HIV infection of naturally occurring and genetically reprogrammed human regulatory T-cells

Abstract

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.

Figure 1. Identification and Phenotype of Treg Cells

(A) Purified CD4⁺ T-cells were stained with anti-CD45RO-FITC and anti-CD25-PE antibodies. The naïve, memory, and Treg subsets were identified as shown in boxes. (B) Purified CD4⁺ T-cells were first stained with a pure antibody against the cell surface molecule shown in the figure, followed by antimouse IgG conjugated with allophycocyanin, followed by CD25-PE and CD45RO-FITC. Gates were set for Treg, memory, and naïve T-cells as shown in (A). These results are representative of one out of five donors analyzed.

Figure 2. Cytokine Secretion by Treg T-cells

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.

Figure 3. Proliferation and Suppressive Capacity of Treg Cells

(A) Sorted Treg and memory T-cells were labeled with CFSE and then activated through suboptimal anti-CD3 (100 ng/ml) and anti-CD28 (1 μg/ml) antibodies. Day 6 postactivation, cells were fixed and CFSE expression was analyzed by flow cytometry. (B) Resting naïve or memory CD4⁺ T-cells (1.5 × 10⁵ T-cells) were labeled with CFSE and cocultured with either unlabeled purified Treg, naïve, or memory T-cells at 1:1 ratio in 96-well plates coated with suboptimal anti-CD3 (100 ng/ml) and anti-CD28 (1 μg/ml) antibodies. At day 4 postactivation, cells were fixed and analyzed for CFSE expression and cell size by flow cytometry. (C) Regions were set based on 2-fold reduction in CFSE mean intensity of naïve or memory T-cells as gated on (B), and plotted as number of cell divisions. Results represent three separate experiments from three different donors.

Figure 4. HIV Infection of Treg Cells

(A) Sorted Treg and memory T-cells were activated using plate-bound anti-CD3 (3 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies and concurrently infected with R5.HIV or VSV-G.HIV at a MOI of 5 (based on prior virus titration using Hut78/CCR5 cells). The percentage of infected cells was determined by GFP expression at 3 d postinfection by flow cytometry. (B) Supernatants from Treg and memory T-cells infected with R5.HIV cultures were collected at different time points and HIV p24 levels were measured by ELISA. (C) Treg cell death was assessed by analyzing infected cells at days 3 and 7 postinfection based on forward- and side-scatter analysis and in some experiments using propidium iodine staining analysis with flow cytometry. All of the infection results are representative of one out of five separate experiments with reproducible results. Statistical significance was determined using the Student's two-tailed t test. * p < 0.05.

Figure 5. Phenotype of FoxP3 Transduced T-cells

Purified CB naïve CD4⁺ T-cells were activated through the TCR and transduced with either HDV.FoxP3 or HDV. Cells were expanded for 14 d in IL-2-containing medium and stained with (A) anti-mCD24, anti-GITR, and anti-CD25, (B) anti-mCD24 and anti-CCR4, or (C) anti-mCD24 and anti-CCR5 antibodies. Gates were set on the mCD24-positive population (transduced), and expression of surface molecule was analyzed. The expression of these markers in the CD24-negative portion of both cultures was identical (data not shown). These results are representative of T-cells isolated from five different donors and transduced independently.

Figure 6. Cytokine Production by FoxP3-Transduced T-cells

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 Figure 5. 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.

Figure 7. Proliferation and Suppression by FoxP3-Expressing Cells

(A) Purified CD4⁺ naïve and memory T-cells were transduced with either HDV.FoxP3 or HDV as described. The transduced T-cells were labeled with CFSE and activated with anti-CD3 (100 ng/ml) and anti-CD28 (1 μg/ml) antibodies. Day 6 postactivation, cells were fixed and analyzed for CFSE expression by flow cytometry. (B) Resting CD4⁺ T-cells (1.5 × 10⁵) were labeled with CFSE and cocultured at 1:1 with either unlabeled sorted HDV.FoxP3-expressing or HDV-transduced CB naïve T-cells and activated with anti-CD3 (100 ng/ml) and anti-CD28 (1 μg/ml ) antibodies. At 4 d postactivation, cells were stained with mCD24-PE as a marker for infection. (C) Naïve and memory T-cells isolated from adult blood were transduced with HDV.FoxP3 or HDV. A coculture suppression experiment was set up with resting purified autologous CD4⁺ T-cells as described above. Region was set on mCD24 negative CFSE⁺ cells (target resting CD4⁺ T-cells) as shown in (B), and CFSE expression was analyzed 6 d poststimulation by flow cytometry. The results are representative of three separate experiments.

Figure 8. HIV Infection of FoxP3-Expressing T-cells

(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. (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.

Figure 9. FoxP3 Expression in Purified CD4⁺CD25^hi (Treg), Naïve, and Memory T-cells from HIV-Infected and Healthy Individuals

(A) RNA was isolated from sorted Treg, naïve, and memory T-cells from HIV-positive (n = 24) and HIV-negative (n = 11) subjects, followed by cDNA synthesis. FoxP3 expression was quantified by TaqMan real-time PCR. The FoxP3-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 FoxP3 expression of Treg compared to naïve T-cells (FoxP3-high, n = 13, FoxP3 difference >10-fold; FoxP3-low, n = 11, FoxP3 difference <10-fold; HIV-negative, n = 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 U test and shown on top of each figure.