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. 2009 Dec;83(24):12925-33.
doi: 10.1128/JVI.01352-09. Epub 2009 Oct 14.

Human regulatory T cells are targets for human immunodeficiency Virus (HIV) infection, and their susceptibility differs depending on the HIV type 1 strain

Maria E Moreno-Fernandez  1 , Wildeman Zapata, Jason T Blackard, Genoveffa Franchini, Claire A Chougnet
Affiliations

Human regulatory T cells are targets for human immunodeficiency Virus (HIV) infection, and their susceptibility differs depending on the HIV type 1 strain

Maria E Moreno-Fernandez et al. J Virol. 2009 Dec.

Abstract

Regulatory T cells (Treg) are a subpopulation of CD4(+) T cells characterized by the suppressive activity they exert on effector immune responses, including human immunodeficiency virus (HIV)-specific immune responses. Because Treg express CXCR4 and CCR5, they represent potential targets for HIV; however, Treg susceptibility to HIV infection is still unclear. We therefore performed an extensive study of Treg susceptibility to HIV, using lab strains and primary isolates with either CCR5 or CXCR4 tropism. Furthermore, we quantified HIV infection at early and late time points of the virus life cycle. We found that Treg were clearly susceptible to HIV infection. Circulating Treg were not preferentially infected with HIV compared to effector T cells (Teff) in vivo. Conversely, in vitro infection with either CCR5-using (R5) or CXCR4-using (X4) viruses occurred with different dynamics. For instance, HIV infection by R5 viruses (lab strains and primary isolates) resulted in lower levels of infection in Treg compared with Teff at both early and late time points. In contrast, X4 viruses induced higher levels of infection in Treg compared to Teff at early time points, but this difference disappeared at the late time points of the virus life cycle. Our results suggest that the relative susceptibility of Treg to HIV infection compared to Teff varies, depending on both viral and host factors. These variations may play an important role in HIV pathogenesis.

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Figures

FIG. 1.
FIG. 1.
Percentages of infected Treg and Teff are similar in vivo. CD25− and CD25+ cells were isolated from seven HIV-infected donors using magnetic beads and stained with anti-CD25, anti-CD127, and anti-p24Gag antibodies and analyzed by flow cytometry. Percentages of HIV p24Gag+ cells in each cell subset are shown.
FIG. 2.
FIG. 2.
Phenotypic characterization of isolated Teff and Treg. Purified CD4+ T cells were stained with anti-CD127-PE and anti-CD25-APC antibodies. The CD25hi CD127lo Treg and CD25lo CD127hi Teff subsets were identified, as shown in the left panel. Freshly isolated Treg and Teff were stained with anti-FOXP3-FITC to confirm their phenotype. Data from a representative experiment of nine independent experiments are shown. Percentages indicate the levels of expression of each marker defined in relation to the unstained cells.
FIG. 3.
FIG. 3.
Treg are less susceptible than Teff to the HIV R5 strain. (A) HIV p24Gag levels in culture supernatants were measured by ELISA. p24Gag levels from one representative experiment of infection by R5 YK-JRCSF of four different experiments are shown. HIV p24Gag levels from AZT-treated cells were measured at day 7. ND, not detectable. (B) p24Gag levels in Treg were expressed as a percentage of the p24Gag levels produced by the Teff from the same donor. P values indicate the differences between Treg and Teff. (C) A representative experiment of intracellular p24Gag staining at day 7 postinfection is shown. (D) Treg and Teff from seven healthy donors were infected, and HIV proviral DNA levels were measured by nested real-time HIV-LTR-Alu PCR 24 h postinfection; HIV DNA levels were normalized based on CD3 quantification and log10 transformed.
FIG. 4.
FIG. 4.
Treg are more susceptible than Teff to the HIV X4 strain. (A) HIV p24Gag levels in culture supernatants were measured by ELISA. p24Gag levels from one representative experiment of infection by X4 NL4-3 are shown. HIV p24Gag levels from AZT-treated cells were measured at day 7. (B) p24Gag levels in Treg were expressed as a percentage of the p24Gag levels produced by the Teff from the same donor. P values indicate the differences between Treg and Teff. (C) A representative experiment of intracellular p24Gag staining at days 3 and 7 postinfection is shown. (D) Treg and Teff from seven healthy donors were infected, and HIV proviral DNA levels were measured by nested real-time HIV-LTR-Alu PCR 24 h postinfection; HIV DNA levels were normalized based on CD3 quantification and log10 transformed.
FIG. 5.
FIG. 5.
Treg susceptibility to HIV primary isolates resembles their susceptibility to HIV lab strains. Treg and Teff were infected by R5 viruses (clade C, YK-JRCSF, YU2, and HIV301965; and clade A, KNH1088), the dualtropic isolate 92USSN20 (clade A), or X4 viruses (clade A, 92UG029; clade BF, 93BR019; clade B, 92HT599; and NL4-3). HIV infection was analyzed at 24 h postinfection by nested real-time HIV-LTR-Alu PCR. Similar levels of CD3 DNA were detected under all conditions. Integrated HIV levels in Treg were expressed as percentages of the levels detected in the Teff from the same donor and represent each virus. Results show the mean (± standard error) percentages for the three donors studied.
FIG. 6.
FIG. 6.
Treg express similar levels of CCR5 and CXCR4 and secrete similar levels of MIP1-α, MIP1-β, and RANTES compared to Teff. (A) CCR5 expression; (B) CXCR4 expression. Sorted Treg and Teff were activated with anti-CD3/CD28 beads for 3 days and then infected with HIV-1. Teff and Treg were stained with anti-CD4, anti-CCR5, and anti-CXCR4 antibody at the time of infection or 3 days postinfection and analyzed by flow cytometry. The percentage of positivity for each marker was defined in relation to the isotype control. Results are shown as means ± standard deviations (n = 4 or 5). (C) Supernatants were collected 3 days postactivation, and chemokine levels were analyzed using a Luminex assay.
FIG. 7.
FIG. 7.
Decreased Treg proliferation affects their susceptibility to infection by R5 viruses but not by X4 viruses. Teff and Treg proliferation was measured by labeling the cells with CFSE before stimulation with anti-CD3/CD28 beads in the presence of 100 U/ml IL-2. (A) Levels of CFSE expression were analyzed by flow cytometry 3 days postactivation as well as 3 and 5 days after infection. The results shown are from one experiment, representative of nine separate experiments. (B and C) The percentage of p24Gag+ cells was measured in the highly proliferating (CFSE low) Treg and Teff at day 5 postinfection with the R5 strain YK-JRCSF (B) and at day 3 postinfection with the X4 strain NL4-3 (C).
FIG. 8.
FIG. 8.
HIV-infected Treg remain suppressive. Purified Treg and Teff were separately activated for 3 days using anti-CD3/CD28 beads (1 cell per 3 beads) in the presence of 100 IU/ml IL-2. Treg were infected or not with YK-JRCSF virus at an MOI of 1 and incubated for 2 h at 37°C. Teff were labeled with CFSE. Treg and Teff were cocultured at a 1:1 ratio for 3 days. The suppressive capacity of Treg was determined by comparing the CFSE MFI between Teff cultured alone to those cultured with Treg. Results shown are from one experiment, representative of two separate experiments.
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