Significantly skewed memory CD8+ T cell subsets in HIV-1 infected infants during the first year of life

Clinical Immunology (2009) 130, 280–289

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

Significantly skewed memory CD8 + T cell subsets in HIV-1 infected infants during the first year of life☆ Nazma Mansoor a,1 , Brian Abel a,1 , Thomas J. Scriba a , Jane Hughes a , Marwou de Kock a , Michele Tameris a , Sylvia Mlenjeni a , Lea Denation a , Francesca Little b , Sebastian Gelderbloem a , Anthony Hawkridge a , W. Henry Boom c , Gilla Kaplan d , Gregory D. Hussey a , Willem A. Hanekom a,⁎ a

South African Tuberculosis Vaccine Initiative, Institute of Infectious Diseases and Molecular Medicine and School of Child and Adolescent Health, University of Cape Town, Anzio Road, Observatory, 7925, South Africa b Department of Statistical Sciences, University of Cape Town, Rondebosch, South Africa c Tuberculosis Research Unit, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, Ohio, USA d Public Health Research Institute, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA Received 7 May 2008; accepted with revision 10 September 2008 Available online 8 November 2008 KEYWORDS CD4; CD8; Memory; HIV-1; Infants

Abstract HIV-1 infection causes a severe T cell compromise; however, little is known about changes in naive, memory, effector and senescent T cell subsets during the first year of life. Tcell subsets were studied over the first year of life in blood from 3 infant cohorts: untreated HIVinfected, HIV-exposed but uninfected, and HIV-unexposed. In HIV-infected infants, the frequency of CCR7+CD45RA+ naive CD8+ T cells was significantly decreased, while the frequency of CCR7−CD45RA− effector memory CD8+ Tcells was increased, compared with the control cohorts. A larger population of CD8+ T cells in HIV-infected infants displayed a phenotype consistent with senescence. Differences in CD4+ Tcell subset frequencies were less pronounced, and no significant differences were observed between exposed and unexposed HIV-uninfected infants. We concluded that the proportion of naive, memory, effector and senescent CD8+ T cells during the first year of life is significantly altered by HIV-1 infection. © 2008 Elsevier Inc. All rights reserved.

Background

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This work was supported by the Elizabeth Glaser Pediatric AIDS Foundation and the TBRU of the NIH (NO1-AI70022). ⁎ Corresponding author. University of Cape Town Health Sciences, Anzio Road, Observatory, 7925, South Africa. Fax: +27 21 406 6081. E-mail address: [email protected] (W.A. Hanekom). 1 These authors contributed equally to this work.

Acquired cell-mediated immunity (CMI) is critical for control and clearance of many human infections [1]. The main mediators of CMI, CD4+ and CD8+ T cells, exist in the peripheral circulation either as naive or as antigen-experienced cells. The latter consist of heterogenous populations that may be classified according to immediate or delayed effector function, expression of homing receptors, cytokine se-

