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AIDS:
doi: 10.1097/QAD.0b013e32834ed8df
Basic Science

Myeloid dendritic cells isolated from tissues of SIV-infected Rhesus macaques promote the induction of regulatory T cells

Presicce, Pietroa,*; Shaw, Julia M.b,*; Miller, Christopher J.c; Shacklett, Barbara L.b,**; Chougnet, Claire A.a,**

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Author Information

aDivision of Molecular Immunology, Cincinnati Children's Hospital Research Foundation, and Department of Paediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio

bDepartment of Medical Microbiology and Immunology, School of Medicine

cDepartment of Pathology, Microbiology and Immunology, California National Primate Research Center, School of Veterinary Medicine, University of California, Davis, California, USA.

*These authors contributed equally to this work.

**These authors contributed equally to this work.

Correspondence to Claire A. Chougnet, PhD (ML#7021), Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229, USA. Tel: +1 513 636 8847; fax: +1 513 636 5355; e-mail: claire.chougnet@cchmc.org

Received 15 August, 2011

Revised 30 September, 2011

Accepted 3 November, 2011

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.AIDSonline.com).

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Abstract

Objective: To determine whether the ability of primary myeloid dendritic cells (mDCs) to induce regulatory T cells (Treg) is affected by chronic simian immunodeficiency virus (SIV) infection.

Design: Modulation of dendritic cell activity with the aim of influencing Treg frequency may lead to new treatment options for HIV and strategies for vaccine development.

Methods: Eleven chronically infected SIV+ Rhesus macaques were compared with four uninfected animals. Immature and mature mDCs were isolated from mesenteric lymph nodes and spleen by cell sorting and cultured with purified autologous non-Treg (CD4+CD25 T cells). CD25 and FOXP3 up-regulation was used to assess Treg induction.

Results: The frequency of splenic mDC and plasmacytoid dendritic cell was lower in infected animals than in uninfected animals; their frequency in the mesenteric lymph nodes was not significantly altered, but the percentage of mature mDCs was increased in the mesenteric lymph nodes of infected animals. Mature splenic or mesenteric mDCs from infected animals were significantly more efficient at inducing Treg than mDCs from uninfected animals. Mature mDCs from infected macaques induced more conversion than immature mDCs. Splenic mDCs were as efficient as mesenteric mDCs in this context and CD103 expression by mDCs did not appear to influence the level of conversion.

Conclusions: Tissue mDCs from SIV-infected animals exhibit an enhanced capability to induce Treg and may contribute to the accumulation of Treg in lymphoid tissues during progressive infection. The activation status of dendritic cell impacts this process but the capacity to induce Treg was not restricted to mucosal dendritic cells in infected animals.

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Introduction

Natural regulatory T cells (Treg) develop in the thymus as a distinct lineage expressing CD4, CD25, and the transcription factor FOXP3. These cells have a critical role in the establishment and maintenance of physiological tolerance through the suppression of auto-immune responses [1]. In addition to thymically derived ‘classical’ Treg, a growing number of studies have described the peripheral conversion of CD4+CD25 T cells into CD25+FOXP3+ Treg [2–8]. These induced or adaptive Treg are generated during induction of oral tolerance and in response to many inflammatory processes, including persistent infections [9].

Simian immunodeficiency virus (SIV) infection of Rhesus macaques results in early and persistent accumulation of Treg in the lymph nodes [10,11]. Similar increases in Treg frequency and number have been observed in macaque and human lymphoid and mucosal tissues during chronic-stage SIV/HIV infection [12–17], yet the causes and consequences of this increase remain unclear. Indeed, several studies have proposed that Treg protect the host by mitigating the adverse affects of immune activation [18,19] and HIV infection of CD4+ non-Treg [20]; however, other findings have suggested that Treg actively suppress virus-specific immune responses, inadvertently promoting viral persistence [21–24]. Treg accumulation in tissues during HIV/SIV may be mediated by multiple mechanisms, including increased survival [12,14,25], trafficking [16,25], proliferation [13,14,16], and extra-thymic/peripheral conversion [26,27]. Understanding the contributions of these various mechanisms will provide a more accurate picture of Treg dynamics in the context of HIV/SIV infection and may aid in the development of targeted immuno-therapies and vaccines.

Antigen-presenting cells (APCs) have been shown to mediate peripheral Treg conversion in mice and humans. In the gut, in which immunity to intestinal pathogens must be tightly coordinated with tolerance to food and commensal antigens, Treg induction by murine CD103+ dendritic cells appears particularly efficient [28,29]. Several authors have reported that murine CD103+ gut dendritic cells are required for de-novo Treg conversion through mechanisms involving transforming factor-β (TGF-β), retinoic acid and indoleamine 2,3-dioxygenase (IDO) [7,30,31]. Moreover, CD103+ dendritic cells in human mesenteric lymph nodes (MLNs) are potent inducers of allogeneic Treg [32].

We hypothesized that the increased frequency of Treg in lymphoid tissue of SIV-infected macaques could be due to enhanced dendritic cell-mediated conversion. To test this hypothesis, we isolated immature and mature myeloid dendritic cells (mDCs) from MLNs and spleen (SPL) of SIV-infected and uninfected Rhesus macaques, cultured them with autologous CD4+CD25 non-Treg for 4 days, and examined levels of CD25 and FOXP3 in T cells. Our results suggest that SIV infection promotes dendritic cell-mediated Treg conversion in both the SPL and MLN. Interestingly, a higher level of conversion was observed for the mature dendritic cell population; however, Treg induction did not correlate with CD103 expression by dendritic cell.

