Thirty-four control children with 1 study visit each were enrolled. Forty-seven percent of these children were female, and their median age was 24.2 months (Table 1). As expected, the control children had a significantly higher median CD4+ T-cell percentage (33.4%, range, 17%–48%) (Fig. 1A) and lower CD8+ T-cell percentage (23.6%, range, 0.4%–35%) (Fig. 1B) than HIV-infected children, consistent with few, if any, of the control children being HIV infected.
T-Cell Subsets at Baseline
Before HAART initiation, EM cells constituted the largest mean percentage of CD4+ T cells in HIV-infected children (37.2%), followed by effector (27.4%), naive (19.4%) and central memory CD4+ T cells (4.6%) after adjusting for age and sex (Figs. 2A, B; see Supplemental Table S2a, Supplemental Digital Content, http://links.lww.com/QAI/A566). In contrast, naive cells were the largest subset in control children and comprised one-third of CD4+ T cells. Although statistically significant differences in CD4+ T-cell subsets between age categories and sex were observed among HIV-infected and control children, these differences were less than 1% (see Supplemental Figure S3 and Supplemental Table S3a, Supplemental Digital Content, http://links.lww.com/QAI/A566).
A majority of CD8+ T cells were of the effector phenotype in both HIV-infected (46.6%) and control children (50.7%) (Fig. 3; see Supplemental Table S2b, Supplemental Digital Content, http://links.lww.com/QAI/A566). Although naive cells comprised the next largest percentage of CD8+ T cells in control children (24.2%), EM CD8+ T cells were the second largest percentage in HIV-infected children (39.8%). Central memory cells constituted a substantially smaller percentage of CD8+ T cells in the circulation of both HIV-infected and control children at less than 0.1%, reflecting recruitment to the bone marrow.22
Consistent with increased cellular immune activation during HIV infection, the percentages of activated CD4+ and CD8+ T cells in HIV-infected children were 3- and 2-fold higher, respectively, than those of control children (Fig. 4; see Supplemental Figure S5, Supplemental Digital Content, http://links.lww.com/QAI/A566). Additionally, the memory capacity of CD8+ T cells, as measured by expression of IL-7Rα, was 27 percentage points lower in HIV-infected children than in control children. Similar to CD4+ T cells, differences among CD8+ T-cell subsets by age categories and sex were less than 1% for HIV-infected and control children except for naive CD8+ T cells, which were 2.75% (95% CI: 0.16 to 8.51) higher in female compared with male control children (see Supplemental Figure S4 and Supplemental Table S3b, Supplemental Digital Content, http://links.lww.com/QAI/A566).
Changes in CD4+ T-Cell Subsets After HAART
Almost 3-quarters (73%) of HIV-infected children achieved at least 20% CD4+ T cells within 6 months of HAART initiation. After adjusting for age and sex, the mean percentage of total CD4+ T cells increased significantly within 3 months of starting HAART and continued to increase through 9 months of treatment, reaching 27.5% of the T-cell population, after which no statistically significant changes occurred (Fig. 1C; see Supplemental Table S2a, Supplemental Digital Content, http://links.lww.com/QAI/A566). The rate at which total CD4+ T-cell percentages increased from baseline to 9 months after HAART did not differ between age groups (see Supplemental Figure S6a, Supplemental Digital Content, http://links.lww.com/QAI/A566).
During the first 6 months of HAART, naive and central memory CD4+ T cells increased significantly and maintained percentages similar to those of control children for the remainder of study follow-up (Fig. 2B). There were no differences in the rates of naive and central memory CD4+ T-cell increases after HAART initiation between age groups (see Supplemental Figures S6b and S6c, Supplemental Digital Content, http://links.lww.com/QAI/A566). EM cells decreased in HIV-infected children on HAART, although not to percentages observed in control children (Fig. 2A), and the rate of decrease did not differ with age (see Supplemental Figure S6d, Supplemental Digital Content, http://links.lww.com/QAI/A566). Effector cell percentages remained similar to those of control children (Fig. 2A). Changes in the mean absolute counts of CD4+ T-cell subsets showed patterns similar to CD4+ T-cell subset percentages after children began HAART (see Supplemental Table S4a, Supplemental Digital Content, http://links.lww.com/QAI/A566). These findings suggest increases in CD4+ T cells were driven primarily by gains in naive CD4+ T cells.