1521-6616/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2008.09.006

Skewed memory CD8+ T cells in HIV+ infants cretion profile, proliferation potential and longevity or survival characteristics [2,3]. Effector memory T cells (TEM) are characterised by the surface phenotype CD45RA−CCR7− CD62Llo. Following antigenic challenge, these cells migrate to inflamed tissues and demonstrate potent effector function. They proliferate poorly and are short-lived. TEM that re-express CD45RA are postulated to be terminally differentiated, and termed TEMRA [2,3]. By contrast, central memory T cells (TCM), characterised by the surface phenotype CD45RA−CCR7+ CD62Lhi, are long-lived in the host, home to lymph nodes, and proliferate efficiently to differentiate into effector cells [4–7]. Tcells may be further classified by expression of CD28, a costimulatory molecule which is associated with better functional capacity of the cells [8], and CD57, which is thought to be a marker of replicative senescence [9]. HIV infection results in severe disruption and depletion of the mucosal, lymphatic and peripheral T lymphocyte compartments. Systemic changes in the entire CD4+ and CD8+ T cell populations have been reported in HIV-1 infected adults [10–12], children [13–15], and infants [13,15,16]. An increased activation state of these T cells occurs during HIV infection [17,18] and may be directly linked to T cell differentiation [19]. Adult HIV-1 infection is characterised by a loss of naive CD4+ and CD8+ T cells and an expansion of CD27− and/or CD28− T cells, and similar observations have been reported for HIV-infected infants and children [14,20,21]. Further, a skewing in T cell phenotype from TCM to TEM (loss of CCR7 and CD62L expression) has been reported in HIV infected adults and children [22], while CCR7 expression has been shown to correlate inversely with HIV-1 viral load [23,24]. Limited knowledge exists about longitudinal phenotypic changes during the first year of life in peripheral blood T cells from antiretroviral treatment (ART)-naive infants perinatally infected with HIV-1, and from HIV-uninfected infants born to infected mothers. HIV-infected infants have a ten-fold increase in mortality, compared with HIV-unexposed infants. This is partly because infection prior to the full development of the immune system results in devastating destruction [25,26], and opportunistic infections may be lethal. Importantly, infants exposed to HIV-1 but not infected have mortality rates up to three times greater than those born to HIV-uninfected mothers [27]. The severity of maternal HIV disease has been linked to increased mortality and morbidity in uninfected infants. Although environmental factors may contribute significantly to this increased mortality, immunological factors may also contribute, as immunological differences between exposed, uninfected infants and unexposed infants have been shown: high level T cell activation has been reported in uninfected neonates born to infected mothers [13,28–30]. Our aim was to compare the phenotype and differentiation status of naive and memory T cell populations longitudinally during the first year of life in infants infected by HIV, exposed to HIV but not infected, and in unexposed infants. We show that CD8+ T cells derived from HIV-infected infants were markedly skewed towards an effector phenotype, rather than the predominant naive phenotype observed in uninfected infants. Moreover, a greater frequency of CD8+ T cells from HIV-infected infants displayed a senescent phenotype, compared with CD8+ T cells from uninfected infants.

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Methods Study population Infants born to HIV-infected and non-infected women from the Worcester region of the Western Cape, South Africa, were enrolled before the age of 3 months. HIV-1 infection status was determined in infants by PCR, and each was classified into 1 of 3 groups: non-HIV-infected born to a non-HIV-infected mother (HIV−), non-HIV-infected born to an HIV-infected mother (Exposed HIV−), or HIV-infected (HIV+). During the study period, roll-out of antiretroviral therapy (ART) as part of the mother-to-child HIV transmission prevention programme commenced. Toward the end of the project, roll-out of ART for treatment pediatric HIV-1 infection was also initiated. Infants who received ART were excluded from the analysis. The protocol was approved by the Research Ethics Committee of the University of Cape Town, and by the Institutional Review Board of the University of Medicine and Dentistry of New Jersey. Good clinical practice and ethical guidelines of the US Department of Health and Human Services and the South African Medical Research Council were followed in the conduct of the project; this included written informed parental consent.

Blood collection EDTA-anticoagulated peripheral blood was collected from the infants at 3, 6, 9, and 12 months of age. In addition to the immunological analysis reported here, a complete blood count and HIV-1 viral load (EasyQ HIV1, version1.2 bioMérieux NucliSens®) were performed at each time point.

Flow cytometric and hematology analysis Whole blood was transported at room temperature to the laboratory and processed within 4 hours of collection. Fifty microlitres of whole blood were incubated with fluorescenceconjugated antibodies against CD3-PE, CD4-allophycocyanin, CD8-PerCP, CD45-FITC, CD45RA-FITC, CCR7-PE, CD62L-PE, CD28-PE, and CD57-PE (all obtained from BD Biosciences), in different combinations, at room temperature for 30 min. Red cells were then lysed with FACS lysing solution (BD Biosciences), and white cells fixed in 1% para-formaldehyde. All cells were acquired on a FACSCalibur flow cytometer (BD Biosciences), equipped for four colour detection, using Cellquest (BD Biosciences). Analysis was completed using Flowjo (Treestar) software. A complete blood count was performed on all infants, allowing calculation of absolute numbers of CD4+ and CD8+ T cell subsets, as CD45 was used in flow cytometric panels to delineate total lymphocyte populations. A rigorous gating strategy was employed which was consistently applied to all participants. All antibodies in the panels were optimally titrated before use in the study.

