Early and delayed benefits of HIV-1 suppression: timeline of recovery of innate immunity effector cells
Azzoni, Livioa,*; Chehimi, Jiheda,*; Zhou, Lanb; Foulkes, Andrea Sc; June, Rayforda; Maino, Vernon Cd; Landay, Alane; Rinaldo, Charlesf; Jacobson, Lisa Pg; Montaner, Luis Ja
From the aThe Wistar Institute, Philadelphia, Pennsylvania
bDepartment of Statistics, Texas A&M University, College Station, Texas
cDivision of Biostatistics, University of Massachusetts School of Public Health and Health Sciences, Amherst, Massachusetts
dBiological Research and Development, BD Biosciences, San Jose, California
eDepartment of Immunology/Microbiology Rush University Medical Center, Chicago, Illinois
fDepartment of Infectious Diseases and Microbiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania
gDepartment of Epidemiology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland.
*These authors contributed equally to the manuscript.
Received 5 July, 2006
Revised 9 October, 2006
Accepted 30 October, 2006
Correspondence to Luis Montaner, DVM, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA. E-mail: firstname.lastname@example.org
Objective: The kinetics of recovery for innate immune effectors following antiretroviral therapy are unknown.
Design and methods: Multiple sequential cryopreserved samples (viremic and ART-suppressed) from 66 patients enrolled in the Women's Interagency HIV Study or Multicenter AIDS Cohort Study cohorts (median follow-up, 700 days) were analyzed to determine natural killer, dendritic and T-cell changes by flow cytometry. Functional parameters were also measured in a subset of samples. Changes over time were analyzed by mixed-effect modeling based on a linear spline with a single knot at 270 days.
Results: Following viral suppression, a rapid rise in CD4 and white blood cell counts and a decline in T-cell activation were confirmed. However, natural killer cell subsets increased after 270 days of therapy, with a negative effect by baseline CD4%. CD123+ plasmacytoid but not myeloid dendritic cells showed a trend to increase during the first 270 days with a positive effect of baseline CD4%; plasmacytoid dendritic cell-induced interferon-α production significantly increased by end of follow-up.
Conclusions: The kinetics of natural killer and plasmacytoid dendritic cell recovery are markedly different from those of T-cell subsets, indicative of early and delayed benefits of suppressive regimens.
Antiretroviral therapy (ART)-mediated viral suppression is associated with an increase in CD4 T-cell counts by up to 150 cells/μl in the first year  even in the absence of increased anti-HIV immunity. The first phase of CD4 expansion is rapid, greater in subjects with lower starting CD4 cell count , and consists mostly of CD45RO+ memory cells, following redistribution from lymphoid tissue and decreased activation-induced apoptosis. Suppressive ART also leads to decreased expression of activation molecules on T cells (CD38 or HLA-DR), recovery of CD28+ T cells and partial recovery of the T-cell receptor (TCR) Vβ repertoire . A second, slower CD4 growth phase follows, related to generation of naive cells [2,4] and diversification of the T-cell receptor repertoire, with expansion of CD45RA+CD62L+ CD4 and CD8 T cells, and reduction of CD8 memory cells (reviewed in ). However, long-term suppressive ART may not result in recovery of pre-infection CD4 cell counts , as immune reconstitution may be inversely correlated to the degree of impairment at baseline . Functionally, more than 6 months of ART result in recovery of T-cell responses to recall antigens [8,9] of a magnitude directly related to pre-therapy CD4 T-cell counts [6,10–12]. T-cell cytokine production is recovered within 8 weeks of suppressive treatment . In contrast, the extent and rates of the recovery of innate effectors upon suppressive ART remain to be determined.
Dendritic cells (DC) initiate T and B-cell responses to pathogens via antigen presentation  and production of cytokines [e.g., interleukin (IL)-12 and interferon (IFN)-α]. Depletion and functional impairment of DC subsets has been documented in HIV infection, including decreased ability to stimulate T cells [15,16] and produce IFN-α, facilitating the onset of opportunistic infections [16–19]. Little is known about the longitudinal effects of ART on DC function, or the role of pre-treatment state on subsequent recovery. Limited, cross sectional studies suggest that ART leads to incomplete recovery of IFN-α production by plasmacytoid DC (PDC), myeloid DC (MDC) frequency and allogeneic mixed lymphocyte responses (MLR) [16,19,20].
