Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling.

Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the interleukin 2 (IL-2) receptor ß-chain, and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.

Elite suppressors harbor low levels of integrated HIV DNA and high levels of 2-LTR circular HIV DNA compared to HIV+ patients on and off HAART.

Elite suppressors (ES) are a rare population of HIV-infected individuals that are capable of naturally controlling the infection without the use of highly active anti-retroviral therapy (HAART). Patients on HAART often achieve viral control to similar (undetectable) levels. Accurate and sensitive methods to measure viral burden are needed to elucidate important differences between these two patient populations in order to better understand their mechanisms of control. Viral burden quantification in ES patients has been limited to measurements of total DNA in PBMC, and estimates of Infectious Units per Million cells (IUPM). There appears to be no significant difference in the level of total HIV DNA between cells from ES patients and patients on HAART. However, recovering infectious virus from ES patient samples is much more difficult, suggesting their reservoir size should be much smaller than that in patients on HAART. Here we find that there is a significant difference in the level of integrated HIV DNA in ES patients compared to patients on HAART, providing an explanation for the previous results. When comparing the level of total to integrated HIV DNA in these samples we find ES patients have large excesses of unintegrated HIV DNA. To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC. We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells. Our findings suggest that measuring integration provides a better surrogate of viral burden than total HIV DNA in ES patients. Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population.

Human immunodeficiency virus integrates directly into naive resting CD4+ T cells but enters naive cells less efficiently than memory cells.

Resting CD4(+) T cells restrict human immunodeficiency virus (HIV) infection at or before reverse transcription, resulting in slower kinetics of reverse transcription. In a previous study, we showed that, despite this restriction at reverse transcription, HIV integration occurs in resting CD4(+) T cells, albeit with slower kinetics. In that study, the resting T cells were a mixture of memory and naïve cells. Here we asked whether the more quiescent naïve cell subset could be directly infected by HIV and, if so, whether the level of integration in naïve cells was comparable to that in memory cells. We found that HIV integrates in the naïve subset of resting CD4(+) T cells without prior activation of the cells. The level of integration (proviruses/cell) in naïve cells was lower than that in memory cells. This difference between naïve and memory cells was observed whether we inoculated the cells with R5 or X4 HIV and could not be explained solely by differences in coreceptor expression. The presence of endogenous dendritic cells did not change the number of proviruses/cell in memory or naïve cells, and deoxynucleoside pools were equally limiting. Our results instead indicate the existence of a novel restriction point in naïve T cells at viral fusion that results in reduced levels of fusion to naïve CD4(+) T cells. We conclude that HIV can integrate into both naïve and memory cells directly. Our data further support our hypothesis that integrated proviral infection of resting T cells can be established without T-cell activation.

The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope.

Current gene transfer protocols for resting CD4(+) T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4(+) T cells without activation. We recently demonstrated, contrary to the prevailing belief, that wild-type human immunodeficiency virus (HIV) integrates into resting CD4(+) T cells. Based on that finding, we investigated whether a commonly used, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentiviral gene therapy vector could also integrate into resting CD4(+) T cells. To investigate this, we inoculated resting CD4(+) T cells with lentiviral particles that were pseudotyped with VSV-G or CXCR4-tropic HIV Env and assayed binding, fusion, reverse transcription, and integration. We found that the VSV-G-pseudotyped lentiviral vector failed to fuse to resting CD4(+) T cells while HIV Env-pseudotyped lentiviral vectors fused, reverse transcribed, and integrated in resting cells. Our findings suggest that HIV Env could be used effectively for the delivery of therapeutic genes to resting CD4(+) T cells and suggest that fusion may be the critical step restricting transduction of resting CD4(+) T cells by lentiviral gene therapy vectors.

Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR.

In this review, we compare four assays that are currently used to measure HIV integration and discuss their strengths and weaknesses. We then outline advances that have been made toward development of a more robust, more sensitive, quantitative HIV integration assay suitable for clinical use. The assay that we have developed uses repetitive-sampling Alu-gag PCR. The detailed protocol describes our assay step-by-step, the creation of an integration standard cell line and accompanying standard curve, as well as the quantitation of integration and calculation of associated error estimates. Finally, we speculate on fundamental, unresolved issues in HIV latency that can be addressed by measuring HIV integration.

Patients on HAART often have an excess of unintegrated HIV DNA: implications for monitoring reservoirs.

HIV establishes a latent reservoir early in infection that is resistant to anti-retroviral therapy and has a slow rate of decay. It is thought that the majority of HIV DNA in treated patients is integrated since unintegrated HIV DNA appears to be unstable. Thus, to monitor the HIV latent reservoir, total HIV DNA is commonly measured in PBMC from infected individuals. We investigated how often total approaches integrated HIV DNA in treated patients. To do this, we first assessed how accurate our integration assay is and determined the error in our measurements of total and integrated HIV DNA. We demonstrated an excess of total over integrated HIV DNA was present in a subset of patients, suggesting that measurements of total HIV DNA do not always correlate to the level of integration. Determining the cause of this excess and its frequency may have important implications for understanding HIV latent reservoir maintenance.

A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients.

Many studies report the level of total viral DNA in HIV-infected patients, but few studies report the level of integrated DNA. It is important to measure integrated DNA in HIV-infected patients because the information could shed light on the effectiveness of antiretroviral therapy, especially intensified therapy, when viral loads may remain undetectable. In order to develop an integration assay for patient samples, we enhanced the sensitivity of our prior integration assay. To do this, we exploited a technique that we developed, called repetitive sampling, and optimized reaction conditions for rare event detection, rather than large dynamic range. We also designed our primers to match more conserved regions of HIV. The result is a new, sensitive, quantitative assay that allows us to measure integrated DNA in HIV-infected patients. When we applied our integration assay to patient PBMCs, we found that the use of HAART is associated with reduced levels of integrated DNA.