Statistical considerations The nonparametric Kruskal–Wallis test, with Dunn's Multiple Comparison Test, was used to evaluate differences between study groups, and these statistical tests were performed using Prism 4.03 (GraphPad). A mixed effects linear regres-

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Table 1 Age

3 months

HIV status

HIV+

Exp. HIV−

HIV−

HIV+

Exp. HIV−

HIV−

HIV+

Exp. HIV−

HIV−

HIV+

Exp. HIV−

HIV−

Sample size, n Lymphocytes (×109/l), median (IQR) CD4 (lymphocytes/μl) median (IQR) CD8 (lymphocytes/μl) median (IQR) Viral load Log10 RNA copies/ml median (IQR)

16 8.1 (5.4–10.7)

25 6.4 (5.1–8.2)

23 6.4 (5.2–8.5)

11 5.8 (3.6–9.8)

18 5.6 (4.2–7.4)

21 7.6 (5.2–9.0)

9 5.7 (4.2–6.5)

17 6.5 (4.2–8.1)

22 6.1 (4.5–8.6)

5 7.7 (5.9–9.7)

14 5.1 (3.4–7.1)

19 6.5 (4.5–9.3)

a b c d e

6 months

9 months

2024 2519 2465 1469 1824 (1204–2840) (1940–2874) (2020–3032) (614–2562) (1383–2124)

2763 1266 (1603–3359) a (760–2038)

12 months

2211 2061 1872 1833 (1430–2319) (1611–2605) (1121–2659) (1394–2084)

2190 (1581–2877)

1569 732 1019 1460 640 1293 (1186–2938) (565–1270) b (778–1250) a (879–3057) (462–980) c, d (892–1567)

1464 766 (1270–2478) (461–1748)

1139 (906–1734)

2819 653 1162 (2127–3653) (515–977) b, e (869–2151)

5.7 (4.9–6.2)

4.9 (3.8–5.5)

N/A

4.9 (2.4–5.7)

N/A

N/A

5.6 (4.8–5.9)

N/A

N/A

N/A

N/A

N/A

HIV+ and HIV− (p b 0.05). HIV+ and Exp. HIV− (p b 0.001). HIV+ and Exp. HIV− (p b 0.01). Exp. HIV− and HIV− (p b 0.01). Exp. HIV− and HIV− (p b 0.05).

N. Mansoor et al.

Skewed memory CD8+ T cells in HIV+ infants sion model was used to compare the time profiles of the CD8 T cell populations (Supplementary Fig. 5). Maximum likelihood estimation was used so that the models coped with missing data arising from infant drop-out because observations at each time point influence estimates of treatment or other effects at every other time point due to the specification of the covariance pattern [31]. Where data was not normally distributed, values were log transformed.

Results Participant characteristics Among 298 infants born to HIV+ mothers enrolled, 20 were ultimately identified as HIV+ by a positive viral amplification test. The number of patients in the HIV+ group decreased gradually over the 12 month period (Table 1): 8 infants died early during the study, conducted during a period when ART was not routinely available in South Africa, and 4 infants were lost to follow-up. Three infants received ART, resulting in exclusion from analysis. Twenty five of the 278 noninfected infants born to HIV-infected mothers were included in the “Exposed HIV−” group. Eleven of these infants were lost to follow-up over their first year or life. Twenty three infants born to HIV-negative mothers (termed “HIV−”) were also enrolled. Of these, 4 were ultimately lost to follow-up. At 3 months of age, blood of only 16 of 20 HIV+ infants was examined.

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Total, CD4 and CD8 lymphocyte counts and viral load There were no differences in total lymphocyte counts between the groups, at all time points (Table 1). The frequencies of CD4+ lymphocytes in the HIV+ group were lower than in the other groups (Fig. 1A), while absolute numbers differed between the HIV+ and the HIV− groups at 6 months of age (Table 1). The frequencies of CD8+ T cells were significantly higher in the HIV+ group, compared with the other 2 groups (Fig. 1B), and absolute numbers were higher in HIV+ infants than in the other 2 groups at 3 months. As expected, the ratios of CD4+ to CD8+ Tcell frequencies were significantly reduced in the HIV+ group, compared with the other groups (Fig. 1C). Participants from the HIV+ group had a detectable plasma HIV-1 viral load, which showed a characteristic gradual decline over the first year of life (Table 1).

