HIV inhibits CD4+ T-cell proliferation by inducing indoleamine 2,3-dioxygenase in plasmacytoid dendritic cells

Abstract

Infection with the human immunodeficiency virus type-1 (HIV) results in acute and progressive numeric loss of CD4(+) T-helper cells and functional impairment of T-cell responses. The mechanistic basis of the functional impairment of the surviving cells is not clear. Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme that inhibits T-cell proliferation by catabolizing the essential amino acid tryptophan (Trp) into the kynurenine (kyn) pathway. Here, we show that IDO mRNA expression is elevated in peripheral blood mononuclear cells (PBMCs) from HIV(+) patients compared with uninfected healthy controls (HCs), and that in vitro inhibition of IDO with the competitive blocker 1-methyl tryptophan (1-mT) results in increased CD4(+) T-cell proliferative response in PBMCs from HIV-infected patients. We developed an in vitro model in which exposure of PBMCs from HCs to either infectious or noninfectious, R5- or X4-tropic HIV induced IDO in plasmacytoid dendritic cells (pDCs). HIV-induced IDO was not inhibited by blocking antibodies against interferon type I or type II, which, however, induced IDO in pDCs when added to PBMC cultures. Blockade of gp120/CD4 interactions with anti-CD4 Ab inhibited HIV-mediated IDO induction. Thus, induction of IDO in pDCs by HIV may contribute to the T-cell functional impairment observed in HIV/AIDS by a non-interferon-dependent mechanism.

Figure 1

Increased IDO in PBMCs from HIV⁺ patients impairs T-cell proliferative responses. (A) IDO mRNA expression, measured by real-time quantitative PCR, in PBMCs from HIV⁺ patients who were or were not receiving ART (HIV⁺ ART and HIV⁺ no ART, respectively) and HCs; horizontal bars within boxes correspond to the median; box limits correspond to the 25th and 75th percentiles; vertical lines extend to the 10th and 90th percentiles. (B) Direct correlation between IDO mRNA expression and viral load (log₁₀ copies/mL) in HIV⁺ patients; *= for patients with undetectable viral load a value of 50 copies/mL (detection limit) has been used. (C-D) Proliferative response to PHA (C) and activating antibodies recognizing CD3 and CD28 (D) in PBMCs from HIV⁺ patients and HCs in cultures without (open bars) and with (solid bars) 1-mT; number of viable cells was measured by bioreduction colorimetric assay; relative cell number was calculated for each sample as ratio between stimulated (with PHA or anti-CD3/CD28) and unstimulated culture, in presence or absence of 1-mT; mean values ± SE are shown. (E) The increase of proliferation was calculated as a ratio between proliferative response in presence and in absence of 1-mT; values higher than 1 indicate that proliferation was increased in presence of 1-mT. 1-mT–induced increase in proliferation in HIV⁺ patients receiving ART compared with HIV⁺ patients not receiving ART (no ART); horizontal bars within boxes correspond to the median; box limits correspond to the 25th and 75th percentiles; vertical lines extend to the 10th and 90th percentiles.

Figure 2

Proliferation of CD4⁺, but not CD8⁺ T cells from HIV⁺ patients is increased by 1-mT. (A-B) Frequency of nondividing cells, measured by CFSE dilution after 6 days of culture, in CD4⁺ T cells of PBMCs from HIV⁺ patients stimulated with PHA (A) or anti-CD3/28 (B) in presence or absence of 1-mT. (C) Individual results for 1 of 22 HIV⁺ patients and 1 of 3 HCs tested are shown for CD4⁺ T-cell proliferation in response to both PHA and anti-CD3/28 stimulation. (D-E) Frequency of nondividing cells, measured by CFSE dilution after 6 days of culture, in CD8⁺ T cells of PBMCs from HIV⁺ patients stimulated with PHA (D) or anti-CD3/28 (E) in presence or absence of 1-mT. (F) Individual results for 1 of 22 HIV⁺ patients and 1 of 3 HCs tested are shown for CD8⁺ T-cell proliferation in response to both PHA and anti-CD3/28 stimulation. For panels A-B and D-E, mean values (n = 22) ± SE are shown.

Figure 3

Infectious or AT-2 HIV induces IDO mRNA expression and enzymatic activity in PBMCs of HIV-uninfected donors. (A) IDO mRNA was measured by quantitative real-time PCR in PBMCs from HIV-uninfected donors cultured for 24 hours in presence of MVs, AT-2 HIV_MN, AT-2 HIV_Ada, infectious HIV_MN, and infectious HIV_Ada (solid bars); a series of experiments was performed using CTLA-4-Fc and CD28-Fc (open bars); mean values (n = 6) ± SE are shown. (B) Ratios between the concentration of kynurenine (μM) and tryptophan (mM) were measured by HPLC in supernatants from PBMCs cultured with microvesicles (control), AT-2 HIV, or AT-2 HIV and 1-mT for 24 and 48 hours; mean values (n = 5) ± SE are shown.

Figure 4

pDCs express IDO in PBMCs exposed to HIV. PBMCs from HIV-uninfected donors were cultured with microvesicles (control; open bars) or AT-2 HIV (solid bars) for 24 hours, and IDO protein expression was detected by flow cytometry in CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD4⁺), monocytes (CD4⁺CD3⁻CD8⁻BDCA2⁻), and pDCs (CD4⁺CD123⁺BDCA2⁺). (A) Mean IDO-PE MFI (n = 5) ± SE gated on each cell type is shown. (B) One representative experiment of 5 tested is shown; left panel shows gate on pDCs among CD4⁺ cells; right panel shows histograms for MFI on isotype control (black), and IDO PE on microvesicle-treated (control; green) and AT-2 HIV-treated (red) cells. (C) Intracellular staining for IDO in pDCs enriched by magnetic separation with anti-BDCA4–coated beads and cultured with or without AT-2 HIV.

Figure 5

IFN-β and IFN-γ mRNA expression is increased in PBMCs exposed to AT-2 HIV. IFN-β (A) and IFN-γ (B) mRNA was measured by quantitative real-time PCR in PBMCs from HIV-uninfected donors cultured for 24 hours in presence of MVs, AT-2 HIV, or AT-2 HIV and mAb anti-CD4; mean values (n = 3) ± SE are shown. (C) Intracellular staining for IDO, gated on pDCs (BDCA2⁺CD123⁺), in PBMCs cultured with or without AT-2 HIV, rIFN-α, rIFN-β, or rIFN-γ.

Figure 6

Blockade of CD4, but not of type I or type II IFN, inhibits AT-2 HIV-induced IDO. (A) IDO mRNA expression was measured in PBMCs from HIV-uninfected donors after exposure to AT-2 HIV in presence or absence of Ab against IFN-α, IFN-β, and/or IFN-γ or CD4; expression was normalized on the AT-2 HIV-treated condition (100%) for each experiment; mean values (n = 3) ± SE are shown. (B) IDO protein was detected by flow cytometry in pDCs (CD4⁺CD123⁺BDCA2⁺) from PBMCs cultured in presence of microvesicles (control; green), AT-2 HIV (red), or AT-2 HIV + anti-CD4 mAb (blue); 1 representative experiment of 3 tested is shown. (C) Ratios between the concentration of kynurenine (μM) and tryptophan (mM) were measured by HPLC in supernatants from PBMCs cultured with AT-2 HIV in presence or absence of Ab against CD4; kyn/Trp ratios were normalized on the AT-2 HIV-treated condition (100%) for each experiment; mean values (n = 3) ± SE are shown.