ T cells persist in patients with progressive disease. Although proliferative responses are typically absent in untreated patients, there are several recent reports that the prevalence of significant proliferative responses to HIV antigens is as high as 30 to 69% of those receiving effective antiretroviral therapy (1, 4, 5, 11, 27, 31, 39). In addition, a number of recent reports have documented the persistence of HIV-specific CD4+ T cells in the majority of patients by intracellular cytokine staining following stimulation with HIV antigens in cross-sectional cohorts (3, 34, 39, 41, 54). Further, when both proliferation and frequency have been measured, the absence of HIV-specific CD4+ T-cell proliferation was not attributable to the complete deletion of HIV-specific CD4+ T cells (34, 39, 48, 54). Thus, there is now general agreement that HIV-specific CD4+ T cells persist in patients with progressive disease yet the ability of these cells to proliferate in vitro is impaired.

However, a number of fundamental questions regarding the functions of HIV-specific CD4+ T cells remain. Among these, the mechanism(s) by which proliferation of HIV-specific CD4+ T cells is diminished or absent in the majority of untreated patients remains poorly understood. Possible explanations for diminished in vitro proliferation include decreased frequencies of HIV-specific CD4+ T cells, diminished antigen-specific T-cell responsiveness, cytokine secretion, anergy, and replicative senescence, among others. Proliferation detected in traditional [3H]thymidine incorporation assays is a downstream result of many different processes. The use of these assays has not permitted the analysis of antigen presentation, exclusion of proliferation by non-CD4+ T-cell subsets, T-cell activation, cytokine secretion, or the presence of antigen-specific CD4+ T cells.

In this study, we further explore the effects of viremia on HIV-specific CD4+ T-cell responses and the importance of these responses in predicting the ability to restrict HIV replication. These observations were obtained from a cohort of 15 patients that while on therapy maintained strong HIV-specific CD4+ T-cell responses equivalent to those of LTNP. Proliferation in response to HIV and non-HIV antigens was measured by standard [3H]thymidine incorporation assays and carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution. In addition, the frequencies of cells that produce gamma interferon (IFN-{gamma}) or interleukin-2 (IL-2) in response to Gag, Pol, and Nef peptide pools were monitored during an interruption of therapy. In each case, proliferative responses to HIV antigens were rapidly inhibited during viremia and were typically associated with diminished IL-2 production. Proliferation of HIV-specific CD4+ T cells during viremia was recovered by addition of exogenous IL-2. These observations suggest that, during viremia, diminished in vitro proliferation of HIV-specific CD4+ T cells is likely related to diminished IL-2 production. These results also suggest that relatively high frequencies of HIV-specific CD4+ T cells persist in the peripheral blood during viremia, retain the ability to activate and produce cytokine, are not replicatively senescent, and proliferate when IL-2 is provided exogenously.


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METHODS AND METHODS
 
Study population. HIV type 1 (HIV-1) infection in study participants was confirmed via HIV-1/2 immunoassay. All subjects signed informed consent and participated in protocols approved by a National Institute of Allergy and Infectious Diseases (NIAID) investigational review board. Proliferative responses to HIV antigens of patients receiving care in the NIAID HIV Clinic were measured by standard [3H]thymidine incorporation assays (see below) at each visit. Patients on antiretroviral therapy were selected for study if they maintained proliferative responses to HIV p24 antigen equivalent to those of LTNP (>2,000 net cpm [ncpm]) and were willing to consent to a therapy interruption and leukapheresis. Those patients receiving IL-2 therapy did not receive doses of IL-2 within the 6 months prior to study. In the subset of patients that have appeared in previous publications all numbers remain consistent with those most recently published to permit cross-referencing. Samples from patients 222, 224, and 203 in the present study were also used in a recent study of preferential infection of HIV-specific CD4+ T cells and are referred to as patients 13, 14, and 16, respectively (13).

