We have previously established an in vitro
model of HIV latency following incubation of resting CD4+ T-cells with the CCR7 ligands, CCL19 [11
]. We have shown here that, in this in vitro
model of HIV latency, there was no spontaneous production of infectious virus and that the block in the virus life cycle and the response to activating stimuli closely mirrors findings by other groups in ex vivo
resting CD4+ T-cells from HIV-infected patients on cART [20
We found that in CCL19-treated latently infected cells MS RNA was detected in the nucleus, but not in the cytoplasm, in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both nucleus and cytoplasm. MS RNA encodes the positive regulators Rev and Tat that are crucial for the efficient expression of US RNA in the cytoplasm [30
]. Therefore, the lack of US RNA expression and viral production in CCL19-treated infected CD4+ T-cells may be explained by the absence of MS RNA in the cytoplasm. The absence of MS RNA in the cytoplasm could potentially be secondary to a block in nuclear export of viral mRNA or destruction of MS RNA in the cytoplasm. We were unable to distinguish between these two possibilities; however, others have previously described that in CD4+ T cells from patients on cART, there is a block in export of MS RNA to the cytoplasm secondary to low levels of polypyrimidine tract binding protein in resting CD4+ T-cells [22
]. We have recently compared gene expression using Illumina microarrays in resting CD4+ T-cells with and without CCL19 [11
], and found no difference in the expression of PTB in the presence or absence of CCL19 (data not shown). These data suggest that PTB may also be functional in this chemokine model of HIV latency, but further experiments will be required to demonstrate this directly.
Production of virus from CCL19-treated infected CD4+ T-cells was clearly demonstrated following activation with multiple different stimuli. The combination of IL-7 and prostratin resulted in the highest levels of RT production (Figure ). Prostratin stimulates HIV through PKC -mediated release of active nuclear factor κB (NF-κB) [24
]. Previous studies have shown that inadequate or low nuclear levels NF-κB and nuclear factor of activated T cells (NFAT) may contribute to the maintenance of latency in resting CD4+ T-cells (reviewed in [32
]). IL-7 has been shown to effectively induce HIV replication ex vivo
in both CD8 depleted PBMCs and resting CD4+ T-cells from patients on cART [23
]. IL-7 can activate both the PI3K and the STAT 5 pathways which could both potentially enhance virus transcription [35
]. Activation of PI3K could increase virus transcription via enhanced production of NF-kB [37
] while phosphorylated STAT5 has been shown to bind and transactivate viral transcription in ex vivo
primary CD4+ T-cells; in the HeLa cell line co-transfected with STAT5 expression vectors and an HIV LTR construct that expresses firefly luciferase construct; and in the latently infected cell line (U1) [34
IL-7 may also potentially contribute to the maintenance of HIV latency via homeostatic proliferation of resting CD4+ T-cells [5
], but proliferation alone would not explain our findings that IL-7 can induce virus production from latently infected cells [42
]. Furthermore, we found that IL-7 alone had no effect on T-cell proliferation of purified resting CD4+ memory T-cells which were used in this model, as measured by Ki67 staining and dilution of carboxyfluorosceinsuccinate (CFSE) (data not shown). The exact mechanism of action of IL-7 in our CCL19-induced model of latency remains unclear.
TNFα resulted in quite potent virus reactivation in our model which is consistent with findings in latently infected primary CD4+T cells that were transduced with the prosurvival molecule Bcl-2 [43
] and in multiple latently infected cell lines [44
]. In contrast, in another primary latency model using non-polarised cells that were activated, infected and allowed to rest, TNFα did not result in any virus reactivation [6
]. In these two previous studies using primary T-cell models of latency, a similar concentration of TNFα, 10 ng/ml, was used as we have used in this study although the response rates were quite different with a percent maximal stimulation of 40%, 20% and 0% in our, the Yang [43
] and Bosque [6
] models respectively. The differences in detection of reactivation are unlikely to be explained by the frequency of latently infected cells as in our model of chemokine induced latency, on average 1% of cells contain integrated DNA, which is similar to the Yang model [43
] but is far lower than the frequency of latently infected cells using the Bosque model, where the frequency of latently infected cells approached 30-50% [6
]. To our knowledge, reactivation of latent infection with TNF-a has not been assessed in resting CD4+ T-cells from patients on suppressive cART and these experiments would add further insight to our understanding of the currently available different models of latency in primary T-cells.
Others have demonstrated the synergism obtained by treatment with a combination of prostratin and the HDACi vorinostat in both a cell line and primary cell model of latent HIV infection [46
]. Herein we also demonstrated the additive effects in activation of HIV replication by combining the PKC activator prostratin with IL-7. We have not yet evaluated the effects of IL-7 with other HDACi in this model, but this will certainly be of interest given the well known safety profiles of drugs such as IL-7 and vorinostat. Strategies that activate latent HIV in infected individuals on cART are likely to include combinatorial approaches and our model provides a robust tool for screening such approaches.