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Memory Responses in Human Immunodeficiency Virus Type 1-Infected Individuals with Long-Term Viral Load Suppression Are Independent of CD4 Cell Nadir

Division of Infectious Diseases, Department of Medicine, University of Miami School of Medicine, Miami, Florida

Received 19 April 2004/ Returned for modification 21 June 2004/ Accepted 6 October 2004

	ABSTRACT

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The persistence of memory responses in suppressive highly active antiretroviral therapy (HAART) has been an area of controversy. By using a previously described proliferation assay that augments specific responses, peripheral blood lymphocytes (PBL) from 61 human immunodeficiency virus type 1-seropositive individuals with CD4 counts of >300/mm³ and suppressed viral burdens were studied for response to p24 antigen as a function of time of viral load suppression on HAART. In the majority of cases, proliferative responses could be measured in PBL from patients with plasma viral load suppression. No differences could be found in proliferative responses from PBL between individuals with a low and those with a high CD4 cell nadir. PBL that did not respond to either Casta antigen or p24 were found to have a higher percentage of naïve cells than did PBL that responded well to antigen. These data support the contention that, after long-term viral load suppression, PBL from infected individuals have memory cell populations that can respond to antigenic stimulation under inducible conditions.

	INTRODUCTION

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Highly active antiretroviral therapy (HAART) has significantly modified the interaction between host immunity and the virus. The response to HAART has been dramatic even in those individuals with low CD4 counts. The excitement associated with this immunologic recovery has been tempered by an inability to eradicate the infection and prevent disease progression in many cases. One area of interest is the role of an individual's CD4 cell nadir in predicting disease recovery or progression. It has been shown that individuals started on HAART with a low CD4 cell nadir do not recover CD4 cell levels to those of uninfected individuals (e.g., see reference 16). Furthermore, the CD4 cell nadir may negatively predict disease progression for individuals with comparable CD4 cell counts (12, 18). Since the ability to respond to immunologic challenge is dependent on memory cells and the ability to develop these cell populations from an existing or replenished naïve cell pool, it is possible that peripheral blood lymphocytes (PBL) from individuals with recovering CD4 counts from low CD4 cell nadirs are not as responsive as PBL from those with higher nadirs. There have been many studies evaluating proliferative responses to human immunodeficiency virus (HIV) antigens after long periods on suppressive HAART, but the results have been equivocal (1, 2, 5, 8, 22, 24, 25, 27). In an earlier study we demonstrated that memory cell proliferative responses to p24 antigen could be measured in a sensitive and specific manner when lipopolysaccharide (LPS) was used to drive T-cell proliferation through interactions with adherent monocytes (15). In the present study we use this assay to evaluate memory cell proliferative responses to p24 in PBL from HIV-infected individuals with suppressed viral loads (VLs) and CD4 counts greater than 300/mm³. The effect of duration of VL suppression and CD4 cell nadir on responsiveness to antigen is evaluated.

	MATERIALS AND METHODS

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Human subjects. Sixty-one HIV type 1 (HIV-1)-infected patients who attended the Outpatient HIV Clinics at the University of Miami-Jackson Memorial Hospital Medical Complex were recruited to participate in this study. Prior to enrollment all individuals consented to participation in the study (approved by the institutional human subjects review board of the University of Miami School of Medicine). Criteria for participation were an absolute CD4 count of >300/mm³ and a nondetectable VL (<400 copies/ml [c/ml]). Forty-two of the participants had a VL of <50 c/ml, 14 had a VL of <200 c/ml, and 5 had a VL of <400 c/ml. All participants were on HAART consisting of a protease inhibitor-containing regimen, a nonnucleoside reverse transcriptase inhibitor-containing regimen, or a triple nucleoside reverse transcriptase inhibitor regimen.

All participants had asymptomatic HIV disease at the time of enrollment. Medical record abstraction provided information on demographics, time of VL suppression (VL < 400 c/ml), and CD4 cell nadir. The CD4 cell nadir is the lowest documented CD4 count that an individual with HIV disease had ever reached. As such, those individuals with CD4 counts of <200/mm³ at any time in their past were known to have a CD4 nadir that was low (<200/mm³). The exact nadir was not available in all cases. The CD4 nadir for some individuals (8 of 61) could not be discerned because their prior clinic history was unavailable. Since one of the goals of this work was to demonstrate long-term persistence of memory cell responses, data from these individuals were included in Fig. 1 and 2.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Long-term p24 antigen-specific proliferative responses persist in virally suppressed individuals. The specific proliferation is shown as a function of duration of VL suppression by using the usual LPA (A) and the LPS-adherence assay (B). Closed and open symbols represent significant and nonsignificant responses, respectively, as described in Materials and Methods. PBL from individuals with low, high, and unknown CD4 cell nadirs are represented by squares, circles, and triangles, respectively.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Proliferative response to Casta and p24 antigen. The 7-day specific proliferation is shown for the PBL studied in Fig. 1 to Casta antigen (1.0 µg/ml) and p24 antigen (1.0 µg/ml) in the presence of LPS (0.01 µg/ml) and overnight adherence. The open symbols represent those PBL that respond well to antigen (triangles) or poorly to antigen (circles) with resting samples available for phenotype analysis. The closed squares represent the samples that were intermediate between the poor responders and the good responders. The dashed line defines those PBL that did not respond to p24 in this assay as described in the text.