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Methods

Animals and viral infection

Colony-bred Rhesus macaques (Macaca mulatta) were obtained from the California National Primate Research Center (Davis, California, USA). Animals were housed and handled in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care International, and the study was reviewed and approved by the animal care and use committees at UC Davis. Care and use of animals were in compliance with institutional (National Institutes of Health) guidelines. Prior to the study, all animals were determined to be seronegative for SIV, simian T-cell lymphotropic virus type 1, and simian retrovirus. Samples were obtained from four mature multiparous cycling female macaques infected via vaginal inoculation with SIVmac251 (5000 TCID50) and seven mature males infected via penile inoculation with SIVmac251 (104 or 105 TCID50). The virology of three of these animals has been published [33]; the others are part of a control group for an unpublished vaccine study. None of the animals received antiretroviral therapy or immune modulators before euthanasia. Real-time PCR (sensitivity: 125 copies/ml) was used to determine plasma SIV RNA levels. Four uninfected animals were included in the study as negative controls. Clinical data at the time of euthanasia are shown in Table 1. Initial experiments were focused on conversion in spleen only due to the paucity of available tissues and did not include dendritic cell phenotyping, resulting in 7 SPL and 6 MLN samples for phenotypic analysis, and 9 SPL and 6 MLN samples for conversion experiments. The number of animals for which both dendritic cell phenotyping data and conversion data were available was 5 SPL and 6 MLN sample pairs.

Table 1
Table 1
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Cell isolation and culture

Single-cell suspensions from MLNs and SPL were generated the day of euthanasia. Each lymph node was dissected and cells were detached from the surrounding membrane using a scalpel. SPL tissue was diced and dissociated into a homogenous cell suspension using a pestle. MLN and SPL cell suspensions were passed through 70 μm cell strainers, washed in Roosvelt Park Memorial Institute 1640 containing 15% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 IU/ml streptomycin, and 2 mmol/l glutamine, and red blood cell lysis performed as needed using ammonium chloride/potassium carbonate/Ethylenediaminetetraacetic acetic. Cells were rested overnight at 37°C and 5% CO2. After overnight culture, viability was consistently above 95% (trypan blue exclusion test).

Spleen CD4+ T cells were purified from cell suspensions (300 × 106 cells on average) by negative selection (CD4+ T Cell Isolation Kit nonhuman primate, Miltenyi Biotec; Auburn, CA). CD25 non-Treg (<1.0% FOXP3+ cells postisolation) were purified from isolated CD4+ T cells using CD25 MicroBeads for nonhuman primates (Miltenyi Biotec).

Dendritic cells were separated from an average of 300 × 106 MLN or SPL cells on a Cytomation MoFlo Cell Sorter (Beckman Coulter, Brea, California, USA). Cells were stained in phosphate-buffered saline containing 2% FBS using fluorochrome-conjugated antibodies. 4’,6-diamidino-2-phenylindole (1 μg/ml, final) was added to cells prior to sorting in order to exclude dead cells. mDCs were defined as Lineage (CD3CD14CD20NKG2DEpCam) but HLA-DR+ and CD11c+. Mature (CD83+) and immature (CD83) mDCs were sorted into separate tubes.

Dendritic cell/non-Treg co-cultures were established in 48-well plates at a ratio of 1 : 10 (5 × 104 dendritic cell : 5 × 105 non-Treg), previously determined to be optimal for FOXP3 induction. Kinetic experiments indicated that FOXP3 induction peaked after 4 days of culture. These experimental conditions were used throughout the study.

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Cell sorting and flow cytometry

The following antibodies (Abs) were used for isolation and characterization of dendritic cell: anti-lineage [CD14 (TuK4, Invitrogen; Carlsbad, California, USA); anti-CD20 (2H7, eBioscience; San Diego, California, USA); anti-CD3e (SP34, BD Pharmingen; San Diego, California, USA); anti-EpCam (9C4, BioLegend; San Diego, California, USA); anti-NKG2D-FITC (1D11, eBioscience)] and anti-CD83-PerCPCy5.5 (HB15e, BioLegend). Anti-HLA-DR-PE-Cy7 (L243, BD Pharmingen), anti-CD11c-AF700 (3.9, eBioscience), and anti-CD123-PE (7G3, BD Pharmingen) were included in the phenotyping panel in addition to anti-CD103 (2G5.1, AbDSerotec; Kidlington, UK), which was labeled using the Pacific Blue mouse IgG2a Zenon Labeling Kit (Invitrogen). APC-conjugated anti-HLA-DR (L243) and PE-conjugated anti-CD11c (3.9) were included in the dendritic cell-sorting panel. All antibodies were titrated prior to use.

The following Abs were used for phenotypic characterization of Treg and non-Treg: anti-CD3-Pacific Blue (SK7), anti-CD4-PerCP-Cy5.5 (L200), and anti-CD25-PE-Cy7 (MA251) (all from BD Pharmingen), and anti-FOXP3-AF647 (PCH101) (eBioscience). In order to analyze T-cell proliferation, non-Treg were labeled before culture with 0.312 μmol/l carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, Oregon, USA).

Cells were treated with 20 μg/ml of human IgG to block Fc receptors, stained for surface markers 30 min at 4°C in PBS, washed, and fixed in 1% paraformaldehyde. For intracellular staining, cells were fixed and permeabilized using the FOXP3 staining buffer set (eBioscience) as per the manufacturer's protocol. Samples were analyzed on a BD LSR-II Flow Cytometer. At least 250 000 events were recorded for each sample. Doublets were excluded on the basis of scatter properties and dead cells excluded using LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen). Data were analyzed using FlowJo software (TreeStar Inc., Ashland, Oregon, USA).