Natural killer (NK) cells are important innate immune effectors, lysing tumor cells and cells infected by viruses or intracellular parasites and bacteria, through direct or antibody-mediated mechanisms reviewed in [21–23]. NK cells also secrete antiviral cytokines (e.g. IFN-γ) that promote T-helper type 1 differentiation. CD16+ CD56+ CD8+/ CD16+ CD56+ CD8– NK cells are depleted in chronic HIV infection, in correlation with CD4+ T-cell depletion and increase of CD8+/CD38+/HLA-DR+ lymphocytes [24–27]. Furthermore, cytokine-induced IFN-γ production by NK cells is similarly impaired in asymptomatic, viremic and ART-suppressed, HIV-infected subjects , suggesting profound, irreversible changes in cellular immunity may occur at early stages of HIV-1 infection [29,30].
We analyzed changes in innate immune effector subsets in relation to CD4 cell counts following viral suppression, using a mixed effects modeling approach . We report the first description of NK and DC subset changes in relation to baseline values and pre-ART CD4 cell counts, and provide models to predict time-related changes on a population basis.
66 HIV-1 infected individuals (38 men and 28 women) in the Multicenter AIDS Cohort Study (MACS) and Women's Interagency HIV Study (WIHS) cohorts were selected for analysis. Selected subjects had one viremic and multiple subsequent suppressed time points (HIV RNA < 400 copies/ml to account for older samples; sampling every 6 months). At baseline, 24 individuals had CD4 cell counts < 350 cells/μl and 34 had serum HIV RNA ≥ 10 000 copies/ml (17 of them having both characteristics). A total of 215 longitudinal samples were evaluated (107 from MACS; 108 from WIHS cohort). All samples had matching clinical laboratory information (HIV RNA, CBC, CD4 and CD8 cell counts).
Five-color immunofluorescence staining was performed on cryopreserved peripheral blood mononuclear cells (PBMC) using the following mAb combinations: (1) CD16+56+161 fluorescein (FITC), CD154 phycoerithrin (PE), HLA-DR ECD, CD94 allophycocyanin (APC) and CD3 biotin; (2) CD16 FITC, CD56 PE, HLA-DR ECD, CD161 APC and CD3 biotin; (3) CD154 FITC, HLA-ABC PE, CD3 ECD, CD38 APC and CD8 biotin; (4) Lin-1 FITC, CD40 PE, HLA-DR ECD, CD11c APC and CD123 biotin; (5) Lin-1 FITC, HLA-ABC PE, HLA-DR ECD, CD11c APC and CD123 biotin (all from BD Biosciences, San Jose, California, USA; except for CD3 and HLA-DR ECD, from Beckman Coulter, Fullerton, California, USA). 2 × 105 PBMC/staining (recovery > 50%) were treated with the indicated mAb combinations, followed by APC-Cy7-conjugated streptavidin. Sample analysis was conducted on a CyAn LX (Dako-Cytomation, Fort Collins, Colorado, USA; > 105 events/run); samples from the same donor were thawed, stained and analyzed at the same time, to avoid inter-experimental variations related to reagents or instrument settings. PBMC from healthy donors were used for channel compensation. Post-run analysis was performed using FlowJo (Tree Star software, Ashland, Oregon, USA).
PBMC (duplicate 105/stimulation in 96 well plates) were cultured for 18 h at 37°C with RPMI-1640 (with 10% fetal bovine serum) alone, or with Gram-negative lipopolisaccaride (LPS; 10 μg/ml), PR-8 influenza virus (10 HA units/ml) or CpG-2216 (10 μg/ml). Cell-free supernatants were analyzed for the presence of IL-12 p40 (radioimmuniassay using the mAb pair C11.79/C8.6, as previously described ), IL-10 and IFN-α (enzyme-linked immunosorbent assay; Endogen, Rockford, Illinois, USA).