Stimulation assays. Standard [3H]thymidine incorporation assays for proliferation were performed as previously described (30, 46). Briefly, peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood by sodium diatrizoate density centrifugation (Organon-Teknika, Durham, N.C.). Fresh isolated cells were incubated (100,000 cells/well) in triplicate in the presence of the following antigens: 10 µg of HIV-1IIIB p24 (Advanced Biotechnologies, Columbia, Md.)/ml, a 1/100 dilution of cytomegalovirus (CMV) lysate, CMV control lysate (Biowhittaker, Walkersville, Md.), 2 µg of phytohemagglutinin (PHA; Sigma, St. Louis, Mo.)/ml, or medium. On day 3, cells incubated with PHA were pulsed for 6 h with 1 µCi of [3H]thymidine per well and harvested. Wells containing all other proteins or controls were similarly pulsed and harvested on day 5.

For flow cytometry-based assays of cytokine production or proliferation, PBMC were isolated from apheresis donor packs by sodium diatrizoate density centrifugation. Stimulation assays were performed on thawed cryopreserved samples maintained at -140°C or on fresh isolated PBMC. Cells were washed twice in 10% human AB media and aliquoted at 4 million per stimulation tube. All 6-h assays for intracellular cytokine production were completed as previously described (3, 34, 41). For experiments performed on cryopreserved samples, 40,000 autologous Epstein-Barr virus-transformed B cells (1%) were added to each stimulation tube. Anti-CD28 and -CD49d antibodies were added to all tubes (1 µg/ml; Pharmingen, San Diego, Calif.). To determine the frequency of antigen-specific CD4+ T cells, one of the following protein antigens was added to the appropriate simulation mixtures: 16 µg of HIV-p24/ml, a 1/80 dilution of CMV lysate, or 10 µg of tetanus antigen (Aventis-Pasteur, Swiftwater, Pa.)/µl. Gag, Pol, and Nef pooled peptides (HIVHXB2; NIH AIDS Research and Reference Reagent Program, Rockville, Md.) were added such that the concentration of the individual peptides within each pool was maintained a 2 µg/ml. Staphylococcal enterotoxin B (10 µg/ml, final concentration; Toxin Technology, Inc., Sarasota, Fla.) was used as a positive control. Incubation, fixation, and permeabilization were performed as previously described (34). All cells were simultaneously stained with anti-CD3-fluorescein isothiocyanate, -IL-2-phycoerythrin, and -CD4-peridinin-chlorophyll A protein (PERCP) (Becton Dickinson, San Jose, Calif.) and anti-IFN-{gamma}-allophycocyanin (APC; Pharmingen) for 30 min at 4°C. By flow cytometry between 70,000 and 500,000 CD3+ CD4+ cell events were collected per sample. All IFN-{gamma} and IL-2 virus-specific cell frequencies reported have frequencies in medium controls subtracted. Results are representative of assays performed on two to four occasions from a single leukapheresis. In flow cytometry assays for proliferation, CFSE (Molecular Probes, Eugene, Oreg.) was used according to the manufacturer's protocols. In experiments using CFSE, cells were labeled with CD3-PERCP and CD4-APC.

Major histocompatibility complex class II (MHC-II) DR0101 tetramers covalently linked to the DR0101 restricted HIVpol peptide FRKQNPDIVIYQYMDDLYV (HXB2pol amino acids 171 to 189) were produced as previously described (16). The BirA substrate peptide was cloned in the C terminus of the alpha chain as described previously (10). S2 Schneider cells were cotransfected with pchygro and the pcv vector e-gfpDRa-BSP-DRb-p51. After selection, cells were grown and the supernatant was collected and passed through an affinity L243 column. DR0101 class II monomers were purified, biotinylated, and tetramerized as described previously (10). The HIV-5b (DYVDRFYKTLRAE; HXB2gag-p55 amino acids 295 to 307) DR0101 tetramer was constructed by the Beckman Coulter Corporation. Data were collected with a FACSCalibur dual-laser cytometer (Becton Dickinson) and analyzed with FLOWJO (TreeStar, San Carlos, Calif.) software.

Statistical analysis. Days until plasma viral RNA increased above 5,000 copies/ml were compared by the log rank method. Comparison of the LTNP and non-LTNP group p24 geometric means was made by Student's t test. The median paired differences from on treatment to off treatment, as well as with and without IL-2, were tested for significance by the Wilcoxon signed rank test. Adjustment of P values for multiple testing was done by the Bonferroni method.


