Although several previous studies have indicated that Treg can serve as a potential target cell population for HIV-1 infection in vivo, Treg and HIV interactions remain poorly understood. We therefore used a more systematic approach to study Treg susceptibility to infection, comparing lab strains with R5 or X4 tropism, as well as confirming the data with primary isolates. Collectively, our data convincingly demonstrate that Treg are infected in vitro by infectious HIV viruses, but their susceptibility depends on the tropism of the virus. Treg were less susceptible to R5 viruses, using both lab strains and primary isolates, compared to Teff, whereas Treg were more susceptible to X4 viruses.
Importantly, we have shown that Treg were less susceptible to several primary R5 isolates from different clades as well as to R5 lab strains, although the levels of infection vary greatly depending on the virus used. These data suggest that Treg could be relatively protected from HIV infection during the acute phase of mucosal transmission, which is thought to predominantly involve R5 viruses (26
). Of note, Estes et al. showed that 13% of lymph node FOXP3+
cells were infected 2 weeks after vaginal infection by the R5 SIVmac251
. Although a direct comparison of the infection levels to non-Treg was not made, such reported frequency of Treg infection is lower than that reported by the same group for lamina propria CD4+
cells (30% cells harboring SIV RNA) in a similar infection model (21
). These results suggest that the decreased susceptibility to R5 HIV infection that we found in vitro reflects what happens in vivo. This important hypothesis will have to be confirmed in future studies of acute SIV infection. Our results are also in agreement with a previous study that used an R5 lab strain to infect memory and naïve Treg, as well as memory non-Treg (3
). However, conflicting data have also been reported: i.e., that Treg are more susceptible to HIV infection and are more prone to HIV-induced death than memory non-Treg when high virus concentrations (MOI of 5) are used (23
). Such discrepant results could be explained by the fact that many critical experimental parameters differed in these studies. Indeed, activation methods, cell purification techniques, and virus concentrations were different between the latter study and ours. However, it should be noted that the type of cell activation may not represent the most critical parameter, because we did not find differences in HIV susceptibility when cells were stimulated with PHA and IL-2 instead of anti-CD3/CD28 beads. Discrepant results were also found when interactions between FOXP3 and HIV LTR were studied in FOXP3-transfected CD4+
T cells. For instance, one study showed that FOXP3 limits HIV-1 LTR and human T-cell leukemia virus type 1 transcription by interfering with activation of NFAT and CREB pathways. The inhibitory effect was not absolute, and low-level LTR activation persisted (12
). In contrast, Cron et al. (28
) reported increased LTR activity in FOXP3-expressing cells, using a different LTR from the one used in the aforementioned study. Because the LTR sequence can vary significantly between HIV strains (30
), it is possible that FOXP3 interactions with distinct HIV LTRs may have different functional consequences, which would explain this apparent discrepancy.
In contrast to infection with R5 viruses, Treg were more susceptible to infection by X4 viruses than Teff. To our knowledge, our study is the first one to assess primary Treg susceptibility to infection by full-length X4 viruses. Importantly, similar results were obtained with primary isolates or the lab strain NL4-3. Our results are also supported by data provided by other experimental systems. Indeed, in vivo infection of humanized DKO mice by the highly pathogenic dualtropic R3A HIV isolate led to an increased level of infection in Treg compared to non-Treg (15
). Furthermore, overexpression of FOXP3 in CD4+
T cells enhanced NL4-3 LTR activity, by modulating chromatin structure as well as enhancing NF-κB activity (14
Variations in levels of p24Gag
production by Treg and Teff could arise from differences in multiple steps of the virus life cycle, as p24Gag
release is at the end of a complex developmental program that takes place in a cycling cell. To identify the potential mechanisms underlying the differences between Treg and Teff, we first analyzed the expression of HIV receptors by these two cell subsets. Similar expression levels of CD4 and a trend toward higher levels of CCR5 per cell were found in activated Treg compared with activated Teff, a result in agreement with previous studies (8
). Treg produced similar levels of CCR5 ligands to Teff. Our data thus suggest that an altered balance of HIV receptors and CCR5 ligands is not the main mechanism explaining decreased Treg susceptibility to R5 HIV. Similar differences between Treg and Teff were found when infection levels were analyzed at an early time point postinfection. One potential explanation of our data could be the decreased activation state of Treg compared to Teff, as the activation state of the target cell markedly affects the efficiency of the early steps of HIV replication (34
). The majority of the Treg underwent proliferation during the 7-day culture (Fig. ), but they clearly proliferated less than Teff. Therefore, to better understand the contribution of decreased proliferation to decreased infection of Treg by R5 viruses, we analyzed the percentage of p24Gag+
cells in the highly proliferating Treg and Teff. Similar levels of infection were found, a result that suggests the absence of intrinsic differences in the susceptibility of proliferating Treg and Teff to this virus. Considering all our data together, decreased proliferative capacity of Treg likely constitutes a major mechanism explaining their decreased susceptibility to infection by R5 viruses.
Despite the fact that Treg proliferated less than Teff, they were more infected than Teff, showing that proliferation is not the only mechanism regulating Treg infection by X4 viruses. Interestingly, a higher percentage of infection was observed in the highly proliferating Treg compared to that in the proliferating Teff, suggesting that some cellular factors may potentiate X4 replication in Treg. Although not statistically significant, there was a trend toward Treg expressing higher levels of CXCR4 than Teff at the time of infection, a result in agreement with previous studies (23
). This characteristic could have mediated their higher susceptibility to HIV X4, which was particularly striking at the early times after infection. Alternatively, differences in the expression of some cellular factors critical for HIV infection could also be involved. Of interest, Th1 and Th2 cells have been shown to express different levels of APOBEC3G, and these differences determined their susceptibility to infection by HIV, including by Vif-competent viruses (35
). How these cellular factors are expressed in activated Treg is not yet known, and differences in their expression could contribute to the regulation of Treg susceptibility to HIV. An additional level of regulation may involve modulation of HIV LTR activity by FOXP3. Indeed, it was previously shown that FOXP3 could enhance the LTR activity of an X4 virus, by modulating chromatin structure as well as enhancing NF-κB activity (14
). Future experiments will be needed to confirm whether differences in receptor-mediated entry/fusion explain the difference in susceptibility of Treg to different HIV strains, or whether other early steps of the life cycle are also implicated.
Of importance, infected Treg were as suppressive as noninfected Treg, as evidenced by their capacity to inhibit Teff proliferation. Our data are in agreement with our previous studies showing that exposure of Treg to noninfectious HIV did not affect their suppressive capacity (22
). Other studies have also shown that circulating Treg purified from most HIV-infected patients exhibited suppressive activity (1
). However, it should be noted that our assay did not assess the functionality of infected Treg on a per-cell basis as viable infected Treg could not be separated and tested for their function. Nevertheless, these data suggest that Treg remain functional in an HIV- infected host and they likely regulate the homeostasis of the immune system as well as control HIV-specific immune responses.
Our in vitro data, as well as those from other laboratories, suggest that FOXP3 and HIV interactions are tightly regulated by both virus and host factors and that minor changes in either side will greatly impact the outcome of infection. These results may explain why no major differences in the level of infection of Treg and non-Treg were seen in vivo in chronically infected patients, both in our study and in two other recent studies (6
). Indeed, both the level of T-cell activation and the level of virus heterogeneity are known to vary among patients, and changes in these critical parameters are expected to impact infection of Treg by HIV.