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Statistical analysis

GraphPad Prism (GraphPad Software, La Jolla, California, USA) was used to graph and analyze data for statistical significance. Intra-group comparison was analyzed using the Wilcoxon test. Inter-group comparison was analyzed using the Mann–Whitney test. Linear regression analysis was used to test correlations. P values between 0.1 and 0.05 were considered trends; P values of 0.05 or less were considered significant.

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Results

Simian immunodeficiency virus infection reduces the frequency of spleen dendritic cells whereas the frequency of mesenteric lymph node dendritic cells remains unchanged

A loss of circulating mDCs and plasmacytoid dendritic cells (pDCs) has been reported in HIV/SIV-infected animals [34–40]. It has been hypothesized that infection is associated with recruitment of dendritic cells to inflamed lymph nodes, partially explaining their disappearance from blood as infection progresses [39–42]. However, results concerning the frequency of these cells in lymphoid tissues are often conflicting. Such discrepancies arise from differences in the stage of the infection and type of tissue analyzed, as well as the use of different animal models and SIV strains [43–52]. Thus, we first investigated the frequency of dendritic cells in the MLN and SPL of SIV-uninfected and chronically infected Rhesus macaques. SIV-infected macaques trended toward a decreased frequency of mDC (LinHLA-DR+CD123CD11c+) in the SPL compared to uninfected animals (P = 0.1). In contrast, mDC frequency in the MLN was comparable in infected and uninfected macaques (Fig. 1a). Similar results were seen for pDC (LinHLA-DR+CD11cCD123+) in which SIV+ Rhesus macaques exhibited significantly higher levels of SPL pDC than uninfected animals (P = 0.04) and differences in MLN pDC between the two groups were negligible (Fig. 1b). Additionally, within control animals a higher frequency of dendritic cell was observed in SPL compared to MLN. This was true for both mDCs, which trended toward an increased frequency (P = 0.06) (Fig. 1a) and pDCs, in which the difference was significant (P = 0.04) (Fig. 1b). In contrast, no significant difference in dendritic cell frequencies was observed between tissues from the infected group. Notably, SIV infection did not change the median mDC/pDC ratios in MLN or SPL (P > 0.30) (Fig. 1c).

Fig. 1
Fig. 1
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Due to limitations in the amount of tissue available we were unable to study Treg induction by both mDC and pDC. For the current study we chose to focus on the more abundant mDC population.

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Maturation of myeloid dendritic cells is enhanced during simian immunodeficiency virus infection

Because dendritic cell maturation state can influence Treg induction [53,54], we examined mDC expression of CD83, a molecule strongly up-regulated on the surface of mature dendritic cells [55,56]. SIV infection enhanced the frequency of CD83+ dendritic cells in MLN (P = 0.05). A similar trend was observed in SPL, although this did not reach statistical significance (P = 0.15) (Fig. 1d). The frequency of mature mDCs correlated with plasma viral load (r = 044, P = 0.01) (Fig. 1e) and duration of infection (r = 0.39, P = 0.02) (Fig. 1f) but did not correlate with CD4 cell count or age (data not shown).

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Myeloid dendritic cells isolated from simian immunodeficiency virus-infected macaques induce autologous Treg

Several studies have reported an accumulation of Treg in lymphoid tissues during HIV/SIV infection [12–17,57]. However, the origin of these cells remains unclear. Recently, Banerjee et al.[2] showed that among APCs, mature mDCs are the most potent inducers of Treg. We therefore hypothesized that enhanced mDC-mediated conversion might play a role in Treg accumulation in lymphoid tissues during HIV/SIV infection.

To test this hypothesis, we sorted mature CD83+ mDC and immature CD83 mDC from the MLN and SPL of uninfected or SIV-infected animals (see figure, Supplemental Digital Content 1, sorting strategy, http://links.lww.com/QAD/A192). In parallel, autologous CD4+CD25FOXP3 non-Treg were isolated from spleen. Depletion of FOXP3+ cells was evaluated postpurification and found to be equal in the two groups of animals (data not shown). Immature or mature mDCs from the SPL or MLN were then cultivated with autologous CD4+CD25FOXP3 non-Treg for 4 days.

Non-Treg cultivated alone expressed neither CD25 nor FOXP3 (Fig. 2a); however, when cultivated with MLN mDCs, FOXP3 and CD25 were both up-regulated on a distinct subset of cells. This induction was significantly higher in co-cultures containing mDCs from infected animals than those from uninfected macaques and was observed for both mature (P < 0.01) and immature (P = 0.05) dendritic cells (Fig. 2b). Similarly, increased conversion was found in cultures with SPL mDC from infected macaques, mature or not (P < 0.01 for both) (Fig. 2c). When purified CD4+CD25+ Treg (FOXP3 expression > 80%) were cultured with MLN or SPL mDCs, they did not undergo proliferation (not shown). It therefore appears likely that the CD25+FOXP3+ cells present after 4 days of co-culture originated from conversion of CD4+CD25 non-Treg rather than from expansion of contaminating CD4+CD25+ Treg. Intriguingly, in SIV-infected animals mature mDCs induced more Treg than immature dendritic cells. This effect was a strong trend for MLN mDC (0.07) and significant for SPL mDC (P < 0.01) (Fig. 2d), suggesting that the activation level of mDC strongly influences regulation of the immune response.