Mixed lymphocyte reaction
Mixed lymphocyte reaction was performed using cord blood lymphocytes as responders, following the original method of Young and Steinman . The results were expressed as stimulation index (SI).
Forty-nine variables were studied (see Table 1). Analysis objectives were exploratory, as a prelude to the validation of results with larger sample sizes and fewer variables; therefore, a two sided unadjusted level = 0.05 test was employed.
Baseline to last available time point differences were evaluated using the Wilcoxon signed rank test. Correlations of outcomes with viral load were evaluated using the Spearman's test.
Longitudinal study on 17 variables (see Table 2) was performed using mixed effects models (MEM).
Mixed effects model
The primary goal was to model the changes from baseline in each of 17 variables as a function of time. Natural logarithmic (loge) transformation of selected variables was employed to reduce skewness. Separate models were fitted for each outcome . Random subject-specific intercepts were assumed to account for potential within subject correlation. Fixed effects included time on study, baseline CD4 percentage and baseline values of the outcome under consideration. Additional exploratory models were fitted with interaction effects between time and baseline CD4 percentage; and time and baseline of the outcome.
To allow for differential linear effects as observed with CD4 cell count changes, a b-spline model with one interior knot at time 270 days was selected for the effect of time. The use of a single knot at 270 days, selected on the basis of our observed data on CD4 changes over time, is supported by several recent reports [4,6,35]. Wald tests were employed to test for main effects of time before and after 270 days and differences in the slopes over time by baseline values. Plots of residuals were generated to confirm model assumptions. Statistical analyses were performed using R v2.1.1 (R Project for Statistical Computing: http://www.r-project.org/).
To characterize the effect of time and interactions between time and baseline values on the response Y, we used the equation:
Equation (Uncited)Image Tools
Equation (Uncited)Image Tools
is the response of individual i at time t, and xi0 is this person's baseline CD4%. Approximate f(t;yi0;xi0) with linear splines, choosing a knot at tk = 270 days.
Two models were fitted.
1. Primary analysis:
Equation (Uncited)Image Tools
2. Exploratory analysis:
Equation (Uncited)Image Tools
Biphasic rise in CD4 cell count and concurrent decrease in T-cell activation following viral suppression
In concordance with prior reports from the MACS and other cohorts [2,36,37], we observed a CD4 T-cell count increase from baseline by 144 ± 191 cells//μl at the first suppressed time point available (mean = 422 days) and 186 ± 226 cells/μl at the last available sample (mean = 753 days). Results from the MEM analysis confirmed the observed significant rise in CD4 cell count (Fig. 1a) with rapid change in the first 270 days (P = 0.0109), and a subsequent slower rise. The observed CD4 cell increment (see Table 1) was inversely correlated with baseline CD4 cell count or percentage (r = −0.6732; P < 0.0001), and positively correlated with baseline viral load (r = 0.3551; P = 0.0084), confirming faster ART-induced CD4 cell rise in patients with advanced disease. As expected , baseline white blood cell counts (WBCC) exhibited a strong positive correlation to CD4 cell counts (r = 0.61; P < 0.0001); significant WBCC increases were observed with ART (P = 0.0153, Table 1); accordingly, MEM only showed a significant WBCC increase until day 270 (see Fig. 1b). Our MEM analysis also confirmed prior studies [39–41], indicating a decline in CD38 expression (mean fluorescence intensity; MFI) on CD8+ T cells in the first 270 days of viral suppression (Fig. 1c, Table 2), with an inverse relationship between baseline activation and CD38 MFI decrease. Significant reduction of CD38 expression was also observed in CD4+ T cells (Tables 1 and 2). The analysis of cognate molecules involved in NK/DC interaction indicated that expression (MFI) of HLA-Class I (KIR/KAR cognate), but not CD154 (CD40 cognate), was significantly decreased in response to ART in both CD4 and CD8 cells (Table 1). By confirming previously described changes in T-cell subsets and activation, these observations acted to validate our analytical approach and dataset.