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RESULTS
 
Ability to restrict HIV replication during an interruption of therapy. A cohort of 15 patients receiving antiretroviral therapy that had strong CD4+ T-cell proliferative responses to HIV antigens while on antiretroviral therapy were recruited (Table 1). For 12 of these patients therapy was to be resumed only when plasma viral RNA exceeded 5,000 copies/ml or CD4+ T-cell counts declined 25% from baseline on two consecutive weekly determinations. To obtain a sample of patients undergoing more-frequent interruptions of therapy, three additional patients that were part of a second cohort alternating 2 months of receiving therapy with 1 month without therapy were included in this analysis. Interruption of therapy was associated with a modest but statistically significant decrease in total CD4+ T-cell count (median, 698 cells/µl on therapy versus 570 cells/µl off therapy; P = 0.05; Table 1). During therapy interruption, the median plasma viral RNA of all 15 patients at the time of apheresis was 22,196 copy eq/ml of plasma (range, 2,185 to 70,060 copy eq/ml of plasma). Two patterns of increases in plasma viral RNA were typically observed. In 12 of the 15 patients, plasma viral RNA increased within the first 3 weeks of therapy interruption and declined only once therapy was resumed (Fig. 1). However, three patients maintained restriction of HIV replication over a more prolonged interval. Plasma viral RNA of patient 228 increased to 400 copies/ml during the first 60 days and diminished to below 50 copies/ml before slowly increasing until therapy was resumed at day 409. In patients 219 and 229, no initial increase in plasma viral RNA occurred in the first 60 days. Patient 219 remained off therapy for 54 weeks. Patient 229 first exceeded 5,000 copies of HIV RNA per ml of plasma on week 28, and RNA concentrations remained just below this value. He had been off therapy for a total of 85 weeks and remained off therapy at the end of this study. Overall, the duration of restriction of HIV RNA below 5,000 copies/ml (median, 28 days) was not different from that for a separate cohort of 29 patients not selected on the basis of HIV-specific immune responses (median, 22 days; P = 0.12) (11). This frequency of patients that maintained plasma HIV RNA concentrations below 5,000 copies/ml beyond 12 weeks (3 of 15) is very similar to the frequency of 10 to 20% observed in other cohorts (11, 36, 38; reviewed in reference 22). Thus, in this cohort of patients highly selected for maintenance of strong HIV-specific CD4+ T-cell responses, prolonged restriction of HIV replication was not observed in the majority of patients.


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  		TABLE 1. Clinical characteristics of patients



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  		FIG. 1. Plasma viral RNA and peripheral blood CD4+ T-cell counts of 15 patients during therapy interruptions. Shaded areas indicate intervals on therapy. (A) Twelve-patient cohort that resumed therapy when plasma viral RNA exceeded 5,000 copy eq/ml on 2 consecutive weekly determinations. (B) Three patients alternating 2 months on therapy with 1 month off therapy. Arrow, apheresis time point during the off-therapy interval that was used for study.

HIV-specific proliferative responses during an interruption of therapy. Proliferative responses measured by standard [3H]thymidine assays were performed at the time of aphereses. Because these assays are highly variable, the time points selected for study typically represented extremes of both viremia and suppression of HIV replication by antiretroviral therapy in a given patient. In addition, [3H]thymidine incorporation assays were performed in a separate laboratory under code to eliminate observer bias. While on therapy, each of the patients maintained strong p24-specific proliferative responses. These values were not significantly different from those from a cohort of 16 LTNP that typically maintain <50 copies of HIV RNA/ml of plasma (geometric mean, 6,471 ncpm [95% confidence interval {CI}, 4,454 to 9,400 ncpm] in therapy interruption patients versus 5,191 ncpm [95% CI, 3,853 to 6,995 ncpm] in LTNP; P = 0.37) (34). Despite strong HIV-specific CD4+ T-cell proliferative responses prior to therapy interruption, an increase in plasma virus was associated with a dramatic decrease in proliferative responses to p24 antigen, as measured by [3H]thymidine incorporation in all patients (median, 8,155 ncpm [range, 1,671 to 20,195 ncpm] on therapy versus 554 ncpm [range, 100 to 1,982 ncpm] off therapy; P < 0.001) (Fig. 2). Data from a patient from whom samples at multiple time points were available are shown in Fig. 2A. Although the proliferative response of this patient appears to decline just prior to an interruption of therapy, this decline corresponds to a twofold decrease, which is within the expected range of variability of this assay. The proliferative response to p24 antigen of each of the patients was regained only after antiretroviral therapy was resumed. Thus, consistent with our prior results for 8 patients (34), in each of the 15 patients in the present study proliferation of HIV-specific CD4+ T cells was rapidly inhibited during viremia.