Fig. 2
Fig. 2
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Numerous studies have highlighted the extent of Treg conversion in the gut [58–60]. We therefore compared the ability of mature MLN and SPL dendritic cells to induce Treg. Surprisingly, a comparable level of Treg induction was observed by mDC from MLN and SPL (P = 0.31) (Fig. 2e). Thus, dendritic cells from both tissues may actively promote Treg accumulation in the context of SIV.

The percentage of CD25+FOXP3+ T cells induced by mDC from SIV-infected macaques did not correlate with plasma viral load or CD4 cell count. In contrast, there was a trend toward an inverse correlation between the duration of infection and the percentage of induced CD25+FOXP3+ T cells (r = 0.24, P = 0.06) (see figure, Supplemental Digital Content 2, correlation analyses, http://links.lww.com/QAD/A192). This result suggests that exposure to SIV over time may either impair the capacity of dendritic cell to induce Treg or alternately, the ability of non-Treg to properly respond to dendritic cell-generated signals.

In order to further characterize the induced Treg, we labeled the isolated CD4+CD25 non-Treg with CFSE prior to culture with mDC. Because the efficiency of Treg induction by MLN and SPL mDC was similar, we combined these data for statistical purposes. In contrast to control animals, the majority of induced FOXP3+ cells in cultures from SIV-infected animals proliferated (P < 0.001) (Fig. 3a and b), offering a potential explanation for the higher percentage of CD25+FOXP3+ cells found in these cultures.

Fig. 3
Fig. 3
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In addition to proliferation, cell death may impact Treg frequency in our cultures. Non-Treg cultivated alone showed the highest level of mortality, suggesting that culture with dendritic cell improved their viability (not shown); nonetheless, no obvious differences in cell death were observed in vitro for uninfected and infected animals (Fig. 3c). These results were upheld when MLN and SPL were considered separately (not shown).

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CD103 expression in myeloid dendritic cells is not correlated with induction of Treg

Iliev et al. recently described CD103+ dendritic cells isolated from human MLN as consistent inducers of allogeneic Treg [32]. CD103 is an integrin involved in the retention of T cells and dendritic cells in the gastrointestinal tract [61,62]. CD103+ dendritic cells produce retinoic acid, which induces Treg in association with TGF-β [7,30,31]. Although mDCs were not sorted based on CD103 expression, we examined this molecule by flow cytometry in mature and immature mDCs from MLN and SPL. The frequency of CD103+ dendritic cells was highest in mature dendritic cells in both animal groups, whereas immature dendritic cells expressed only very low levels of CD103 (<6%) (data not shown). We therefore asked whether an increased frequency of CD103+ mDC in MLN and SPL of SIV+ macaques could explain their higher level of Treg conversion. Unexpectedly, CD103 trended toward higher expression in mature MLN mDC from uninfected compared to infected animals (P = 0.08), and the percentage of SPL CD103+ mDC was equivalent between the two groups. Moreover, whereas uninfected Rhesus macaques showed a strong trend toward increased CD103 expression in MLN mDC compared to SPL (P = 0.06), infected macaques maintained similarly low levels in both tissues (Fig. 4a). Thus, expression of CD103 by mDC did not appear to be predictive of their capacity to induce Treg. We directly tested this hypothesis by analyzing the association between the percentage of CD103+ mDC in each cellular preparation from the infected macaques and their ability to induce Treg. Although there did appear to be a positive correlation, we did not find a statistically significant relationship for either MLN (Fig. 4b) or SPL (Fig. 4c).

Fig. 4
Fig. 4
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Discussion

Treg have been shown to accumulate in lymphoid tissues during chronic SIV infection, potentially contributing to the suppression of antiviral T-cell responses. Dendritic cells play a well established role in initiating adaptive immune responses to viral pathogens, including SIV; however, dendritic cells are also known to mediate peripheral tolerance, in part through the induction of Treg [28,63]. Because dendritic cells actively migrate to lymph nodes during SIV/HIV infection, we hypothesized that trafficking of dendritic cells during SIV/HIV infection may favor the induction of Treg within lymphoid tissues. Whereas we saw a decreased percentage of dendritic cells in the SPL of infected Rhesus macaques, levels in the MLN were comparable to those in uninfected controls, and mDCs from both the MLN and SPL of SIV-infected animals exhibited a more mature phenotype based on CD83 expression. These results are in agreement with previous studies showing altered maturation and activation of dendritic cells during HIV/SIV infection both in blood and lymphoid tissues, including the presence of semi-mature dendritic cells [26,47,48,64–67]. Semi-mature dendritic cells express high levels of co-stimulatory molecules, but do not secrete cytokines necessary for the development of effector T cells; rather, they are often associated with the induction of tolerance [53]. Additionally, high circulating levels of lipopolysaccaride typically found in chronic HIV and SIV infection [68,69] may result in dysfunctional dendritic cells, as prolonged in-vitro exposure to lipopolysaccharide produced ‘exhausted’ dendritic cells that could not prime T-helper 1 differentiation but instead secreted immunosuppressive IL-10 [70]. Therefore, although the mDCs we studied were phenotypically mature, they could have been functionally impaired or otherwise skewed toward Treg induction.