Functional reconstitution of plasmacytoid dendritic cells
We analyzed the frequency of PDC and MDC based on the expression of CD123 or CD11c on Lin–/HLA-DR+ cells. For each subset, we also evaluated the expression of CD40 (involved in T-cell and possibly NK cell cross-talk), and HLA class I molecules as markers of maturation/activation. As shown in Fig. 2, we observed a significant increase in PDC (mean, 0.04 ± 0.21%) between baseline and last available time point (panel d), whereas, in contrast with previously published cross-sectional data , no significant change in MDC was observed (Table 1, Fig. 2j). MEM did not indicate a significant effect of time on PDC frequency; however, we detected a significant positive interaction effect between baseline CD4% and time for both PDC and MDC for t < 270 days (PDC, P = 0.008; MDC, P = 0.043) suggesting that subjects with higher baseline CD4% experience a stronger rise during the first 270 days (see Table 2); this supports the presence of a ‘DC recovery’ subgroup within our cohort.
The analysis of CD40 and MHC-class I molecule expression on DC subsets indicated a decrease in MHC-I expression in both subsets and of CD40 on PDC, consistent with a decrease in activation (see Table 1, Fig. 2b). A significant decrease of MHC-I expression on PDC and an interaction between MHC-I expression on PDC and baseline CD4% was observed, further supporting the potential existence of differences in kinetics of innate immune reconstitution between individuals at different stages of disease (Table 2, Fig. 2c).
To assess PDC functionality, we measured IFN-α production in response to influenza virus (Fig. 2e) and the TLR-9-specific CpG-2216 (Fig. 2f) on a subset of 29 subjects (MACS cohort only, due to WIHS sample yield restrictions). No MEM analysis was performed on these data due to their limited size. At baseline, stimulation with inactivate influenza strain PR-8 resulted in production of 67.5 ± 78.3 pg/ml of IFN-α; with a significantly higher levels of PR-8 induced IFN-α production at endpoint (143.1 ± 103.7pg/ml, P = 0.0016; Fig. 2 and Table 1). Similarly, CpG-2216-induced production of IFN-α (40.5 ± 53.8 pg/ml at baseline) was significantly higher at endpoint (91.2 ± 100.7 pg/ml, P = 0.0105). Repeated measure ANOVA comparison between first and last available suppressed time points indicated that only IFN-α production at the last, but not the first suppressed time was significantly different from baseline. Interestingly, the individual gain in PR-8 induced IFN-α production following ART (IFN-αendpoint – IFN-αbaseline) was positively correlated with baseline CD4 levels (Spearman r = 0.428, P = 0.026), consistent with a similar observation of a significant effect of time and CD4% on PDC frequency as noted earlier. MLR activity showed a significant rise with ART (P = 0.004, Table 1) consistent with a functional gain as described in previous studies . Consistently, APC cytokine responses following LPS stimulation also suggested a gain of function [higher levels of induced IL-12p40 (P = 0.0786, Fig. 2k) and IL-10 secretion (P = 0.0376, Fig. 2l)].
Taken together, our data support an increase in PDC frequency and PDC/MDC function after viral suppression, which may be more pronounced in subjects with higher baseline CD4 cell counts.
Delayed recovery of mature NK subsets
Cross-sectional studies indicate that NK cells are depleted in viremic HIV-1-infected adults and children [28,42]. We assessed NK cell frequency based on expression of CD161, CD56 or CD16, in the absence of CD3 (see Fig. 3, panels d–i). Consistent with prior observations, NK cell frequency increased from baseline (4.8 ± 2.8% of PBMC) by an average of 0.14% (first suppressed time point) and 0.33% (last suppressed time point). The MEM analysis indicated that NK cells increased significantly only after 270 days (P = 0.0029, Fig 3a and Table 2), supporting a delayed recovery as compared to CD4 cell count. The analysis of the CD56+ NK cell population and the mature CD3–/CD161bright/CD56+/CD16+ subset yielded similar results (CD56+: P = 0.008 for t > 270 days, Fig. 3b; CD3–/CD161bright/CD56+/CD16+: t > 270 days, P = 0.013, Fig. 3c. See also Table 2). As observed for DC subsets, a positive interaction of baseline CD4% with time was observed, indicating that subjects with higher CD4 counts might show earlier recovery of NK cells. Analysis of the expression of CD94, CD154 and HLA-DR within NK did not show statistically significant changes (Table 1). Taken together, results indicate that the expansion of circulating NK cell subsets is observed only after long-term viral suppression.