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  		FIG. 2. HIV-specific proliferative responses during an interruption of therapy. (A) Peripheral blood CD4+ T-cell count, plasma viral RNA, and responses to PHA and p24 antigen in [3H]thymidine assays of patient 226. (B) In vitro [3H]thymidine incorporation in response to p24 antigen during therapy interruption for all 15 patients.

HIV-specific CD4+ T cells persist during viremia despite loss of proliferative responses. Because of the possibility that diminished proliferation in response to HIV antigens was due to changes in the numbers of HIV-specific CD4+ T cells during viremia, the frequency of HIV-specific CD4+ T cells was also examined by intracellular cytokine staining following HIV antigen stimulation. Our prior efforts to analyze the frequency of HIV-specific CD4+ T cells during an interruption of therapy were limited by the use of whole-protein antigens. These antigens required the use of fresh PBMC, which did not permit repetition of experiments and which permitted a complete analysis of changes in frequency of HIV-specific cells during an interruption of therapy in only three patients. More importantly, use of whole-protein antigens requires protein uptake, processing, and presentation in a 6-h assay and vastly underestimates the true frequency of antigen-specific cells.

To obtain a more detailed and reproducible analysis of the frequency of HIV-specific cells during an interruption of therapy, the frequency of CD4+ T cells that produce IFN-{gamma} and/or IL-2 in response to Gag, Pol, or Nef overlapping peptide pools was measured. In the majority of patients, the frequency of Gag-specific CD4+ T cells was highest, representing up to 1.0% of the peripheral blood CD4+ T cells, prior to an interruption of therapy. The total percentage of HIV-specific IFN-{gamma}-producing cells in some patients was as great as 2.5% (Fig. 3). The three patients that demonstrated some capacity to restrict HIV replication by maintaining plasma HIV RNA concentrations below 5,000 copies/ml for 196 to 400 days (patients 219, 228, and 229) were not distinguished by higher frequencies of HIV-specific CD4+ T cells. During an interruption of therapy, there was a significant increase in the frequency of IFN-{gamma}-producing cells specific for Gag (median, 0.26% on therapy versus 0.53% off therapy; adjusted P < 0.01), but not for Pol (median, 0.05% on therapy versus 0.07% off therapy; adjusted P > 0.5) or Nef (median, 0.06% on therapy versus 0.06% off therapy; adjusted P > 0.5). Thus, although in vitro proliferation in response to HIV antigens was suppressed during viremia, HIV-specific CD4+ T cells persisted in the peripheral blood, in some cases at very high frequencies.



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  		FIG. 3. Frequency of HIV-specific IFN-{gamma}-producing CD4+ T cells during therapy interruption. The percentages of peripheral blood CD4+ T cells that produce IFN-{gamma} in response to HIV Gag, Pol, or Nef overlapping peptide pools are shown. ARV + and ARV -, on and off antiretroviral therapy, respectively.