The results of our co-culture experiments indicated that mDCs from SIV-infected Rhesus macaques were significantly more efficient at inducing Treg than mDCs from uninfected animals and it was the mature rather than the immature mDCs that were most efficient at driving Treg induction. Immature dendritic cells are classically associated with the induction of tolerance; however, several studies have identified activated or semi-activated dendritic cells as efficient at inducing or expanding Treg [2,71–73], a function that may help mitigate host tissue damage due to an over-exuberant immune response. In lymph nodes of untreated HIV-infected individuals Krathwohl et al.[26] found a high percentage of CD83+IL-12 semi-mature dendritic cells which induced FOXP3 expression in allogeneic T cells ex vivo. Mature monocyte-derived dendritic cells exposed to high doses of HIV in vitro also induced an immunosuppressive phenotype in allogeneic T cells, characterized by increased expression of FoxP3 and Blimp-1[74]. Furthermore, expression of co-stimulatory molecules on mature dendritic cells is necessary for the maintenance of self-tolerance [75]. Thus, the activation state of dendritic cells in lymphoid tissues during HIV/SIV infection may support an environment favorable to Treg induction rather than the activation of effector T cells.

Interestingly, our results show that induction of CD25 and FOXP3 was not limited to the gut-associated lymphoid tissues of infected animals, as splenic dendritic cells were as efficient as MLN in this context. Neither do our results support a role for CD103 in dendritic cell-mediated Treg induction, as we did not find a significant correlation between CD103 expression by mature dendritic cells and their capacity to induce CD25 and FOXP3 in autologous T cells; however, this may be due to the low number of animals available for these analyses. It is possible a larger study would reveal a statistically significant relationship. Altogether, our data suggest that SIV infection promotes dendritic cell-mediated Treg induction in all lymphoid compartments, a finding consistent with previous studies showing increased Treg frequency in multiple lymphoid tissues of chronically infected Rhesus macaques, including the spleen, peripheral lymph nodes and colon [12,15].

Although we did not directly examine the molecular mechanism(s) involved in dendritic cell-mediated Treg induction, previous studies indicate possible roles for TGF-β and IDO. TGF-β, a cytokine known to be involved in Treg conversion, is increased in lymphatic tissues during SIV and HIV infection and appears to be generated by Treg present there [11,76]. TGF-β-producing Treg may then educate dendritic cells to become tolerogenic through a phenomenon termed ‘infectious tolerance’ [77,78], thereby perpetuating a cycle of immune-suppression. The tryptophan-catabolizing enzyme, IDO, has been implicated in SIV/HIV-mediated immune dysfunction [15,17,79] and several groups have described a role for IDO in driving Treg induction [5,71,72]. Manches et al.[27] found that pDCs exposed to HIV in vitro induced allogeneic Treg through an IDO-dependent mechanism. However, Kwa et al. recently reported higher IDO levels in mDC from MLN of SIV+ Rhesus macaques compared to pDCs. It is possible TGF-β and IDO contributed to mDC-mediated conversion in our cultures; however, further study will be needed to fully elucidate the mechanisms involved.

This study did not assess the suppressive capacity of the CD25+FOXP3+ cells generated in vitro due to difficulties in purifying a sufficient number of viable CD25+ Treg post co-culture; an additional group of animals would have been required to address this question. Results of previous studies indicate that similarly induced Treg are functionally suppressive [22,27,74]; nonetheless, this important question will need to be addressed in future experiments.

To our knowledge, our study is the first to show that SIV infection increases the capacity of dendrtic cells to induce FOXP3 expression in autologous T cells ex vivo. These data thus expand our knowledge of how dendritic cells may influence Treg frequencies within lymphoid tissues, and provide a potential mechanism underlying increased Treg frequencies found in tissues during progressive SIV/HIV infection. Regardless of the role played by Treg in HIV/SIV pathogenesis, a better understanding of the mechanisms regulating Treg dynamics could provide new opportunities for the development of targeted immuno-therapies designed to either boost anti-HIV responses or limit hyper-immune activation.

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Acknowledgments

The authors thank Linda Fritts, Linda Hirst and the veterinary staff at the California National Primate Research Center for assistance with these experiments, as well as Carol Oxford and Bridget McLaughlin for their expert help in cell sorting and flow cytometry.

P.P. and J.M.S. performed experiments, analyzed data, and wrote the manuscript. C.J.M., B.L.S., and C.A.C. designed the experiments and wrote the manuscript.

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Conflict of interest

The investigation was conducted in a facility constructed with support from the Research Facilities Improvement Program (grant C06 RR-12088-01) from the National Center for Research Resources, National Institutes of Health. The LSR-II violet laser was upgraded with funding from the James B. Pendleton Charitable Trust. These studies were supported in part by the California National Primate Research Center through a Pilot Project award funded by Base Grant NCRR-RR000169 and NIH P01 AI8227 to C.J.M. P.P. and C.A.C. are supported by NIH R01 AI068524. B.L.S. and J.M.S. are supported by NIH R01 AI057020.

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References

1. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008; 133:775–787.

2. Banerjee DK, Dhodapkar MV, Matayeva E, Steinman RM, Dhodapkar KM. Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients. Blood 2006; 108:2655–2661.

3. Beswick EJ, Pinchuk IV, Das S, Powell DW, Reyes VE. Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+ CD25+ FoxP3+ regulatory T cells. Infect Immun 2007; 75:4334–4341.

4. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 2003; 198:1875–1886.

5. Curti A, Pandolfi S, Valzasina B, Aluigi M, Isidori A, Ferri E, et al. Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25- into CD25+ T regulatory cells. Blood 2007; 109:2871–2877.

6. Iliev ID, Mileti E, Matteoli G, Chieppa M, Rescigno M. Intestinal epithelial cells promote colitis-protective regulatory T-cell differentiation through dendritic cell conditioning. Mucosal Immunol 2009; 2:340–350.

7. Sun CM, Hall JA, Blank RB, Bouladoux N, Oukka M, Mora JR, et al. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. J Exp Med 2007; 204:1775–1785.