Memory phenotype of CD8+ T cells To investigate the memory phenotype of CD8+ T cells, we determined the expression of CD45RA and CCR7, or of CD45RA and CD62L, by flow cytometry (Fig. 2). The frequency of naive CD8+ T cells (CD8+ TNaive; CD45RA+CCR7+) was approximately 3-fold lower in HIV+ infants at 3 months of age, compared with infants from the other 2 groups (Fig. 2C; Supplementary Fig. 1A). This frequency decreased in all groups over the next 9 months, but HIV+ infants had consistently lower frequencies

Figure 1 Frequencies of total CD4+ Tcells (A) and CD8+ Tcells (B), among total Tcells and CD4/CD8 ratios (C), in the peripheral blood of infants from the HIV+, HIV− Exposed, or HIV− groups, over the first year of life, measured by flow cytometry. Boxes in boxplots represent medians and interquartile ranges, with the total range as the whiskers. Differences: ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001, Kruskal–Wallis test, with Dunn's Multiple Comparison Test.

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Figure 2 Frequencies and absolute numbers of CD8+ T cell memory populations defined by CD45RA and CCR7, or CD45RA and CD62L, expression in peripheral blood of the 3 groups of infants, measured by flow cytometry. (A) Gating strategy: lymphocytes were selected for by setting a lymphocyte gate according to FSC and SSC profiles, and CD8 T cells included by selecting CD8+ cells. The phenotype of CD8+ T cells was assessed by gating on the CD8+ T cells, and analysing either CD45RA/CCR7 staining or CD45RA/CD62L staining. (B) Dotplots shown for CCR7 and CD45RA staining from 3 representative infants. (C–D) Median frequencies of each subset. (E–F) Median absolute numbers of each subset. CD62L/CD45RA staining is only depicted when this differed from CCR7/CD45RA staining. Differences: ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001, Kruskal–Wallis test, with Dunn's Multiple Comparison Test. The distributions are shown in Supplementary Figures 1 and 2.

Skewed memory CD8+ T cells in HIV+ infants throughout (Fig. 2C; Supplementary Fig. 1A). Differences between groups were not as prominent when the absolute number of CD8 + T Naive cells was evaluated (Fig. 2E; Supplementary Fig. 2A); however, the HIV− group had an increased CD8+ TNaive subset, compared with the Exposed HIV− group, at 6 and 12 months. Very similar patterns were observed when CD62L was substituted for CCR7 in the analysis (data not shown). The frequency of effector memory CD8+ T cells (CD8 TEM; CD45RA−CCR7−) was significantly higher in the HIV+ group at all time points, compared with the other 2 groups, and increased over time in all groups (Fig. 2D, left panel; Supplementary Fig. 1B). The absolute numbers of CD8+ TEM cells followed a similar pattern (Fig. 2F, left panel; Supple-

285 mentary Fig. 2B). Mixed effects linear regression modelling of this data also demonstrated significant differences in the naive and effector CD8 T cell subsets between the HIVinfected group and the control groups (Supplementary Fig. 5). At 3 months of age, HIV+ infants also had a significantly higher frequency of CD8+ TEM cells that re-expressed CD45RA (TEM45RA), compared with the Exposed HIV− group (Fig. 2D, middle panel; Supplementary Fig. 1C). At later time points there were no differences in the frequency of these cells for the 3 groups, but absolute numbers were higher in HIV+ infants throughout the first year of life (Fig. 2F, middle panel; Supplementary Fig. 2C). Again, when CD62L was substituted for CCR7, similar patterns were observed (data not shown).

Figure 3 Frequencies and absolute numbers of CD4+ T cell memory populations defined by CD45RA and CCR7, or CD45RA and CD62L, expression in peripheral blood of the 3 groups of infants, measured by flow cytometry. (A) Dotplots from 3 representative infants. (B– C) Median frequencies of each subset. (D–E) Median absolute numbers of each subset. CD62L/CD45RA staining is only depicted when this differed from CCR7/CD45RA staining. Gating strategy: lymphocytes were selected for by setting a lymphocyte gate according to FSC and SSC profiles, and CD8 T cells included by selecting CD8+ cells. The phenotype of CD8+ T cells was assessed by gating on the CD8+ T cells, and analysing either CD45RA/CCR7 staining or CD45RA/CD62L staining. Differences: ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001, Kruskal–Wallis test, with Dunn's Multiple Comparison Test. The distributions are shown in Supplementary Figures 3 and 4.