Effects of viremia on responses to non-HIV antigens. To examine whether the effect of viremia on in vitro proliferation was HIV specific, responses to other non-HIV antigens were measured. CD4+ T-cell proliferation in response to CMV, tetanus antigen, HIV p24 protein, and the Gag peptide pool in a subset of 10 patients was measured by CFSE dilution and flow cytometry. Consistent with the data from conventional [3H]thymidine assays, proliferation of HIV-specific CD4+ T cells was diminished during an interruption of therapy in each of the patients tested (Fig. 4). During viremia, there was a statistically significant decrease in the percentage of CD3+ CD4+ CFSElow cells in cultures stimulated with p24 protein (17.4% on therapy versus 6.6% off therapy; P = 0.003) or Gag peptides (13.6% on therapy versus 3.9% off therapy; P = 0.008). Overall, there was no statistically significant change in CD4+ T-cell proliferation in response to CMV (mean, 32% CFSElow on therapy versus 29.2% off therapy; P > 0.5) or tetanus antigens (mean, 11.5% on therapy versus 12.4% off therapy; P > 0.5). These results suggested that the effect of viremia in suppressing in vitro CD4+ T-cell proliferation during an interruption of therapy was HIV specific.



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  		FIG. 4. CD4+ T-cell proliferation in response to HIV or non-HIV antigens during an interruption of therapy. PBMC were stained with CFSE on day 0 and stimulated with the indicated antigen. The percentages of CD3+ CD4+ lymphocytes present on day 6 that had undergone at least one division based on CFSE dilution are shown.

Antigen-specific IL-2 production. Production of IL-2 is thought to be critical to the ability of CD4+ T cells to survive and proliferate in vitro. Given that the diminished proliferation of CD4+ T cells during viremia was HIV specific, it remained possible that diminished proliferation of antigen-specific cells was related to the frequencies of IL-2-producing cells for a given antigen. Because the frequency of HIV-specific CD4+ T cells that produce IL-2 is low, it has not been studied previously in detail. The frequencies of CD4+ T cells that produce IL-2 in response to Gag, Pol, or Nef pooled peptides and CMV antigens in all 15 patients prior to and during an interruption of therapy were studied. The majority of either HIV- or CMV-specific IL-2-producing CD4+ T cells were a subset of IFN-{gamma}-producing cells (Fig. 5). A summary of the frequency of Gag-specific IFN-{gamma}+ or IL-2+ cells is shown in Fig. 6. During an interruption of therapy, there was an increase in total (all IFN-{gamma}+ plus all IL-2+) Gag-specific CD4+ T cells (median, 0.18% of cells on therapy versus 0.311% of cells off therapy; P < 0.001). There was also an increase in the percentage of IFN-{gamma}+ IL-2- Gag-specific cells (median, 0.03% on therapy versus 0.30% off therapy; P < 0.01). Despite the increase in total Gag-specific cells, the frequency of IL-2-producing cells in 9 of the 15 patients actually decreased. Because the frequency of Gag-specific IL-2-producing cells prior to therapy interruption was very low and close to the limit of detection of this assay in a number of patients, the decrease in Gag-specific IL-2+ cells was only at the borderline of statistical significance (median, 0.06% on therapy versus 0.05% off therapy; P = 0.06; Fig. 6). Nonetheless, a marked and reproducible decrease in the frequency of Gag-specific IL-2+ CD4+ T cells was observed in those patients with measurable frequencies prior to an interruption (Fig. 5 and 6). This effect was not observed in CMV-specific cells of these patients. Overall, significant decreases in the frequency of CMV-specific IL-2-producing cells were not observed (median, 0.45% [range, 0.07 to 2.13%] on therapy versus 0.29% [range, 0.00 to 3.37%] off therapy; P > 0.5; data not shown). These results suggested that, at least in a subset of patients, IL-2 production in response to HIV antigens may be blunted during viremia and could play a role in diminished HIV-specific CD4+ T-cell proliferation.



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  		FIG. 5. HIV- or CMV-specific CD4+ T-cell IFN-{gamma} and IL-2 production. Density plots of cells following 6-h stimulation with a Gag peptide pool or CMV antigen are shown. Numbers in quadrants indicate the percentages of gated CD3+ CD4+ lymphocytes.



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  		FIG. 6. Frequencies of HIV-specific IFN-{gamma}- and IL-2-producing CD4+ T cells during therapy interruption. The percentages of peripheral blood CD3+ CD4+ cells that produce the indicated cytokines in response to the HIV Gag peptide pool are shown. The percentage of cytokine-producing cells in medium controls is subtracted. ARV + and ARV -, on and off antiretroviral therapy, respectively.