Changes in CD8+ T-Cell Subsets After HAART
Although the mean percentage of total CD8+ T cells in HIV-infected children decreased significantly after 6 months of HAART, this percentage remained higher (34%) than in control children (24%) for the remainder of follow-up (Fig. 1C). As observed among CD4+ T-cell subsets, naive CD8+ T-cell percentages in HIV-infected children increased significantly after HAART initiation but maintained mean percentages between 10% and 15% over study follow-up, slightly lower than 24% of CD8+ T cells among control children (Fig. 3; see Supplemental Table S2b, Supplemental Digital Content, http://links.lww.com/QAI/A566). After HAART, percentages of central memory and effector cells remained similar in HIV-infected and control children, comprising <0.1% and 50%, respectively, of the total CD8+ T-cell population. In contrast, the mean EM CD8+ T-cell percentage in HIV-infected children decreased significantly during HAART but remained higher than that observed among control children, with EM T cells largely responsible for the decline in total CD8+ T cells. Similar changes in absolute CD8+ T-cell counts were observed in response to HAART (see Supplemental Table S4b, Supplemental Digital Content, http://links.lww.com/QAI/A566). Sex and age were not associated with differences in the rates of change in any CD8+ T-cell subsets among HIV-infected children (see Supplemental Figures S7a-S7e, Supplemental Digital Content, http://links.lww.com/QAI/A566).
Cellular Activation and Memory Capacity
Activated CD4+ T cells decreased to less than half of baseline levels 6 months after starting HAART and approached levels observed in control children (Fig. 4). Activated CD8+ T cells demonstrated a similar decline after HAART initiation but remained slightly higher than was observed in control children (Fig. 4; see Supplemental Table S3b, Supplemental Digital Content, http://links.lww.com/QAI/A566). Memory capacity of CD8+ T cells, as measured by IL-7Rα expression, more than doubled after 6 months of HAART, but remained lower than the mean percentage observed in control children. Sex and age were not associated with changes in activated CD4+ or CD8+ T-cell subsets or IL-7Rα expression by CD8+ T cells (see Supplemental Figures S8a-8c and Supplemental Table S3, Supplemental Digital Content, http://links.lww.com/QAI/A566).
The cellular immune activation status and levels of memory T-cell subsets in HIV-infected Zambian children were substantially altered after starting HAART. As expected, untreated HIV-infected children had lower percentages of naive T cells and higher percentages of EM and activated T cells than control children, consistent with changes in T-cell composition during chronic HIV infection and persistent immune activation.23,24 HAART significantly reduced levels of cellular immune activation and EM CD4+ T cells, and promoted reconstitution of naive T cells and IL-7Rα-expressing CD8+ T cells. Interestingly, CD4+ and CD8+ effector T-cell percentages did not differ between HIV-infected and control children. However, increased CD8+ effector T-cell percentages at HAART initiation were significantly associated with an increased risk of mortality in this cohort of children,25 possibly reflecting T-cell exhaustion and loss of polyfunctional cytokine responses commonly observed in HIV-infected individuals.26
Most HIV-infected children require lifelong treatment with HAART, as the major barrier to cure is the development of a cellular reservoir that harbors latent infectious virus.27 Current approaches for HIV cure include purging the latent reservoir through re-activation of resting cells28; thus, understanding both the relative contributions and maximum counts and percentages of cellular subsets is important for assessing reservoir size, calculating HIV clearance kinetics, and estimating the potential impact of cure strategies. The cellular distribution of the proviral reservoir in HIV-infected children has not been thoroughly investigated, but evidence suggests it is comprised of resting memory CD4+ T cells, including long-lived central memory T cells29 that predominantly home to lymphoid organs.30 Whether the increase in central memory T cells observed in Zambian children represents increased memory cell levels that typically develop with aging or redistribution of central memory T cells in circulating blood relative to those sequestered in secondary lymphoid organs is unclear. In healthy children younger than 10 years, the proportions of naive T cells decrease as the immune system matures and encounters antigens, whereas memory T-cell proportions increase.5 Since control children were enrolled for only a single visit, our ability to differentiate between phenomena associated with aging, such as antigen exposure, and HAART duration was limited when comparing cellular subsets among HIV-infected children. We attempted to control for this by adjusting for age in statistical models and believe a large part of the considerable changes in T-cell subsets can be attributed to HAART, as observed upon stratification of cell subset trajectories by age and sex. Additionally, the proportion of central memory T cells that developed in response to HIV and non-HIV antigens was not determined.
