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. 2008 Aug 13;3(8):e2961.
doi: 10.1371/journal.pone.0002961.

HIV-induced type I interferon and tryptophan catabolism drive T cell dysfunction despite phenotypic activation

Adriano Boasso  1 , Andrew W Hardy, Stephanie A Anderson, Matthew J Dolan, Gene M Shearer
Affiliations

HIV-induced type I interferon and tryptophan catabolism drive T cell dysfunction despite phenotypic activation

Adriano Boasso et al. PLoS One. .

Abstract

Infection by the human immunodeficiency virus (HIV) is characterized by functional impairment and chronic activation of T lymphocytes, the causes of which are largely unexplained. We cultured peripheral blood mononuclear cells (PBMC) from HIV-uninfected donors in the presence or absence of HIV. HIV exposure increased expression of the activation markers CD69 and CD38 on CD4 and CD8 T cells. IFN-alpha/beta, produced by HIV-activated plasmacytoid dendritic cells (pDC), was necessary and sufficient for CD69 and CD38 upregulation, as the HIV-induced effect was inhibited by blockade of IFN-alpha/beta receptor and mimicked by recombinant IFN-alpha/beta. T cells from HIV-exposed PBMC showed reduced proliferation after T cell receptor stimulation, partially prevented by 1-methyl tryptophan, a competitive inhibitor of the immunesuppressive enzyme indoleamine (2,3)-dioxygenase (IDO), expressed by HIV-activated pDC. HIV-induced IDO inhibited CD4 T cell proliferation by cell cycle arrest in G1/S, and prevented CD8 T cell from entering the cell cycle by downmodulating the costimulatory receptor CD28. Finally, the expression of CHOP, a marker of the stress response activated by IDO, was upregulated by HIV in T cells in vitro and is increased in T cells from HIV-infected patients. Our data provide an in vitro model for HIV-induced T cell dysregulation and support the hypothesis that activation of pDC concomitantly contribute to phenotypic T cell activation and inhibition of T cell proliferative capacity during HIV infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HIV induces increased CD69 and CD38 on T cells in a type I IFN-dependent manner.
PBMC from HIV-uninfected donors were cultured for 24 and 48 hours in presence of control microvescicles, HIV alone or in presence of blocking antibodies against the cellular receptor for IFN-α (anti-IFNAR). CD38 and CD69 expression were analyzed by flow cytometry on gated CD3+CD4+ and CD3+CD8+ cells (CD4 and CD8 T cells, respectively). (A) and (C) show flow cytometry contour plots of CD69 and CD38 expression for one example experiment for CD4 and CD8 T cells, respectively. (B) and (D) show bar graphs summarizing mean fluorescence intensity (MFI) of CD38 and CD69 in CD4 and CD8 T cells, respectively (48 hours only). Mean values±standard error calculated on 5 independent experiments are shown in the bar graphs.
Figure 2
Figure 2. rIFN-α induces increased CD69 and CD38 on T cells.
PBMC from HIV-uninfected donors were cultured for 24 (upper panels) and 48 hours (lower panels) in presence or absence of recombinant IFN-α (rIFN-α). CD38 and CD69 expression were analyzed by flow cytometry on gated CD3+CD4+ and CD3+CD8+ cells (CD4 and CD8 T cells, respectively). Flow cytometry contour plots of CD69 and CD38 expression for one example experiment for CD4 (left panels) and CD8 T cells (right panels).
Figure 3
Figure 3. HIV exposure impairs T cell proliferative responses, contribution of IDO-mediated tryptophan catabolism.
CFSE-labeled PBMC from HIV-uninfected donor were cultured for 24 hours in presence of control microvescicles, HIV alone, or in presence of the IDO inhibitor 1-methyl-D-tryptophan (1mT). After 24 hours anti-CD3 was added to the cultures and cells were analyzed by flow cytometry after 72 hours. (A) Flow cytometry histograms showing CFSE dilution for one example experiment for CD4 (left panels) and CD8 T cells (right panels) are shown. Upper panels show the comparison between control-pretreated cells (green line) and HIV-pretreared cells (red line); bottom panels show the comparison between HIV-pretreared cells (red line) and cells pretreated with HIV in presence of 1mT (Blue line). One representative of 5 independent experiments is shown. (B) Bar graphs showing division index (number of cell divisions/total cell number) and proliferation index (number of cell divisions/number of divided cells) of CD4 (left panels) and CD8 T cells (right panels) pretreated with AT-2 HIV or mock and stimulated with anti-CD3 in presence (solid bars) or absence (open bars) of 1mT. Mean values±standard error calculated on 5 independent experiments are shown.
Figure 4
Figure 4. HIV-induced IDO suppresses both CD4 and CD8 T cell proliferation in a 2-step experiment.
(A) CD4 and CD8 T cell proliferation is shown as the increase in the number of viable cells measured using a bioreduction assay. Relative cell number was calculated for each sample as ratio between stimulated (with anti-CD3 and anti-CD28) and unstimulated cells cultured in the three different conditioned media (CM). (B) CD4 and CD8 T cell apoptosis is shown as frequency of annexin V+ cells measured by flow cytometry. Apoptosis of cells cultured in the three different conditioned media (CM) is shown for both unstimulated and stimulated (with anti-CD3 and anti-CD28) cells. In all cases mean values±standard error calculated on 8 independent experiments are shown.
Figure 5
Figure 5. Effect of HIV-induced IDO on CD4 and CD8 T cell cycle progression in a 2-step experiment.
(A) Flow cytometry dot plots showing BrdU incorporation and DNA staining with 7-AAD for one example experiment for CD4 and CD8 T cells stimulated with anti-CD3 and anti-CD28 in the three different conditioned media (CM); red boxes indicate gates for cells in G0/G1, S and G2 phase; numbers represent the percentage of T cells in S phase for each condition. (B) Cyclin D1 (marker of G1 phase) and cyclin E1 (marker of S phase) mRNA expression in CD4 and CD8 T cells stimulated in the three different conditioned media (CM). Relative mRNA levels are calculated as ratio between stimulated (with anti-CD3 and anti-CD28) and unstimulated cells. (C) CD28 mRNA expression in unstimulated CD8 T cells cultured in the three different conditioned media (CM) over a 72 hours period. In all bar graphs mean values±standard error calculated on 8 independent experiments are shown.
Figure 6
Figure 6. CHOP mRNA expression is upregulated in CD4 and CD8 T cells by HIV-induced IDO in vitro and in HIV-infected patients in vivo.
(A) CHOP mRNA expression in unstimulated CD4 and CD8 T cells cultured in the three different conditioned media (CM). Mean values±standard error calculated on 8 independent experiments are shown. (B) Plots showing CHOP mRNA expression in CD4+ and CD8+ cells isolated from PBMC of uninfected healthy controls (HC) and HIV-infected patients (HIV+) with plasma virus levels below (VL<50) or above (VL>50) the detection threshold. Each symbol represents one individual patient or donor: HC are indicated with squares; HIV-infected patients undergoing HAART are indicated with circles (independently of plasma virus load) and HIV-infected patients not undergoing HAART are indicated with triangles (independently of plasma virus load). Horizontal bars represent mean values for HC, HIV+ VL<50 and HIV+ VL>50, respectively.
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