PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of aidMary Ann Liebert, Inc.Mary Ann Liebert, Inc.JournalsSearchAlerts
AIDS Research and Human Retroviruses
 
AIDS Res Hum Retroviruses. 2009 December; 25(12): 1313–1328.
PMCID: PMC2828165

Systemic Dendritic Cell Mobilization Associated with Administration of FLT3 Ligand to SIV- and SHIV-Infected Macaques

Abstract

Reports indicate that myeloid and plasmacytoid dendritic cells (mDCs and pDCs), which are key effector cells in host innate immune responses, can be infected with HIV-1 and are reduced in number and function during the chronic phase of HIV disease. Furthermore, it was recently demonstrated that a sustained loss of mDCs and pDCs occurs in SIV-infected macaques. Since loss of functional DC populations might impair innate immune responses to opportunistic microorganisms and neoplastic cells, we explored whether inoculation of naive and SIV- or SHIV-infected pigtailed macaques with the hematopoietic cytokine FLT3-ligand (FLT3-L) would expand the number of mDCs and pDCs in vivo. After the macaques received supraphysiologic doses of FLT3-L, mDCs, pDCs, and monocytes increased up to 45-fold in blood, lymph nodes, and bone marrow (BM), with DC expansion in the BM preceding mobilization in blood and lymphoid tissues. FLT3-L also increased serum levels of IL-12, at least transiently, and elicited higher surface expression of HLA-DR and the activation markers CD25 and CD69 on NK and T cells. During and after treatment of infected animals, APCs increased in number and were activated; however, CD4+ T cell numbers, virion RNA, and anti-SIV/SHIV antibody titers remained relatively stable, suggesting that FLT3-L might be a safe modality to expand DC populations and provide therapeutic benefit during chronic lentivirus infections.

Introduction

Dendritic cells (DCs) are antigen-presenting cells (APCs) that link the innate and adaptive immune systems by recognizing pathogens and initiating humoral and cell-mediated immune responses. Two major types of DCs circulate in human blood: CD11c+ myeloid (mDCs) and CD123+ plasmacytoid (pDCs). In general, mDCs are more competent than pDCs at presenting antigen (Ag) to naive T cells1,2; however, Hoeffel et al.3 recently showed in vitro that human pDCs cross-present HIV-1 epitopes, (as lipopeptides), or after uptake of HIV-1 Ags in apoptotic cells, as efficiently as mDCs. Following recognition of pathogen-associated molecular patterns contained in bacteria and viruses by toll-like receptors (TLRs), both DC subsets secrete cytokines and upregulate the costimulatory molecules CD80 and CD86.4,5 On virus activation pDCs secrete more interferon (IFN)-α per cell than other cell types and are called IFN-producing cells.6 Furthermore, mDCs and pDCs act synergistically to mediate immune responses against HIV-1 and tumor cells and to enhance the development and activation of regulatory T (Treg) and NK cells.711

Studies on mDCs and pDCs typically focus on cells in blood, but both DC subsets in various stages of development are common in murine, macaque (Macaca), and human bone marrow (BM), their site of origin.1215 DCs also accumulate in respiratory mucosae following allergic stimuli and in cerebrospinal fluid during bacterial meningitis, induce antiviral responses in the vaginal mucosa, and can be isolated from human tonsils, thymus, lungs, liver, kidney, colon, and peripheral lymph nodes (LNs).1624 Reduction in the numbers and functions of circulating mDCs and pDCs can occur during viral infections, such as with hepatitis B and C viruses, human T cell leukemia virus, SARS coronavirus, and HIV-1.2531 We and others showed that DC numbers are reduced in blood and secondary lymphoid organs of rhesus (Macaca mulatta) and pigtailed macaques (M. nemestrina) acutely and chronically infected with SIV.3234 Characterization of pDCs in BM of normal pigtailed macaques also revealed that while these cells are less mature phenotypically than their blood counterparts, they were responsive to microbial stimuli.15,35