Exogenous IL-2 recovers proliferation of HIV-specific T cells during viremia. The effects of addition of exogenous IL-2 to cultures were also explored. Prior study of the effects of exogenous IL-2 on HIV-specific CD4+ T cells was limited by proliferation of bystander cells in cultures. To study the effects of exogenous IL-2 in vitro, methods to identify proliferation of HIV-specific cells were required. Although a number of technical obstacles to the production of stable human MHC-II peptide tetramers remain, DR0101 tetramers bound to a p24 peptide (01-05b) and a Pol peptide (51-11) have recently been synthesized. These reagents were used to determine the effects of exogenous IL-2 on HIV-specific CD4+ T cells during viremia. Two patients (219 and 222) carried the DR0101 allele and maintained CD4+ T-cell proliferative responses to the 01-05b and 51-11 peptides while on therapy (Fig. 7). Proliferation of 01-5b-specific CD4+ T cells from patient 219 was very low prior to therapy interruption, likely because of extremely low frequencies of cells specific for this peptide (0.02%; data not shown). Proliferation of 01-5b- or 51-11-specific CD4+ T cells was diminished or absent during viremia (Fig. 7). However, addition of only modest amounts of IL-2 (1 U/ml) caused proliferation of 01-5b- or 51-11-specific CD4+ T cells.



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  		FIG. 7. Exogenous IL-2 rescues proliferation of HIV-specific CD4+ T cells during viremia. A representative density plot of CFSE-stained PBMC from patient 222 on day 6 is shown. Cells were cultured in medium alone or medium containing the indicated stimuli and stained with anti-CD3, anti-CD4, and the MHC-II 01-5b or Pol 51-11 DR0101 tetramer. Numbers in quadrants indicate the percentages of gated CD3+ CD4+ lymphocytes.

The ability of IL-2 to cause proliferation of CD4+ T cells of other specificities during viremia was also explored. Analysis of the effects of exogenous IL-2 was limited by the lack of available class II tetramers for a broad range of specificities and alleles. To further explore the ability of exogenous IL-2 to expand HIV-specific CD4+ T cells from viremic patients, CFSE-labeled cultures stimulated with HIV p24 or CMV antigen were restimulated with Gag peptide pools on day 7 and the frequency of IFN-{gamma}+ CFSElow CD4+ T cells was measured (Fig. 8A). No increase in IFN-{gamma}+ CFSElow CD4+ T cells in CMV-stimulated cultures that were restimulated with Gag peptide pools on day 7 was observed (data not shown), indicating that this assay specifically detected the expansion of Gag-specific CD4+ T cells. Addition of IL-2 did not affect the frequency of Gag-specific CFSElow IFN-{gamma}+ cells in PBMC cultures from the time point on therapy (median, 3.27% without IL-2 versus 4.63% with IL-2; P > 0.5; Fig. 8B). In contrast, addition of exogenous IL-2 to PBMC from viremic patients recovered proliferation of HIV-specific CD4+ T cells (median, 0.61% CFSElow IFN-{gamma}+ cells without IL-2 versus 1.87% with IL-2; P = 0.02). Taken together these results indicated that HIV-specific CD4+ T cells persist at relatively high frequencies during viremia, retain the ability to activate and produce IFN-{gamma}, and retain the ability to divide when IL-2 is provided exogenously.



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  		FIG. 8. Exogenous IL-2 recovers proliferation of Gag-specific IFN-{gamma}-producing CD4+ T cells. (A) Representative density plot of CFSE-stained PBMC from patient 222 on day 6. PBMC were incubated for 6 days in medium alone or medium containing HIV p24 antigen. Ten units of IL-2 was added to cultures where indicated. Cultures were restimulated on day 7 with the Gag peptide pool. Numbers in quadrants indicate the percentages of gated CD3+ CD4+ lymphocytes. (B) Summary of CFSElow IFN-{gamma}+ CD4+ T cells in response to restimulation with the Gag peptide pool in eight patients.