Complete medium consisted of RPMI with antibiotics, L-glutamine, minimal essential medium with nonessential amino acids, minimal essential medium with sodium pyruvate, and 10 mM HEPES plus 10% normal human serum, type AB⁺ (Atlanta Biologicals, Norcross, Ga.).

Cells. Whole blood was collected in EDTA-containing tubes. Following collection PBL were isolated from the blood by density separation according to the manufacturer's protocol (lymphocyte separation medium from ICN Biomedicals, Inc., Aurora, Ohio). The PBL were subsequently frozen at –80°C, in 50% complete medium plus 40% fetal bovine serum plus 10% dimethyl sulfoxide, for later use. Data from the ACTG Immunology Laboratories (23) demonstrated that proliferation to recall antigen of cryopreserved PBL was preserved compared to that of freshly isolated PBL. Furthermore, although the percentages of memory cells displaying CD45RA and CD62L are slightly diminished on thawed cells compared to fresh whole blood (23), numerous studies have used memory phenotypes as reliable markers on frozen-thawed samples (e.g., see references 7 and 28).

Antigen proliferation experiments. Frozen PBL obtained from donors were thawed and plated in quadruplicate for each experimental point in wells (10⁵ cells/well) of a 96-well U-bottomed microtiter tissue culture plate. For the LPS-adherence proliferation experiments the tissue culture plate was incubated in a 5% CO₂ incubator at 37°C for 1 day prior to addition of p24 and/or Casta antigen as dictated by the experiment. When indicated, LPS (0.01 µg/ml) was added to the experimental wells 1 h after the addition of antigen. After addition of reagents the experimental plate was incubated for 6 days in a 5% CO₂ incubator at 37°C. After incubation for 6 days, 1 µCi of [³H]thymidine was added per well and the plate was incubated overnight. Wells were subsequently harvested, and DNA-incorporated radioactivity was counted. In the usual lymphocyte proliferation assay (LPA) the antigen is incubated with PBL for 6 days, after which time radiolabel is added as described above for 1 day before harvesting. Determinations of specific counts were obtained by subtracting nonspecific counts (no antigen) in the absence or presence of LPS, as appropriate for the experiment, from the absolute counts and then normalized to 1,000 CD4 cells/well as determined by the percent CD4 (17). The percent CD4 was obtained on clinical samples from the same blood drawing from which the PBL were obtained. Added reagents were present for the full period of incubation. Specific proliferation is given in counts per minute. Proliferative responses were considered significantly different if the absolute increase in the specific response was greater than 3,000 cpm, the absolute counts were statistically different between the experimental and control groups (P < 0.05, unpaired t test), and the stimulation index (cpm_exp/cpm_control) was >3 (17).

FACS studies. Frozen PBL obtained from donors were thawed, aliquoted at 10⁶ cells per fluorescence-activated cell sorting (FACS) tube, and maintained on ice (4°C). Cells were subsequently washed with FACS buffer (0.02 M HEPES, 0.05% NaN₃, 0.1% bovine serum albumin, pH 7.4) and incubated for 1 h in FACS buffer at 4°C containing 10% normal human serum (AB⁺) to block Fc receptor. Then, after washing, cells were stained with anti-CD4-PE, anti-CCR7-PE-CY7, and anti-CD45RA-fluorescein isothiocyanate. After 1 h of staining on ice, cells were washed two times with cold FACS buffer and fixed with 4% paraformaldehyde in buffered phosphate-buffered saline for 15 min at 4°C. The cells were subsequently run on a Vantage SE instrument (Becton Dickinson, Mountain View, Calif.) with compensation. Fifty thousand events were captured and subsequently gated on the small lymphocyte population and CD4-positive T cells.

Statistics. The unpaired t test was used to determine whether the absolute proliferation to antigen was different than the no-antigen-medium control or no-antigen-LPS control (as dictated by the experiment). The analysis of variance and the Holm t test (10) were used to determine significance between demographic variables in Table 1. The Mann-Whitney rank test was used to determine significance between memory or naïve populations between PBL from low and high responders (10) (Fig. 3).