Because of their importance in initiating immune responses to pathogens and Ags in vaccine formulations, developing novel strategies to mobilize, by activation and/or maturation, DC populations in vivo is a valid goal. CD34+ myeloid and lymphoid progenitor cells have high levels of Fms-like tyrosine kinase 3 (FLT3) receptors on their cell surfaces.36,37 The ligand for FLT3 (FLT3-L) is a cytokine expressed in most tissues, but it is at the highest levels on peripheral blood mononuclear cells (PBMCs). A soluble isoform of FLT3-L is also present in human blood at concentrations typically between 50 and 100 pg/ml.38 FLT3-L is elevated in serum from patients undergoing chemotherapy, and abnormally high levels are associated with hematologic disorders and autoimmune diseases.3941 Initially, murine studies showed that 9 days of subcutaneous inoculation of FLT3-L increased functional splenic DCs up to 17-fold.37,42,43 After human stem cell transplantation, administration of FLT3-L to patients elicited increases in DC numbers and enhanced recovery of DC and NK cell subsets.4446 FLT3-L-induced DC expansion, with subsequent emigration from BM, was first performed in macaques using a 10-day regimen that efficiently led to activated and increased numbers of DCs in blood,47 but Teleshova et al.48 showed that a 7-day regimen was equally effective. Similar experiments using a chimeric FLT3 and granulocyte colony-stimulating factor (G-CSF) receptor agonist, progenipoietin-1 (ProGP), were performed in naive and SIVmac32H-infected macaques.49 ProGP induced as much as a 50-fold expansion of blood DCs, but this increase was insufficient to augment responses to HIV Ags elicited by DNA vaccination and to protect macaques against challenge with SHIV-89.6P.50 In contrast, Kwissa et al.51 inoculated macaques with FLT3-L for 14 days followed by immunization, either alone or with CpG-B DNA, a TLR9 ligand, with plasmid DNA (recombinant vaccinia virus Ankara, rMVA) expressing both SIV and HIV proteins. Their results demonstrated that by day 14 of FLT3-L treatment, activation enhanced expression of CD86 on mDCs and monocytes and, after boosting twice with rMVA, the number of CD8+ T cells expressing IFN-γ increased. This regimen did not prevent infection in all immunized groups after rectal SIVmac251 challenge, but viremia was reduced at 2 and 24 weeks in the best responders, those that received CpG-B DNA and FLT3-L.

Although blood DCs can be activated and expanded during cytokine administration, previous studies did not clarify the extent to which mobilization occurred in macaque tissues, especially in BM where DC progenitors develop from CD34+ stem cells.4750 Since DCs play important roles in both innate and adaptive immunity, loss and/or impairment of these cells could exacerbate immune dysfunction observed during lentivirus disease. To determine whether targeting DCs as a way to enhance their numbers and functional maturity was a viable therapeutic strategy, we examined the kinetics of FLT3-L-induced activation and migration of mDCs and pDCs in blood, BM, and secondary lymphoid tissues in naive and SIV/SHIV-infected macaques.

Materials and Methods

Animals and FLT3-L administration

Adult pigtailed macaques of both sexes were used and included four uninfected macaques, four macaques infected for a mean duration of 213 weeks with SHIV-89.6P,52 and three macaques infected for a mean duration of 191 weeks with one of two SIVmac239-derived mutants: SIVmac239ΔGY(S>P) (macaques CT19 and 98P016) or SIVmac239-AIRPA (99P032).53 Macaque 98P016 was dually infected with SIVsmE660 for 109 weeks; reverse transcriptase polymerase chain reaction (RT-PCR) showed that SIVmac239ΔGY(S>P) predominated in plasma, with SIVsmE660 detected only sporadically (P.N. Fultz, Q. Wei, M. Piatak, J. Lifson, and J. Hoxie, unpublished observations). Animals were inoculated subcutaneously with sterile human recombinant FLT3-L, a gift from Amgen Corporation, for 7 consecutive days (100 μg/kg/day). Before each procedure (Table 1), macaques were anesthetized with an intramuscular injection of ketamine-HCl (10 mg/kg), weighed, their physical conditions were evaluated, and blood was collected. Necropsies were performed and tissues were taken after euthanasia with an intravenous overdose of pentobarbital (Fatal Plus; Vortech Pharmaceuticals). Macaques were housed in BSL2 isolation facilities at UAB in accordance with institutional and Animal Welfare Act guidelines. All procedures were approved by the UAB Institutional Animal Care and Use Committee.