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DISCUSSION
 
These results provide additional insight into the mechanism(s) by which proliferation of HIV-specific CD4+ T cells is diminished in HIV-infected patients. Diminished proliferation of HIV-specific CD4+ T cells in viremic patients might be caused by diminished frequencies of antigen-specific cells within the peripheral blood, diminished ability of cells to respond to antigen or cytokines, or replicative senescence. However, the results of the present study suggest that each of these is unlikely to be the case. Rather, relatively high frequencies of HIV-specific CD4+ T cells persist in the peripheral blood and readily activate, produce IFN-{gamma} in response to HIV antigens, and retain the ability to divide when IL-2 is supplied exogenously. These observations strongly suggest that the mechanism(s) of diminished proliferation of HIV-specific CD4+ T cells in viremic patients is very different from that of the diminished proliferation of HIV-specific CD8+ T cells that was recently described (35). In that report, HIV-specific CD8+ T cells were not suppressed by viremia during a therapy interruption and were not associated with changes in IL-2 production. In addition, the diminished ability of HIV-specific class I tetramer+ CD8+ T cells to divide was quite durable in that it was not overcome by stimulation with anti-CD3/CD28, addition of autologous lymphoblasts, or addition of exogenous IL-2. However, the results of the present study are consistent with a number of recent reports describing experimental animals under conditions of high levels of antigen. Diminished proliferation and IL-2 production of virus-specific CD4+ T cells have been observed during chronic infection of perforin knockout mice during chronic lymphocytic choriomeningitis virus (LCMV) infection (17). A similar pattern of diminished proliferation and IL-2 production in CD8+ T cells has also been observed during chronic infection with LCMV clone 13 and in day 8 effector memory cells at the peak of viremia (52).

The findings presented here may also be consistent with a number of reports on the effects of exogenous IL-2 under conditions of high levels of antigen in vitro or in vivo. It has been previously observed that memory CD4+ T cells undergo initial cytokine-independent and subsequent cytokine-dependent phases of proliferation (14, 23). It is possible that the initial cytokine-independent phase occurs in vivo upon antigen encounter in HIV-infected patients during viremia and that the further proliferation of these cells ex vivo requires addition of exogenous cytokines such as IL-2. Consistent with this interpretation, IL-2 was recently shown to increase proliferation and survival of T cells in LCMV-infected mice when provided during the contraction phase of the cellular immune response after the peak of viremia (6). Although administration of IL-2 to HIV-infected patients causes an increase in total CD4+ T-cell proliferation in vivo (21; reviewed in references 40 and 49), it remains unclear whether it causes an increase in the frequency of HIV-specific CD4+ T cells. There are two recent reports, one cross-sectional and one longitudinal, in which an increase in HIV-specific CD4+ T cells was observed (19, 50). However, we have not observed higher frequencies of HIV-specific CD4+ T cells in patients receiving IL-2 with antiretrovirals than in those receiving antiretroviral therapy alone in our cross-sectional or longitudinal cohorts in prior work (15, 34), in results presented here, or in subsequent work (J. C. Tilton, unpublished observations). Although it is clear that additional data are needed, it is possible that detection of increased frequencies of HIV-specific CD4+ T cells in patients receiving IL-2 therapy may be complicated by infection of these cells by HIV or the concomitant proliferation of non-HIV-specific CD4+ T cells.