Our data represent the first retrospective, long-term longitudinal study (66 subjects, 2040 days follow-up) of innate effector cell recovery in subjects initiating suppressive ART, providing evidence of a differential rate of recovery of DC and NK cells relative to the biphasic growth of CD4 T cells. To overcome the lack of uniform sampling, we applied a linear spline-based mixed-effect modeling (MEM) approach to analyze changes in variable values as a function of time. Our results not only contribute to the definition of innate immune reconstitution under ART, but also validate an analytical approach (linear spline-based MEM) that may facilitate future studies employing archived, unsynchronized samples from repository cohorts.
Overall, significant changes over time were observed in one of three main patterns, in relation to an inflection time point of 270 days from the baseline viremic visit:
1. Significant early rise, followed by slow growth (significant increase, smaller slope) or plateau (or non-significant change). This group includes WBCC and CD4 cell counts.
2. Significant early decrease, followed by a plateau (non-significant change). This group includes activation markers (CD38 expression on CD8+ T cells).
3. Initial lag (no significant change) followed by a significant rise. This behavior is observed in NK cells, including mature NK subsets.
Our analysis evidenced a change in the rate of the CD4 cell count increase (inflection point) after approximately 270 days from the pre-ART viremic time point, in agreement with previous studies placing the change in kinetics anywhere between 50 days and 18 months from ART initiation [2,4,43]. Because of the nature of the cohorts (observational sampling every 6 months, independent of incurring events), our analysis does not address the exact time of ART initiation, but rather models consecutive viremic and suppressed samples treating time as a continuous variable. Since the average time to viral suppression after ART initiation approximates 90 days (our unpublished data), we estimate that all patients with undetectable viral load at the second visit would have been treated for a period of 3 to 6 months. An adjustment of our model to accommodate an earlier inflection point would require the analysis of a more intensively sampled cohort with a known date for ART initiation. Despite this limitation, our results correctly describe anticipated ART-associated changes during the initial response to ART (CD4 count and activation marker changes [4,6,35,39–41]) while identifying the unexpected observation of a delay in NK cell recovery.
Changes in DC subsets were restricted to the observed increase in PDC frequency between baseline and end of follow-up. Our modeling results indicate an effect of baseline CD4 frequency on changes of both MDC and PDC subsets; suggesting a differential PDC recovery potential in patients with different levels of pre-ART CD4 T cells; this finding remains to be confirmed in larger cohorts with direct CD4-based stratification. Our longitudinal results support prior studies showing a partial increase in IFN-α secretion after viral suppression , and link PDC recovery with other parameters of immune activation and immune reconstitution. Other cross-sectional studies suggested an early depletion of PDC  and a lack of recovery on ART : a potential reason for this discrepancy is the fact that our longitudinal cohort represented a more immunological competent group at start of ART (median baseline CD4 = 434 cells/μl). A partial restoration of MDC/APC function, if not frequency in this cohort, is also supported by the increased level of MLR activity together with IL-12 and IL-10 achieved after long-time suppression.