Table 1.
Schedule (Days) a of Tissue Collections from Macaques

Tissue collection and processing

Heparinized blood was collected from the femoral vein; PBMCs were isolated by density gradient centrifugation through lymphocyte separation media (ICN Biomedicals, Inc.). BM was obtained from the medullary cavity of the proximal humerus with 15-gauge BM aspiration needles (Medical Device Technologies) previously flushed with heparin; yellow fat layers were removed from BM in EDTA-treated tubes. LNs and spleen tissue samples were minced and single-cell suspensions were generated by passing the fragments through metal cell strainers. Sections of the superior lobes of lungs were dissected, minced, and digested in medium supplemented with 0.5 mg of collagenase/ml at 37°C for 1 h, then passed through a cell strainer. At necropsy 6-cm sections of jejunum were removed, opened lengthwise, and rinsed with phosphate-buffered saline (PBS). The epithelial layer was removed and incubated in medium containing 0.5 mg of collagenase/ml at 37°C for 20 min. Digested samples were passed through a cell strainer, suspended in media, and passed over two glass wool columns to remove residual epithelial cells. Contaminating red blood cells were lysed hypotonically using an ammonium chloride solution. Mononuclear cells (MCs) were resuspended in RPMI-1640 with 10% fetal bovine serum (FBS(, or were cryopreserved in a dimethyl sulfoxide/FBS solution in liquid nitrogen vapor.

Flow cytometry

Three- and four-color flow cytometry were used to enumerate percentages of different cell populations and to characterize macaque cells in EDTA-treated whole blood or single-cell suspensions of BM, spleen, and LN tissues; cryopreserved MCs were used in some experiments. Lymphocyte subsets were phenotyped as follows: T cells were identified using Pacific Blue-conjugated anti-CD3 (SP34-2) with subsets distinguished by PE-conjugated anti-CD4 (SK3) and PerCp-conjugated anti-CD8 (SK1); NK cells were identified as CD3CD8+ and stained with PE-conjugated anti-CD16 (3G8) or anti-NKG2A (Z199; Immunotech). Activation markers were evaluated using FITC-conjugated anti-CD25 (2A3) or anti-CD69 (FN50). After identifying HLA-DR+, with PerCp-labeled anti-HLA-DR (G46-6), and lineage-negative (Lin) populations using an FITC-conjugated antibody cocktail to Lin markers—CD3epsilon (SP34), CD14 (M[var phi]P9), CD16 (3G8), and CD20 (2H7)—pDCs were distinguished with PE-labeled anti-CD123 (7G3) and mDCs by substituting PE-labeled anti-CD11c (S-HCL-3) and replacing CD16 in the FITC-labeled Lin cocktail with anti-CD8 (SK1). The latter was done because CD8 excludes most NK cells and macaque mDCs sometimes express low levels of CD16.32 Coreceptor and activation/maturation molecules were evaluated using anti-CD40 (5C3) or anti-CD86 (2331) antibodies, both of which were conjugated to allophycocyanin, or biotin-conjugated anti-CD80 (L307.4) and streptavidin-allophycocyanin. Isotype-matched antibody controls for each fluorochrome were always included. MCs were gated based on forward-and-side-scatter characteristics. All antibodies and reagents were purchased from BD Biosciences Pharmingen, unless otherwise noted; acquisitions were performed on a BD-LSRII.

Quantification of SIV viral RNA and SIV antibodies

SIV virion RNA (vRNA) in plasma was quantified at the Quantitative Molecular Diagnostics Core of the AIDS Vaccine Program, SAIC Frederick, NCI (Frederick, MD), as described previously (sensitivity, <100 copies/ml).54 Serum SIV-specific antibody titers were determined using a cross-reactive HIV-2 enzyme immunoassay kit (Bio-Rad).

Quantification of cytokines

FLT3-L concentrations in serum were determined using a Quantikine ELISA kit (R&D Systems) with a lower limit of detection of 7 pg/ml. Concentrations of IFN-α, tumor necrosis factor (TNF)-α, and interleukin (IL)-12p40 in serum samples were determined using cross-reactive human cytokine ELISA kits (Biosource); lower limits of detection were 10, 2, and 4 pg/ml, respectively.

Results

Hematologic changes associated with FLT3-L administration

Although FLT3-L upregulates hematopoiesis and cell proliferation in mice and humans, studies with macaques have provided limited data beyond specific myeloid or lymphoid subsets.42,4548 Previous studies did not examine the effects that subcutaneous inoculation of supraphysiologic doses of FLT3-L had on serum levels of this cytokine in normal macaques. On the day macaques first received FLT3-L (day 0), serum concentrations ranged from 18.1 to 170 pg/ml, with no obvious differences between infected and uninfected animals (Fig. 1A). However, by days 3 to 5 serum levels increased as high as 1370 pg/ml (macaque 97P009) and in individual animals the increase ranged from 7- to 44-fold (day 0 concentration to the highest amount). Serum FLT3-L in most animals rapidly declined after the last inoculation (day 6), with two exceptions: infected macaques CT19 and AV1C maintained high levels that were at or above 1000 pg/ml on day 12.

