HIV Infection of Naturally Occurring and Genetically Reprogrammed Human Regulatory T-cells

A T-cell subset, defined as CD4(+)CD25(hi) (regulatory T-cells [Treg cells]), was recently shown to suppress T-cell activation. We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naïve human CD4(+) T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4(+) and higher levels of activated T-cells have greatly reduced levels of FoxP3(+)CD4(+)CD25(hi) T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset

Identification of a CCR5-Expressing T Cell Subset That Is Resistant to R5-Tropic HIV Infection

Infection with HIV-1 perturbs homeostasis of human T cell subsets, leading to accelerated immunologic deterioration. While studying changes in CD4(+) memory and naïve T cells during HIV-1 infection, we found that a subset of CD4(+) effector memory T cells that are CCR7(−)CD45RO(−)CD45RA(+) (referred to as T(EMRA) cells), was significantly increased in some HIV-infected individuals. This T cell subset displayed a differentiated phenotype and skewed Th1-type cytokine production. Despite expressing high levels of CCR5, T(EMRA) cells were strikingly resistant to infection with CCR5 (R5)–tropic HIV-1, but remained highly susceptible to CXCR4 (X4)–tropic HIV-1. The resistance of T(EMRA) cells to R5-tropic viruses was determined to be post-entry of the virus and prior to early viral reverse transcription, suggesting a block at the uncoating stage. Remarkably, in a subset of the HIV-infected individuals, the relatively high proportion of T(EMRA) cells within effector T cells strongly correlated with higher CD4(+) T cell numbers. These data provide compelling evidence for selection of an HIV-1–resistant CD4(+) T cell population during the course of HIV-1 infection. Determining the host factors within T(EMRA) cells that restrict R5-tropic viruses and endow HIV-1–specific CD4(+) T cells with this ability may result in novel therapeutic strategies against HIV-1 infection

Cytokine Production by FoxP3-Transduced T-cells

<p>CD4⁺ naïve T-cells isolated from CB (CB-naïve) and AB (AB-naïve) and memory T-cells from AB (AB-memory) were transduced with HDV or HDV.FoxP3 as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020198#pbio-0020198-g005" target="_blank">Figure 5</a>. Transduced T-cells were purified through magnetic sorting of mCD24⁺ cells and activated using plate-bound anti-CD3 and soluble anti-CD28 antibodies. Supernatants were collected at 18–24 h postactivation and analyzed for (A) IL-2 production or (B) IFNγ, IL-4, and IL-5 production from HDV or HDV.FoxP3-tranduced naïve T-cells, using CBA assay. The results represent five separate experiments from different donors with similar relative differences in cytokine production.</p

HIV Infection of FoxP3-Expressing T-cells

<div><p>(A) HDV.FoxP3 and HDV-transduced T-cells were activated using plate-bound anti-CD3 (100 ng/ml) and soluble anti-CD28 (1 μg/ml) antibodies or maintained in IL-2-containing medium. Cells were concurrently infected at different MOI of VSV-G.HIV, and infection was determined by GFP expression at 72 h postinfection by flow cytometry.</p> <p>(B) Supernatants were collected at different time points from R5.HIV-infected HDV.FoxP3-expressing or HDV-transduced cell cultures, and HIV p24 levels were measured by ELISA. The percentages of infected cells at days 3, 9, and 16 for HDV.FoxP3 were 2, 10, and 26, and for HDV were 0.8, 10, and 18, respectively.</p></div

FoxP3 Expression in Purified CD4⁺CD25^hi (Treg), Naïve, and Memory T-cells from HIV-Infected and Healthy Individuals

<p>(A) RNA was isolated from sorted Treg, naïve, and memory T-cells from HIV-positive (<i>n</i> = 24) and HIV-negative (<i>n</i> = 11) subjects, followed by cDNA synthesis. <i>FoxP3</i> expression was quantified by TaqMan real-time PCR. The <i>FoxP3</i>-fold difference expression was calculated for CD4⁺CD25^hi (Treg) versus naïve (N), and memory (Mem) versus naïve (N) T-cells. Treg cells sorted from HIV-positive subjects were further subdivided into two groups based on <i>FoxP3</i> expression of Treg compared to naïve T-cells (FoxP3-high, <i>n</i> = 13, FoxP3 difference >10-fold; FoxP3-low, <i>n</i> = 11, FoxP3 difference <10-fold; HIV-negative, <i>n</i> = 9). These groups were stained with anti-CD3, anti-CD4, anti-CD45RO, anti-CD25, and anti-HLA-DR and analyzed by flow cytometry for (B) CD4⁺ T-cell percentage, (C) activated T-cell percentage (CD4⁺HLA-DR⁺), and (D) CD4⁺CD25^hi percentage. Horizontal lines identify means. Statistical significance between groups was determined by Mann–Whitney <i>U</i> test and shown on top of each figure.</p

Cytokine Secretion by Treg T-cells

<p>Sorted Treg and memory T-cells were activated using plate-bound anti-CD3 (3 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies. Supernatants were collected 18–24 h postactivation and analyzed for cytokines using the CBA assay. Results are representative of cytokine secretion from Treg and memory T-cells from three different donors.</p