In contrast to CD4 T cells, the delayed kinetics of NK cell restoration upon viral suppression suggest that NK cell frequency is less sensitive to regulation by active viral replication than CD4+ T cells (e.g., lack of redistribution into circulation), possibly requiring reconstitution of bone marrow production upon long-term viral suppression. The observed recovery is driven by the main mature NK cell subsets (CD161bright/CD56+/CD16+ and CD161bright/CD56–/CD16+ cells). This is in line with prior reports of loss of cytotoxic, perforin-rich CD56bright NK cells in advanced HIV infection [45–47]; further studies using fresh PBMC would be necessary to assess the kinetics of changes in other subsets which have been reported as depleted (CD56dim NK cells ) or expanded (CD161+/CD56– NK cells ) in chronic HIV infection. The extent to which the delay in NK cell reconstitution correlates to a functional recovery of cytotoxicity upon achieving viral suppression remains to be determined, as both viral factors (e.g. direct inhibition of Ca++-dependent granule release by viral proteins ) and accessory cells (e.g. IFN-α production by plasmacytoid DC ) can modulate NK cell-mediated cytotoxicity in HIV infection. However, the study of the functional implications of delayed reconstitution of the cytotoxic NK subset will need to be assessed in studies utilizing fresh PBMC, as repository cryopreserved samples were not suitable for this purpose due to limited cell number. Interestingly, following virus suppression, measured markers of activation within NK subsets (e.g. HLA-DR) did not significantly decrease, suggesting a more stable phenotype for NK cells in contrast to CD38 expression in CD8 T cells. This is particularly interesting in light of recent reports of rapid expansion of the CD56dim/CD16+ NK cell subset in response to acute HIV infection , followed by depletion of the same cells with disease progression: together, these findings suggest that NK cell sensitivity to viral load changes might be limited to acute infection, whereas in chronic infection changes follow slower kinetics. Levels of CD94 also did not change in NK cells, despite cross-sectional data reports indicating a direct association between CD94 and viral replication . As for PDC recovery, this discrepancy may be related to the staging of our cohort, which is composed of patients with a relatively preserved immune system (i.e., no measurable depletion of specific subsets such as CD94+ or activated NK cells).
A limitation of our analysis is the potential inflation of type 1 error due to multiple testing. In general, exploratory analyses require validation of results in larger data sets with selected variables. However, the internal consistency in our findings across multiple variables (PDC, NK and T-cell groupings) lends support to our conclusions and further suggests that early/late benefits of therapy involve changes in multiple variables.
The clinical implications of a delayed recovery of DC function and NK cell expansion remain to be elucidated with regard to common co-morbidities observed in the presence of ART. However, our data strongly supports the suggestion of the existence of delayed benefits of sustained suppressive therapy, subsequent to the rapid increase in CD4 cell count following viral suppression. Our results are also relevant to clinical applications that rely at least in part on NK cell-mediated responses (e.g. immunotherapy with IL-12, IL-15, IL-2, IFN-α, etc.), and suggest a benefit, to be confirmed in larger studies, for considering the recovery kinetics of NK frequencies when evaluating the potential success of such approaches.
Data in this manuscript were collected by the WIHS Collaborative Study Group with centers (Principal Investigators) at New York City/Bronx Consortium (Kathryn Anastos); Brooklyn, NY (Howard Minkoff); Washington DC Metropolitan Consortium (Mary Young); The Connie Wofsy Study Consortium of Northern California (Ruth Greenblatt); Los Angeles County/Southern California Consortium (Alexandra Levine); Chicago Consortium (Mardge Cohen); Data Coordinating Center (Stephen Gange). The WIHS is funded by the National Institute of Allergy and Infectious Diseases with supplemental funding from the National Cancer Institute, the National Institute on Drug Abuse (UO1-AI-35004, UO1-AI-31834, UO1-AI-34994, UO1-AI-34989, UO1-AI-34993, and UO1-AI-42590). Funding is also provided by the National Institute of Child Health and Human Development (UO1-CH-32632) and the National Center for Research Resources (MO1-RR-00071, MO1-RR-00079, MO1-RR-00083).
Data in this manuscript were collected by the MACS with centers (Principal Investigators) at The Johns Hopkins University Bloomberg School of Public Health (Joseph B. Margolick, Lisa Jacobson), Howard Brown Health Center and Northwestern University Medical School (John Phair), University of California, Los Angeles (Roger Detels, Beth Jamieson), and University of Pittsburgh (Charles Rinaldo). The MACS is funded by the National Institute of Allergy and Infectious Diseases, with additional supplemental funding from the National Cancer Institute. UO1-AI-35042, 5-MO1-RR-00722 (GCRC), UO1-AI-35043, UO1-AI-37984, UO1-AI-35039, UO1-AI-35040, UO1-AI-37613, UO1-AI-35041.
Website located at http://www.statepi.jhsph.edu/macs/macs.html.
Sponsorship: This study was supported by a grant from the National Institutes of Health AI51986, AI51225, AI47760, U01AI065279, the Philadelphia Foundation, and funds from the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health. A.S.F. was supported in part by a research award from the NIAID (# AI056983)
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