In early pregnancy, CD4+ and CD8+ Treg subpopulations were generally increased in HIV-infected compared with uninfected women. This included CD4+CD25+FoxP3+, CD8+CD25+FoxP3+, CD4+TGFβ+ and CD4+IL10+ Treg and it was most likely an effect of HIV infection, which is in agreement with previous findings in non-pregnant HIV-infected adults 
. Our findings, however, contrast with a recently published study of pregnant women, which showed lower CD4+CD25+FoxP3+CD127low
Treg frequencies in HIV-infected compared with uninfected women during the 2nd trimester of pregnancy
. The duration of HAART of the HIV-infected subjects differed between the two studies, with most of our subjects starting HAART during pregnancy, while in the study by Kolte et al., most subjects were on HAART before pregnancy. Since HAART has been previously shown to decrease the frequency of circulating Treg in HIV-infected individuals 
, the difference in the duration of HAART may explain the difference between the two study results.
More importantly, in late pregnancy several Treg subpopulations, including CD4+CTLA4+%, CD8+CTLA4+% and CD8+TGFβ+% were lower in HIV-infected compared with uninfected pregnant women, and only CD4+IL10+% remained higher in HIV-infected women. Furthermore, no significant increases in Treg% were detected in HIV-infected women between early and late gestation, whereas in uninfected women, we observed a marginally significant increase of CD8+CD25+FoxP3+% and CD4+TGFβ+% over the course of pregnancy. Previous studies also observed Treg increases during normal pregnancy and ascribed them a role in inducing maternal tolerance of the fetus 
. Blois et al. also determined that CD8+ Treg had a pivotal role as a mediator of the anti-abortogenic effect of progesterone in mice 
. The lack of Treg% upregulation during late gestation in HIV-infected pregnant women may lead to a premature loss of fetal tolerance and premature delivery. In fact, HIV-infected women have a higher incidence of premature delivery compared with the general population 
. This was sometimes, but not always, correlated with the use of protease inhibitor-containing HAART 
. It is conceivable that the use of HAART may exert a downregulatory pressure on Treg subpopulations similar to what was described in non-pregnant HIV-infected adults 
. Furthermore, we found an association, albeit weak, between plasma HIV load and CD4+FoxP3+% in early pregnancy, which also supports the notion that active HIV replication may increase Treg%, while HAART-mediated control of HIV replication may have the opposite effect. The hypothesis that lack of upregulation of specific Treg subpopulations in late pregnancy is associated with premature delivery needs to be further confirmed in larger studies, because it may lead to new therapeutic interventions.
To gather information on the Treg functionality, we studied the relationship of Treg% with VZV-specific lymphocyte proliferation. We chose VZV CMI as a representative measure because of the high prevalence of VZV infection in the US adult population (>90%) and the well-demonstrated role of VZV CMI in protection against VZV reactivation and herpes zoster 
. We found that higher CD4+FoxP3+%, CD8+FoxP3+% and CD8+TGFβ+% were significantly associated with lower VZV-specific proliferation in HIV-infected pregnant women. Interestingly, not all Treg subpopulations were associated with decreased CMI in HIV-infected pregnant women, which suggests different specificities of Treg subpopulations. Furthermore, Treg% were not associated with lower CMI in HIV-uninfected pregnant women. It is possible that the association between high Treg% and low CMI in the context of HIV infection is indirect and that both phenomena are linked to active HIV replication. However, it is also conceivable that the higher frequency of Treg in HIV-infected women during early pregnancy allowed us to detect an inhibitory effect on CMI only in this group of participants. This may be due to a higher ratio between Treg and effector memory T cells in HIV-infected compared with uninfected women and/or broader antigen specificity of certain CD4+FoxP3+, CD8+FoxP3+ and CD8+ TGFβ+ Treg in HIV-infected women.
Among the Treg markers used in this study, CTLA4 seemed to behave differently from others. CD4+CTLA4+% and CD8+CTLA4+% were higher in HIV-uninfected compared with infected pregnant women and this difference reached statistical significance in late pregnancy, whereas most other Treg% were higher in HIV-infected women in early pregnancy. Furthermore, soluble CTLA4 plasma concentrations were significantly higher in HIV-uninfected women both in early and late gestation, whereas IL10 plasma concentrations were higher in HIV-infected women. CTLA4 is expressed both by Treg and memory effector T cells and in both circumstances contributes to down-modulation of the cellular immune response. Memory effector T cells quickly upregulate CTLA4 expression upon cognate antigenic stimulation. Binding of CD80 or CD86 ligands to CTLA4 on effector memory T cells decreases their IL2 production and cell cycle progression 
. In counterpart, CTLA4 expression on Treg allows them to block the activation of naïve T cells. In this study, we did not differentiate between effector memory and Treg expression of CTLA4 
. It is conceivable that some of the CTLA4+ T cells measured in this study were effector memory cells expressing CTLA4, which may explain the different dynamics of CTLA4+ T cells compared with other Treg subpopulations. Conversely, CTLA4+ T cells may represent bonafide Treg and their behavior in pregnancy differs from that of other Treg subpopulations. Further studies are needed to elucidate the role of CTLA4 expression in maintenance of pregnancy and the potential role that CTLA4Ig might play as a therapeutic intervention.
We found that HIV-infected women had elevated CD4+CD38+HLADR+% and CD8+CD38+HLADR+% Tact compared with uninfected women, which was in agreement with previous observations in non-pregnant individuals 
. Insufficient T cell regulation was invoked as a potential contributor to high levels of T cell activation in HIV-infected individuals 
. In this study, we found negative associations between Treg% and Tact%, which reached statistical significance in uninfected pregnant women and were only marginally significant in HIV-infected participants. This is unlikely to be due to deficient Treg function in the context of HIV infection, because we also observed a strong negative association between Treg% and lymphocyte proliferation in HIV-infected women. A more likely explanation is that in the context of HIV infection Treg and Tact share some of the pathophysiologic mechanisms, partially masking the inhibitory Treg effect on Tact. This would be in agreement with the model proposed by Boasso et al. whereby activation of plasmacytoid dendritic cells by HIV infection would increase both regulation and activation 
Plasma concentrations of inflammatory factors, Th1, Th2 and Treg cytokines were consistently higher throughout pregnancy in HIV-infected compared with uninfected pregnant women. High levels of inflammatory factors in non-pregnant HIV-infected adults were previously shown to correlate with increased risk of non-AIDS severe adverse events, such as cardiovascular, hepatic and renal dysfunctions 
. In pregnancy, high inflammatory factors may lead to obstetric morbid conditions, such as premature delivery, pre-eclampsia and liver disorders 
. In HIV-infected pregnant women, like in other HIV-infected non-pregnant adults 
, inflammatory factors, Th1, Th2 and Treg cytokines were positively correlated with Treg%, probably reflecting a common pathophysiologic mechanism. However, in uninfected pregnant women, CD8+FoxP3+% were negatively associated with inflammatory markers and positively associated with Treg cytokines signaling that Treg interactions may differ between HIV-infected and uninfected pregnant women. It is important to confirm these findings and further characterize Treg subpopulations and inflammatory factors that play a direct role in the outcome of pregnancies, which may provide new avenues for therapeutic interventions.