View larger version (25K): [in this window] [in a new window]   FIG. 3. A high naïve CD4 phenotype is associated with PBL that have a low antigenic response. The percentage of CD4+ cells in the naïve subset, CD45+ CCR7+ (A); the central memory subset, CD45RA– CCR7+ (B); and the effector memory subset, CD45RA– CCR7– (C), is shown for individuals who responded poorly to Casta and p24 antigen (low responders) and those who responded well to Casta and p24 antigen (high responders). In panels D, E, and F are shown the absolute number of cells per well for each subset for individuals who responded poorly to Casta and p24 antigen (low responders) and those who responded well to Casta and p24 antigen (high responders). These PBL were chosen from available resting samples according to their response to antigen as shown in Fig. 2. Gating and staining are described in Materials and Methods. Significance was determined using the Mann-Whitney rank-sum test. NS, nonsignificant.

	RESULTS

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VL suppression and p24 responses. Numerous groups have looked at proliferative responses to p24 as a function of time after initiation of HAART. For patients with extended viral suppression, some groups are able to demonstrate a proliferative response to p24 (1, 2, 24, 25) whereas others are not (5, 7, 22, 27). In a previous study (15) we described a sensitive assay for specifically measuring proliferative responses to p24 antigen. In that work it was shown that the proliferative dose-response to antigen was shifted to the left, providing a sensitive readout for proliferative responses to p24 antigen concentrations. This assay required PBL to adhere to the tissue culture plate and incubation with LPS to augment the response. In Fig. 1 this assay is leveraged to demonstrate that PBL from infected individuals with chronically suppressed viral burdens can proliferate to p24. In Fig. 1A is shown the specific proliferation of PBL to p24 (1.0 µg/ml) in the usual LPA whereas Fig. 1B represents the specific proliferation of PBL to p24 after overnight incubation followed by LPS (0.01 µg/ml) and p24 (1.0 µg/ml) additions. In Fig. 1A there were 47 nonresponders (open symbols), 45 of whom fell below the dashed line (150 cpm/1,000 CD4 cells), whereas only one responder (closed symbols) fell below the dashed line. In Fig. 1B there were 26 nonresponders, 18 of whom fell below the dashed line. There were no responders who fell below the dashed line. The dashed line (150 cpm/1,000 CD4 cells) effectively represents a line of demarcation for responders in a sensitive manner (>90% for both assays). Only two nonresponders in Fig. 1B were responders in Fig. 1A, whereas 23 of the nonresponders in the usual LPA (Fig. 1A) were responders in Fig. 1B (35 total responders of 61). Individuals with low CD4 cell nadirs (<200 cells/mm³), high CD4 cell nadirs (>200 cells/mm³), or unavailable CD4 cell nadirs are shown by squares, circles, and triangles, respectively. PBL from individuals with either a high or low CD4 cell nadir responded to p24 in the boosted assay and did so even after long-lasting VL suppression (Fig. 1B). Of those PBL from individuals with a low CD4 nadir, 47% were nonresponders (15 of 32), whereas 38% of those with a high CD4 nadir were nonresponsive (8 of 21) and 38% of those with an unknown CD4 cell nadir were nonresponders (3 of 8), all nonsignificant differences (P > 0.05).

High and low responders have different memory cell distributions. In Fig. 1B there were PBL from infected individuals that did not proliferate to p24 antigen in this assay. This group provided an opportunity to address the question of whether the PBL that did not respond to antigenic stimulation in the LPS-adherence assay shared any common properties that might not be revealed in a less sensitive assay. Since data from non-HIV studies have shown that different memory cell populations proliferate differently to antigen and produce different cytokine profiles (11, 13, 26), we decided to compare memory phenotypes in resting PBL. We looked at two specific subsets of resting PBL defined by their ability to respond well to Casta antigen and p24 (the high responders) or to respond poorly to Casta antigen and p24 (the low responders) in the LPS-adherence assay. We reasoned that, by looking at PBL that could proliferate at the two extremes, we might discern subpopulation differences when the unstimulated CD4⁺ T cells from available stored samples were evaluated by flow cytometry for CCR7 and CD45RA expression. Figure 2 shows proliferation of the PBL to p24 (1.0 µg/ml) and Casta antigen (1.0 µg/ml) for all the samples evaluated in Fig. 1. The closed squares represent the samples that were intermediate between the poor responders and good responders. The open circles show the specific proliferation for available stored PBL that were nonresponsive to antigen, and the open triangles show the specific proliferation for available stored PBL that responded well to antigen. When these two populations were studied by flow cytometry for CD4⁺ memory phenotypes, there was a significantly (P < 0.05) greater percentage of CD4 cells that expressed the naïve phenotype, CD45RA⁺ CCR7⁺, for those donor PBL for the low responders compared to the high-responder PBL (36 compared to 23%, respectively), as shown in Fig. 3A. There were no differences seen in percentages of central memory or effector memory phenotype (Fig. 3B and C). The differences in proliferative response to antigen were not simply a consequence of fewer cells with the memory phenotype. As shown in Fig. 3E and F there is no difference in the absolute number of central or effector memory cells per experimental well. Furthermore, there is no significant difference in the absolute number of naïve cells between the good- and poor-responder PBL (Fig. 3D), demonstrating that it is not the absolute number of naïve cells per well that explains the differences in proliferation between groups.