8. Zahorchak AF, Raimondi G, Thomson AW. Rhesus monkey immature monocyte-derived dendritic cells generate alloantigen-specific regulatory T cells from circulating CD4+CD127-/lo T cells. Transplantation 2009; 88:1057–1064.

9. Curotto de Lafaille MA, Lafaille JJ. Natural and adaptive foxp3+ regulatory T cells: more of the same or a division of labor?. Immunity 2009; 30:626–635.

10. Estes JD, Li Q, Reynolds MR, Wietgrefe S, Duan L, Schacker T, et al. Premature induction of an immunosuppressive regulatory T cell response during acute simian immunodeficiency virus infection. J Infect Dis 2006; 193:703–712.

11. Estes JD, Wietgrefe S, Schacker T, Southern P, Beilman G, Reilly C, et al. Simian immunodeficiency virus-induced lymphatic tissue fibrosis is mediated by transforming growth factor beta 1-positive regulatory T cells and begins in early infection. J Infect Dis 2007; 195:551–561.

12. Nilsson J, Boasso A, Velilla PA, Zhang R, Vaccari M, Franchini G, et al. HIV-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV/AIDS. Blood 2006; 108:3808–3817.

13. Epple HJ, Loddenkemper C, Kunkel D, Troger H, Maul J, Moos V, et al. Mucosal but not peripheral FOXP3+ regulatory T cells are highly increased in untreated HIV infection and normalize after suppressive HAART. Blood 2006; 108:3072–3078.

14. Allers K, Loddenkemper C, Hofmann J, Unbehaun A, Kunkel D, Moos V, et al. Gut mucosal FOXP3+ regulatory CD4+ T cells and Nonregulatory CD4+ T cells are differentially affected by simian immunodeficiency virus infection in rhesus macaques. J Virol 2010; 84:3259–3269.

15. Boasso A, Vaccari M, Hryniewicz A, Fuchs D, Nacsa J, Cecchinato V, et al. Regulatory T-cell markers, indoleamine 2,3-dioxygenase, and virus levels in spleen and gut during progressive simian immunodeficiency virus infection. J Virol 2007; 81:11593–11603.

16. Andersson J, Boasso A, Nilsson J, Zhang R, Shire NJ, Lindback S, et al. The prevalence of regulatory T cells in lymphoid tissue is correlated with viral load in HIV-infected patients. J Immunol 2005; 174:3143–3147.

17. Favre D, Mold J, Hunt PW, Kanwar B, Loke P, Seu L, et al. Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 alters the balance of TH17 to regulatory T cells in HIV disease. Sci Transl Med 2010; 2:32ra36.

18. Chase AJ, Yang HC, Zhang H, Blankson JN, Siliciano RF. Preservation of FoxP3+ regulatory T cells in the peripheral blood of human immunodeficiency virus type 1-infected elite suppressors correlates with low CD4+ T-cell activation. J Virol 2008; 82:8307–8315.

19. Jiao Y, Fu J, Xing S, Fu B, Zhang Z, Shi M, et al. The decrease of regulatory T cells correlates with excessive activation and apoptosis of CD8(+) T cells in HIV-1-infected typical progressors, but not in long-term nonprogressors. Immunology 2008; 128:e366–e375.

20. Moreno-Fernandez ME, Rueda CM, Rusie LK, Chougnet CA. Regulatory T cells control HIV replication in activated T cells through a cAMP-dependent mechanism. Blood 2011; 117:5372–5380.

21. Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF. Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J Virol 2004; 78:2454–2459.

22. Kinter A, McNally J, Riggin L, Jackson R, Roby G, Fauci AS. Suppression of HIV-specific T cell activity by lymph node CD25+ regulatory T cells from HIV-infected individuals. Proc Natl Acad Sci U S A 2007; 104:3390–3395.

23. Kinter AL, Horak R, Sion M, Riggin L, McNally J, Lin Y, et al. CD25+ regulatory T cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIV-specific CD8+ T cells in vitro. AIDS Res Hum Retroviruses 2007; 23:438–450.

24. Weiss L, Donkova-Petrini V, Caccavelli L, Balbo M, Carbonneil C, Levy Y. Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T-cell responses in HIV-infected patients. Blood 2004; 104:3249–3256.

25. Ji J, Cloyd MW. HIV-1 binding to CD4 on CD4+CD25+ regulatory T cells enhances their suppressive function and induces them to home to, and accumulate in, peripheral and mucosal lymphoid tissues: an additional mechanism of immunosuppression. Int Immunol 2009; 21:283–294.

26. Krathwohl MD, Schacker TW, Anderson JL. Abnormal presence of semimature dendritic cells that induce regulatory T cells in HIV-infected subjects. J Infect Dis 2006; 193:494–504.

27. Manches O, Munn D, Fallahi A, Lifson J, Chaperot L, Plumas J, et al. HIV-activated human plasmacytoid DCs induce Tregs through an indoleamine 2,3-dioxygenase-dependent mechanism. J Clin Invest 2008; 118:3431–3439.

28. Belkaid Y, Oldenhove G. Tuning microenvironments: induction of regulatory T cells by dendritic cells. Immunity 2008; 29:362–371.

29. Josefowicz SZ, Rudensky A. Control of regulatory T cell lineage commitment and maintenance. Immunity 2009; 30:616–625.

30. Coombes JL, Siddiqui KR, Arancibia-Carcamo CV, Hall J, Sun CM, Belkaid Y, et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med 2007; 204:1757–1764.

31. Matteoli G, Mazzini E, Iliev ID, Mileti E, Fallarino F, Puccetti P, et al. Gut CD103+ dendritic cells express indoleamine 2,3-dioxygenase which influences T regulatory/T effector cell balance and oral tolerance induction. Gut 2010; 59:595–604.

