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.
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:
Two models were fitted.
1. Primary analysis:
2. Exploratory analysis:
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.
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.
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