The results of the present study also suggest that the frequency of HIV-specific CD4+ T cells is considerably higher than was previously appreciated. Because the frequency of HIV-specific IFN-{gamma}+ CD4+ T cells in response to HIV protein antigens was modest (approximately 0.1% of peripheral blood CD4+ T cells specific for the Gag p55 protein), the possibility remained that the frequency was too low and that depletion of HIV-specific CD4+ T cells played a role in the absence of HIV-specific proliferative responses in vitro (3, 34, 39, 41, 54). However, these earlier studies used protein antigens that require antigen uptake, cleavage, and presentation in relatively brief 6-h assays. In the present study, when overlapping peptide pools were used, the frequency of HIV-specific IFN-{gamma}+ CD4+ T cells was 5- to 10-fold higher than these previous estimates. These frequencies are consistent with one recent report using overlapping peptide pools and gating on CD3+ CD8- cells (3). The frequency of HIV-specific CD4+ T cells is typically below the frequency specific for CMV in a given patient, although this was not the case for patients 225 or 230. Although the mean frequency of CMV-specific IFN-{gamma}-producing CD4+ T cells in the peripheral blood is approximately 3%, Epstein-Barr virus- or varicella-zoster virus-specific frequencies are typically approximately 0.2% (2, 24, 25, 34, 41). This suggests that the frequency of HIV-specific CD4+ T cells may not necessarily be limiting. It should be noted that comparisons with frequencies found in other chronic viral infections are complicated by the fact that most studies of the frequencies of CD4+ T cells in these infections have been performed using whole-protein antigens. In addition, the frequency of HIV-specific CD4+ T cells measured in current assays may be an underestimate given that the peptide sequences of HIV antigens are likely more distantly related to those of the patient than is the case for antigens of members of the herpesvirus family (20).

These results also confirm and extend some previous observations regarding the relationship of measurement of virus-specific CD4+ T-cell responses and restriction of virus replication. In each of the 15 patients studied, in vitro HIV-specific proliferative responses were rapidly inhibited during viremia, consistent with our prior observations for eight patients (34). In a number of other viral infections, diminished in vitro virus-specific proliferative responses during viremia have been reported, suggesting that this effect is not unique to HIV infection. For example, there are several viruses infecting humans, such, as measles virus, CMV, Dengue virus, and hepatitis B and C viruses, for which in vitro proliferative responses are diminished or absent in the acute or chronic phase in viremic individuals (7, 9, 18, 32, 44, 53). Similarly, when 1011 to 1013 replication-defective adenovirus particles were administered to patients in gene therapy trials, strong proliferative responses to adenovirus antigens were inhibited (43). For HIV then, the absence of in vitro HIV-specific CD4+ T-cell proliferative responses in viremic patients may be an effect, but not necessarily a cause, of ongoing viral replication. Although, while on therapy, patients maintained HIV-specific proliferative responses and HIV-specific CD4+ T-cell frequencies equivalent to those of LTNP, a similarly high degree of restriction of virus replication was not observed. Although these results do not suggest that depletion or functional disruption of HIV-specific CD4+ T cells is a critical defect that dictates the loss of restriction of viral replication during chronic infection, it remains possible these effects play a larger role either during acute infection or in end stage disease.

Although speculative, it remains possible that these decreases in proliferative responses in vitro during viremia reflect some diminished function(s) in vivo. Ongoing viral replication clearly has effects on in vitro responses to HIV antigens, although, as noted above, it remains unclear whether this represents an immunological "defect" per se. In addition, although no reproducible effect on non-HIV-specific responses was detected in the present study during a brief interruption of therapy, responses to non-HIV antigens may be diminished during chronic viremia (26, 34). Further, rapid improvements in immune function in vivo, such as immune reconstitution syndromes, have been observed in patients beginning effective antiretroviral therapy prior to increases in CD4+ T-cell counts (reviewed in reference 12). Thus far, progress in dissection of the mechanism(s) of diminished proliferation of HIV-specific and non-HIV-specific CD4+ T cells during viremia has been accelerated by the availability of better reagents and newer flow cytometry-based assays. Further understanding of the mechanism(s) of these effects may provide important insights of direct relevance in vivo on the ways in which HIV disrupts the response to opportunistic pathogens and to HIV itself.


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ACKNOWLEDGMENTS
 
C.I. and J.C.T. contributed equally to this work.


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FOOTNOTES
 
* Corresponding author. Mailing address: LIR, NIAID, NIH, Bldg. 10, Rm. 11B-09, 10 Center Dr., MSC 1876, Bethesda, MD 20892-1876. Phone: (301) 496-8057. Fax: (301) 402-0070. E-mail: mconnors{at}niaid.nih.gov. Back


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Journal of Virology, October 2003, p. 10900-10909, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.10900-10909.2003




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