In the high-responder group all patients had undetectable VLs with stable CD4 counts for greater than 1 year, so it was unlikely that further increases in naïve populations would be significant (e.g., see references 8 and 27). The mean CD4 counts between the high and low responders (600 and 444/mm³, respectively), as well as the time of VL suppression between the high and low responders (22.0 and 23.4 months, respectively), were not significantly different (P > 0.05). Of the high-responder group six of seven had CD4 cell nadirs less than 200/mm³, and of the poor responder group five of seven had CD4 cell nadirs less than 200/mm³.

	DISCUSSION

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In non-HIV-infected individuals long-term cellular memory may persist even in the absence of antigen and play an important role in vaccination strategies (see, for example, reference 9). In HIV disease the system may be somewhat different since HIV itself impacts host immunity and the recovery from HAART is incomplete. Studies demonstrating long-lived CD4 memory responses in HIV-1-infected individuals on a suppressive HAART regimen have been mixed. Some groups are able to measure memory responses to antigenic stimulation in proliferation assays after long periods of VL suppression whereas others are not (1, 2, 5, 8, 22, 24, 25, 27). Data from intracellular cytokine staining have also suggested that the frequency of memory cells to HIV antigen does diminish over time (21). Therefore, it is important to understand whether HIV-infected individuals have persistent long-term memory responses to recall antigen and to measure them accurately. In an earlier study we described an assay that used LPS and monocyte adherence to augment specific proliferative responses to p24 (15). In the study described here we use this assay to evaluate proliferative responses to p24 in a HAART-suppressed cohort of infected individuals with CD4 counts greater than 300 cells/mm³. It was shown in Fig. 1 that the sensitivity of the usual LPA was not sufficient to reveal proliferative responses to p24 in the majority of cases (Fig. 1A), whereas with the LPS-adherence assay the majority of the PBL did proliferate to p24 (Fig. 1B). This was true for individuals who had a low or high CD4 cell nadir. This is in distinction from other studies that have suggested that those with a high CD4 cell nadir have poorer proliferative responses to antigen than do those with low CD4 cell nadirs (6).

In this study the majority of PBL from patients responded to p24 antigen in the LPS-adherence assay. There is a small group of patients whose PBL did not respond to either p24 or Casta antigens in the LPS and adherence assay (Fig. 2), consistent with a functionally anergic population. Interestingly, resting PBL from these individuals had a greater percentage of naïve CD4⁺ T cells than did PBL from infected individuals who responded well to antigenic stimulation (Fig. 3A). When the absolute number of cells was evaluated, there was no difference between high and low responders for both the naïve and memory phenotypes (Fig. 3D to F). These data suggest that the poor proliferation in one group compared to those that respond well to antigen is not simply a consequence of a disproportionate amount of naïve cells but an inherent difference in the memory subsets that proliferate. It is possible that the recovery of naïve cells for these two populations was different even though there was no significant difference in the duration of suppression between groups. However, numerous studies have shown that memory cells account for the early CD4 cell recovery followed by a slow persistent increase in naïve CD4 cells. After 1 to 2 years on suppressive HAART the CD4 count stabilizes as dictated by the naïve and memory cell populations (4, 17, 19).

The importance of using innate immunity to drive proliferative responses may in part stem from producing an environment in which effector cytokines augment and recruit specific proliferative responses not easily seen without this adjunctive step (14, 20). The fact that by using the LPS-adherence assay it was possible to discern a group of nonresponders who shared the property of more naïve cells compared to a set of PBL that proliferated well to antigen suggests that this assay may have use in other systems when the evaluation of immune responsiveness is important. Since immune activation may be predictive of response to HAART (3), it would be interesting to see if there are any correlations between activation parameters and proliferative responses in the LPS-adherence assay.

	FOOTNOTES

 
* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Medicine, University of Miami School of Medicine, P.O. Box 016960 (R21), Miami, FL 33101. Phone: (305) 243-4791. Fax: (305) 243-2362. E-mail: mkolber{at}med.Miami.edu.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kolber, M. A. Articles by Kaul, S. Search for Related Content PubMed PubMed Citation Articles by Kolber, M. A. Articles by Kaul, S.