This is one of the largest studies to assess long-term changes in T-cell subsets in HIV-infected children, particularly in sub-Saharan Africa. Of 13 studies assessing cellular subsets among HIV-infected children, 10 included fewer than 50 children.2,11–14,31–38 Only 2 previous studies assessed T-cell activation status among HIV-infected children before HAART initiation in sub-Saharan Africa, but these studies used cross-sectional study designs.14,31 After HAART initiation, T-cell subsets of European and American HIV-infected children demonstrated patterns of change similar to those of Zambian children.32,38 Among European children starting HAART, both naive and total memory CD4+ T cells increased within 3 months of HAART, but only naive CD4+ T cells sustained increases after 12 months.32 Similarly, naive CD4+ and CD8+ T-cell percentages increased after 144 weeks of HAART in American children, whereas memory cell percentages decreased or remained unchanged.38 The shift in T-cell composition after HAART initiation, most notably the increase in naive T cells and reduction in EM T cells, is consistent with control of HIV-mediated T-cell stimulation and differentiation.39
Although the pattern of T-cell subset changes after HAART initiation was similar between HIV-infected children in Zambia and those in developed countries, Zambian children had lower levels of naive T cells and higher levels of EM T cells. Among HIV-infected American and Spanish children, for example, naive cells constituted 55%–56% and 30%–38% of the CD4+ and CD8+ T-cell populations, respectively, before HAART initiation,33,37,38 whereas the corresponding percentages in HIV-infected Zambian children constituted only 19% and 4%. These differences are underscored by the older median ages of 7–10 years in the American and Spanish cohorts, as older age typically is associated with lower naive T-cell percentages. Similarly, control Zambian children had lower proportions of CD4+ and CD8+ naive T cells than HIV-uninfected American, Brazilian, and Cameroonian children.11,36,40 Naive T cells comprised 54% of CD4+ and 49% of CD8+ T cells in 12- to 24-month old Cameroonian children40 compared with only 34% and 24% in Zambian children. These differences are more pronounced upon comparison with HIV-uninfected American children who had CD4+ and CD8+ T-cell percentages of 75% and 58%, respectively,36 possibly reflecting more frequent exposures to pathogens that induce cellular differentiation in the Zambian children.
A limitation of this work was the inability to measure T-cell subsets for all children at later study visits due to short duration of follow-up. We performed sensitivity analyses to determine the extent to which estimates change after restricting analyses to HIV-infected children with at least 12 months of follow-up and no more than 1 missing visit, resulting in 87 HIV-infected children and 34 control children. However, the estimates did not differ between the original and sensitivity analyses. Moreover, inferences regarding differences between HIV-infected and control children remained the same. Although we were unable to adjust for HIV viral load changes after HAART initiation, the decline in the percentages of CD4+ EM cells and cellular immune activation is consistent with decreased HIV stimulation as EM cells migrate to nonlymphoid tissues to quickly respond to cognate antigens41 and turnover rapidly.42
HAART significantly reduced levels of cellular immune activation and EM CD4+ T cells, and promoted reconstitution of naive T cells and IL-7Rα-expressing CD8+ T cells. To the best of our knowledge, this is the first study to report the relative proportions and sizes of T-cell subsets, specifically the central memory CD4+ T-cell population, in HIV-infected children from sub-Saharan Africa before and after HAART initiation.
The authors thank the participants of this study and the clinicians and study staff who collected data and cared for the participating children. They also thank Fred Menendez and Tricia Niles for their assistance with optimizing the flow cytometry.
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HIV; immune reconstitution; T cells; antiretroviral therapy; children
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