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The frequencies of central memory CD8+ T cells (TCM) in the 3 groups, were extremely low when CCR7 was used to define this subset (CD45RA−CCR7+) (Fig. 2D, right panel; Supplementary Fig. 1D). When CD62L was used instead of CCR7 to define TCM, increased frequencies and absolute numbers of CD8 TCM cells could be detected in the HIV+ group, compared with the other 2 groups, over the first year of life (Fig. 2D, right panel; Supplementary Figs. 1D and 2D).

Memory phenotype of CD4+ T cells Phenotypic differences in CD4+ T cells were far less pronounced than in the CD8+ T cells, with only occasional differences that were significant between HIV+ and the HIV− infants (Fig. 3; Supplementary Figs. 3 and 4). Interestingly, CCR7 appeared to be a better marker to differentiate subsets, compared with CD62L. There were no dramatic differences between Exposed HIV− infants and HIV− infants in any of the CD4+ T cell subsets.

Expression of CD28 and CD57 The absolute number of CD8+ T cells expressing the senescence marker CD57 was higher in the HIV+ group at most time points, compared with the HIV− Exposed group. Surprisingly, expression of this marker tended to be higher in HIV− infants, compared with Exposed HIV− infants (Fig. 4A). The absolute number of CD8+ T cells not expressing the costimulatory molecule CD28 was markedly higher in HIV+ infants, compared with the others, at most time points (Fig. 4B). CD57 and CD28 expression on CD4+ T cells was not different between the groups (data not shown).

Discussion This is a comprehensive report of changes in memory CD4+ and CD8+ T cell subsets in untreated HIV-infected infants (HIV+), which were compared with HIV-exposed but uninfected infants (Exposed HIV−), and with HIV-unexposed infants (HIV−). The data show skewing of predominantly CD8+ T cell populations in HIV-infected infants, compared with the other infant groups. As early as 3 months after birth, the frequency of naive CD8+ T cells was markedly reduced in HIV+ infants. In HIVinfected adults, loss of naive T cells is the result of increased immune differentiation, driven by immune activation, which is characteristic of HIV-1 infection [32–35]. In adults, the naive CD4+ T cell compartment is more severely depleted than the CD8 compartment [11]. We found that loss of naive CD8+ T cells was more prominent in infants, whereas McCloskey et al. reported a significant loss of naive CD4 and CD8 T cells in the first year of life [14], and Gallagher et al. demonstrated a significant decrease in naive CD8 T cells already at 6 weeks of age [36]. Naive T cell loss perturbs T cell homeostasis and consequently, thymic output [11]. Clinical studies have shown that thymic dysfunction is associated with rapid progression of disease in perinatally infected infants [25,37,38]. The numbers and proportions of effector memory CD8+ T cells (TEM) were significantly increased at all time points in

Figure 4 Absolute numbers of CD8+ T cells expressing CD57 (A) and CD28 (B) in peripheral blood of the 3 infant groups, measured by flow cytometry. Boxes in boxplots represent medians and interquartile ranges, with the total range as the whiskers. Gating strategy: lymphocytes were selected for by setting a lymphocyte gate according to FSC and SSC profiles, and CD8 T cells included by selecting CD8+ cells. The phenotype of CD8+ T cells was assessed by gating on the CD8+ T cells, and analysing either CD57 expression or CD28 expression. Differences: ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001, Kruskal–Wallis test, with Dunn's Multiple Comparison Test.

HIV-infected infants. Such maturational skewing is well described in HIV-infected adults [22,39]. The clonal expansion of antigen-specific effector T cells is important for immunity against primary infections, whereas the development and maintenance of memory T cell populations is critical for protective immunity against re-infections. HIV-driven, nonspecific maturation may thus be particularly detrimental to the development of immunological memory in infants. Our results contrast with findings of Jordan et al., who failed to show changes in the distribution of memory and effector T cell populations in children aged 2–14 [22]; however, the latter study investigated very small cohorts of 10 HIV-infected and 4 uninfected children, and all HIV-infected children had received ART. The CD8+ TEMRA population, effector memory cells that have re-expressed CD45RA, was also increased in HIV+ infants, compared with Exposed HIV− infants. This increase has also been reported in adult HIV-1 infection [22], and may reflect chronic immune activation that drives cells into a state of terminal differentiation.