32. Iliev ID, Spadoni I, Mileti E, Matteoli G, Sonzogni A, Sampietro GM, et al. Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells. Gut 2009; 58:1481–1489.

33. Ma ZM, Keele BF, Qureshi H, Stone M, Desilva V, Fritts L, et al.SIVmac251 is inefficiently transmitted to rhesus macaques by penile inoculation with a single SIVenv variant found in ramp-up phase plasma. AIDS Res Hum Retroviruses 2011. doi: 10.1089/aid.2011.0090.

34. Chehimi J, Campbell DE, Azzoni L, Bacheller D, Papasavvas E, Jerandi G, et al. Persistent decreases in blood plasmacytoid dendritic cell number and function despite effective highly active antiretroviral therapy and increased blood myeloid dendritic cells in HIV-infected individuals. J Immunol 2002; 168:4796–4801.

35. Donaghy H, Pozniak A, Gazzard B, Qazi N, Gilmour J, Gotch F, et al. Loss of blood CD11c(+) myeloid and CD11c(-) plasmacytoid dendritic cells in patients with HIV-1 infection correlates with HIV-1 RNA virus load. Blood 2001; 98:2574–2576.

36. Feldman S, Stein D, Amrute S, Denny T, Garcia Z, Kloser P, et al. Decreased interferon-alpha production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors. Clin Immunol 2001; 101:201–210.

37. Grassi F, Hosmalin A, McIlroy D, Calvez V, Debre P, Autran B. Depletion in blood CD11c-positive dendritic cells from HIV-infected patients. AIDS 1999; 13:759–766.

38. Soumelis V, Scott I, Gheyas F, Bouhour D, Cozon G, Cotte L, et al. Depletion of circulating natural type 1 interferon-producing cells in HIV-infected AIDS patients. Blood 2001; 98:906–912.

39. Barron MA, Blyveis N, Palmer BE, MaWhinney S, Wilson CC. Influence of plasma viremia on defects in number and immunophenotype of blood dendritic cell subsets in human immunodeficiency virus 1-infected individuals. J Infect Dis 2003; 187:26–37.

40. Pacanowski J, Kahi S, Baillet M, Lebon P, Deveau C, Goujard C, et al. Reduced blood CD123+ (lymphoid) and CD11c+ (myeloid) dendritic cell numbers in primary HIV-1 infection. Blood 2001; 98:3016–3021.

41. Fonteneau JF, Larsson M, Beignon AS, McKenna K, Dasilva I, Amara A, et al. Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells. J Virol 2004; 78:5223–5232.

42. Muller-Trutwin M, Hosmalin A. Role for plasmacytoid dendritic cells in anti-HIV innate immunity. Immunol Cell Biol 2005; 83:578–583.

43. Barratt-Boyes SM, Wijewardana V, Brown KN. In acute pathogenic SIV infection plasmacytoid dendritic cells are depleted from blood and lymph nodes despite mobilization. J Med Primatol 2010; 39:235–242.

44. Biancotto A, Grivel JC, Iglehart SJ, Vanpouille C, Lisco A, Sieg SF, et al. Abnormal activation and cytokine spectra in lymph nodes of people chronically infected with HIV-1. Blood 2007; 109:4272–4279.

45. Brown KN, Trichel A, Barratt-Boyes SM. Parallel loss of myeloid and plasmacytoid dendritic cells from blood and lymphoid tissue in simian AIDS. J Immunol 2007; 178:6958–6967.

46. Brown KN, Wijewardana V, Liu X, Barratt-Boyes SM. Rapid influx and death of plasmacytoid dendritic cells in lymph nodes mediate depletion in acute simian immunodeficiency virus infection. PLoS Pathog 2009; 5:e1000413.

47. Dillon SM, Robertson KB, Pan SC, Mawhinney S, Meditz AL, Folkvord JM, et al. Plasmacytoid and myeloid dendritic cells with a partial activation phenotype accumulate in lymphoid tissue during asymptomatic chronic HIV-1 infection. J Acquir Immune Defic Syndr 2008; 48:1–12.

48. Kwa S, Kannanganat S, Nigam P, Siddiqui M, Shetty RD, Armstrong W, et al.Plasmacytoid dendritic cells are recruited to the colorectum and contribute to immune activation during pathogenic SIV infection in rhesus macaques. Blood 2011. doi: 10.1182/blood-2011-02-339515.

49. Lehmann C, Lafferty M, Garzino-Demo A, Jung N, Hartmann P, Fatkenheuer G, et al. Plasmacytoid dendritic cells accumulate and secrete interferon alpha in lymph nodes of HIV-1 patients. PLoS One 2010; 5:e11110.

50. Nascimbeni M, Perie L, Chorro L, Diocou S, Kreitmann L, Louis S, et al. Plasmacytoid dendritic cells accumulate in spleens from chronically HIV-infected patients but barely participate in interferon-alpha expression. Blood 2009; 113:6112–6119.

51. Reeves RK, Fultz PN. Disparate effects of acute and chronic infection with SIVmac239 or SHIV-89.6P on macaque plasmacytoid dendritic cells. Virology 2007; 365:356–368.

52. Zhang L, Jiang Q, Li G, Jeffrey J, Kovalev GI, Su L. Efficient infection, activation, and impairment of pDCs in the BM and peripheral lymphoid organs during early HIV-1 infection in humanized rag2-/-{gamma} C-/- mice in vivo. Blood 2011; 117:6184–6192.

53. Lutz MB, Schuler G. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?. Trends Immunol 2002; 23:445–449.

54. Maldonado RA, von Andrian UH. How tolerogenic dendritic cells induce regulatory T cells. Adv Immunol 2010; 108:111–165.

55. Lechmann M, Zinser E, Golka A, Steinkasserer A. Role of CD83 in the immunomodulation of dendritic cells. Int Arch Allergy Immunol 2002; 129:113–118.

56. Zhou LJ, Tedder TF. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc Natl Acad Sci U S A 1996; 93:2588–2592.

57. Bandera A, Ferrario G, Saresella M, Marventano I, Soria A, Zanini F, et al. CD4+ T cell depletion, immune activation and increased production of regulatory T cells in the thymus of HIV-infected individuals. PLoS One 2010; 5:e10788.

58. Siddiqui KR, Powrie F. CD103+ GALT DCs promote Foxp3+ regulatory T cells. Mucosal Immunol 2008; 1 (Suppl 1):S34–S38.

59. Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science 2011; 331:337–341.

60. Broere F, du Pre MF, van Berkel LA, Garssen J, Schmidt-Weber CB, Lambrecht BN, et al. Cyclooxygenase-2 in mucosal DC mediates induction of regulatory T cells in the intestine through suppression of IL-4. Mucosal Immunol 2009; 2:254–264.

61. Gorfu G, Rivera-Nieves J, Ley K. Role of beta7 integrins in intestinal lymphocyte homing and retention. Curr Mol Med 2009; 9:836–850.

62. Cepek KL, Shaw SK, Parker CM, Russell GJ, Morrow JS, Rimm DL, et al. Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin. Nature 1994; 372:190–193.

63. Kushwah R, Hu J. Role of dendritic cells in the induction of regulatory T cells. Cell Biosci 2011; 1:20.

64. Harman AN, Wilkinson J, Bye CR, Bosnjak L, Stern JL, Nicholle M, et al. HIV induces maturation of monocyte-derived dendritic cells and Langerhans cells. J Immunol 2006; 177:7103–7113.

65. Jones GJ, Watera C, Patterson S, Rutebemberwa A, Kaleebu P, Whitworth JA, et al. Comparative loss and maturation of peripheral blood dendritic cell subpopulations in African and non-African HIV-1-infected patients. AIDS 2001; 15:1657–1663.

66. Buisson S, Benlahrech A, Gazzard B, Gotch F, Kelleher P, Patterson S. Monocyte-derived dendritic cells from HIV type 1-infected individuals show reduced ability to stimulate T cells and have altered production of interleukin (IL)-12 and IL-10. J Infect Dis 2009; 199:1862–1871.

67. Wang X, Zhang Z, Zhang S, Fu J, Yao J, Jiao Y, et al. B7-H1 up-regulation impairs myeloid DC and correlates with disease progression in chronic HIV-1 infection. Eur J Immunol 2008; 38:3226–3236.

68. Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, et al. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 2006; 12:1365–1371.

69. Leinert C, Stahl-Hennig C, Ecker A, Schneider T, Fuchs D, Sauermann U, et al. Microbial translocation in simian immunodeficiency virus (SIV)-infected rhesus monkeys (Macaca mulatta). J Med Primatol 2010; 39:243–251.

70. Langenkamp A, Messi M, Lanzavecchia A, Sallusto F. Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat Immunol 2000; 1:311–316.

71. Chung DJ, Rossi M, Romano E, Ghith J, Yuan J, Munn DH, et al. Indoleamine 2,3-dioxygenase-expressing mature human monocyte-derived dendritic cells expand potent autologous regulatory T cells. Blood 2009; 114:555–563.

72. Hill M, Tanguy-Royer S, Royer P, Chauveau C, Asghar K, Tesson L, et al. IDO expands human CD4+CD25high regulatory T cells by promoting maturation of LPS-treated dendritic cells. Eur J Immunol 2007; 37:3054–3062.

73. Verhasselt V, Vosters O, Beuneu C, Nicaise C, Stordeur P, Goldman M. Induction of FOXP3-expressing regulatory CD4pos T cells by human mature autologous dendritic cells. Eur J Immunol 2004; 34:762–772.

74. Shankar EM, Che KF, Messmer D, Lifson JD, Larsson M. Expression of a broad array of negative costimulatory molecules and Blimp-1 in T cells following priming by HIV-1 pulsed dendritic cells. Mol Med 2011; 17:229–240.

75. Lohr J, Knoechel B, Jiang S, Sharpe AH, Abbas AK. The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens. Nat Immunol 2003; 4:664–669.

76. Pal S, Schnapp LM. HIV-infected lymphocytes regulate fibronectin synthesis by TGF beta 1 secretion. J Immunol 2004; 172:3189–3195.

77. Belladonna ML, Orabona C, Grohmann U, Puccetti P. TGF-beta and kynurenines as the key to infectious tolerance. Trends Mol Med 2009; 15:41–49.

78. Waldmann H, Adams E, Fairchild P, Cobbold S. Infectious tolerance and the long-term acceptance of transplanted tissue. Immunol Rev 2006; 212:301–313.

79. Hryniewicz A, Boasso A, Edghill-Smith Y, Vaccari M, Fuchs D, Venzon D, et al. CTLA-4 blockade decreases TGF-beta, IDO, and viral RNA expression in tissues of SIVmac251-infected macaques. Blood 2006; 108:3834–3842.

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Keywords:

dendritic cells; FACS; lymphoid tissue; pathogenesis; regulatory; simian immunodeficiency virus; T lymphocytes

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