Skewed memory CD8+ T cells in HIV+ infants Expression of the costimulatory molecule CD28 and of the senescence marker CD57 was also different between our infant groups. CD8+ T cells of HIV-infected infants displayed a phenotype consistent with senescence, with low CD28 and elevated CD57 expression. HIV-specific CD8+ T cells detected very early after birth, in in utero-infected infants [40–42], may therefore be prone to senescence, and be less functional, especially in the absence of specific CD4+ T cell help [40,43,44]. These phenotypes have been shown to be prevalent in HIV− infected adults with high viremia, where the senescent T cells have also shown reduced proliferative capacity [9,45]. Similarly, adults [46–50] and children [14,21] with high viral replication characteristically have CD8+ T cell downregulation of CD28, which leads to dysregulation of normal T cell function. Interestingly, we found that HIV-exposed but uninfected infants expressed significantly lower levels of CD57 than HIV-1 unexposed infants. This result was surprising, since increased immune activation is expected in exposed HIV-uninfected infants [13,28–30], which should result in greater T cell senescence, compared with unexposed infants. Further study would be necessary to clarify this finding. CCR7 and CD62L expression are often used interchangeably with CD45RA to define memory subsets. We showed that in infants, significant differences between the expression of these 2 markers do exist. For example, populations of central memory CD8+ T cells were detectable only when CD62L was used, and CCR7 was a better marker to define effector memory CD4+ T cells. We do not know why these markers were not equivalently useful to delineate T cell subsets in infants, but this warrants further inspection, and these differences should be taken into account when measuring these subsets in infant studies. The significant drop-out rates, primarily due to death of HIV-infected infants later in the first year of life, constituted a limitation of our study. These death rates were consistent with those observed in other cohorts of African infants not on antiretroviral therapy [26]. We therefore also assessed our results with maximum likelihood mixed effects linear regression modelling, which copes with loss to follow-up. These models showed results that were similar to those without applying the models. In turn, sensitivity of the models was tested by applying three different methods of imputation of missing data and comparing this with analysis of just those patients who had complete data: there was excellent agreement between the results of all four analyses (data not shown). Invariably, results from HIV-uninfected infants remained different from HIV-infected infants. Additionally, we did not prospectively address co-variates that could have had an effect on infant immunity, such as maternal viral load and immune status, maternal therapy, infant nutritional status, and additional infections in infants. Regardless, the T cell phenotype in HIV-infected infants during the first year of life remained strikingly different to the control groups. We therefore believe that this study's results are unique and completely novel, particularly as the current routine availability of antiretroviral therapy precludes this kind of study from ever being repeated. To conclude, the changes in T cell phenotype reported here suggest a gross dysfunction in T cell development and maturation in HIV-infected infants. A reduction of CD4+ T cells and increase in CD8+ T cells, with lower CD4/CD8 ratios,

287 in HIV-infected infants is well documented [25]; however, we now demonstrate, in addition, that subsets of these cells are also significantly altered. Furthermore, the observation that total lymphocyte counts between the 3 groups are similar indicates that there is a re-distribution of the existing T cells from the CD4 to the CD8 compartment, and from naive Tcells to effector Tcells. In contrast, very little difference between HIV exposed but uninfected and HIV-1 unexposed infants was observed. The findings from this study indicate that T cell memory subsets are severely disrupted in HIV-infected infants, and this may have dire implications on the efficacy of childhood vaccination, and may contribute to the increased morbidity and mortality of infected infants. Our novel results also underscore the positive impact that early antiretroviral therapy may have in reversing the abnormal immune profile. This hypothesis is substantiated by recent findings of markedly improved clinical outcome of infants who have started antiretroviral therapy at 6 weeks of age, or at the earliest time point after HIV diagnosis, compared with infants who have started therapy at later ages [51]. Conflict of interest The authors declare no conflicts of interest.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clim.2008.09.006.

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