FIG. 1.
Hematologic parameters during and after administration of FLT3-L to macaques. (A) Serum levels of FLT3-L were quantified by ELISA. Absolute numbers of lymphocytes (B), monocytes (C), and platelets (D) per microliter of blood were determined from CBC and ...

The numbers of circulating lymphocytes (B, T, and NK cells), monocytes, and DCs were determined using complete blood counts and differentials and percentages of each population obtained by FACScan. The total numbers of blood lymphocytes before inoculation of FLT3-L appeared lower in infected animals compared to naive macaques: mean cell numbers/microliter for naive and SIVmac239- and SHIV-89.6P-infected macaques were 5279, 2558, and 2033 lymphocytes, respectively. Whether day 0 lymphocyte numbers from the naive outlier UA2 were included or excluded from its cohort, the mean number of lymphocytes in the naive group was statistically greater (Mann–Whitney U test), but only when compared to the means for the SHIV-89.6P-infected animals (p = 0.029) or for SIVmac239- and SHIV-89.6P-infected animals combined (p = 0.012, UA2 included; p = 0.033, UA2 excluded). Lymphocyte counts in most infected animals were relatively stable during FLT3-L treatment, whereas total lymphocytes in all naive animals fluctuated (Fig. 1B).

Numbers of blood monocytes in infected compared to control animals were marginally lower on day 0, but this population increased in all groups on days 6 to 12 (Fig. 1C). Because thrombocytopenia is a common hematologic manifestation of HIV- and SIV-induced disease,5557 platelet numbers were also evaluated. Before treatment, numbers of blood platelets had a large range in the SIVmac239- and SHIV-89.6P-infected macaques, but none was thrombocytopenic. Combining results from all animals, when day 0 platelet numbers were compared with nadirs that occurred between days 8 and 12, the observed transient decrease was significant (p < 0.001, Wilcoxon test).

Changes in predictors of disease progression in SIV- and SHIV-infected macaques

Because the FLT3-L-induced changes in numbers of PBMCs might influence virus replication in the infected animals, vRNA in plasma was quantified. Most animals had initial viral loads at or below the limit of detection; these levels changed marginally during or after treatment, with three exceptions (Fig. 2A). Macaques CT19 and 97P045 developed transient viremias on days 5 and 8, respectively. Macaque 98P016, infected with both SIVmac239ΔGY(S>P) and SIVsmE660, had 1900 copies of vRNA/ml on day 0, which then increased to 11,000 copies/ml on day 15, before decreasing to pretreatment levels. All SIVmac239- and SHIV-89.6P-infected animals had viral antibody titers that ranged from 400 to 409,600 (reciprocal serum dilutions); no significant changes in titers from those on day 0 were observed during or after FLT3-L treatment, which is consistent with minimal changes in viral loads. Loss of CD4+ T cells is a hallmark of progressive lentiviral disease; before FLT3-L treatment the mean numbers of CD4+ T cells in pigtailed macaques chronically infected with SIVmac239 mutants (702 /μl) or SHIV-89.6P (462 cells/μl) were lower than in naive controls (1499 cells/μl). Comparison of the medians of CD4+ T cells on day 0 to the highest number for each animal revealed that changes in this cell population were not significant (p = 0.212, Mann–Whitney U test) (Fig. 2B).

FIG. 2.
Changes in plasma virion RNA (A) and absolute numbers of CD4+ T cells (B) elicited by FLT3-L. The level of sensitivity for detection of SIV and SHIV virion RNA by RT-PCR was 30 copies/ml as indicated by the horizontal dotted line in (A). Absolute numbers ...

Changes in circulating numbers of mDCs and pDCs associated with FLT3-L

CD34+ myeloid and lymphoid progenitor cells express the FLT3 receptor; therefore, FLT3-L exogenously administered should promote proliferation and differentiation of DC subsets (Fig. 3A). Before injection of FLT3-L, the mean numbers of blood mDCs were lower in SIVmac239- and SHIV-89.6P-infected (50,200 and 67,800 mDCs/ml, respectively) macaques compared to those in naive animals (116,000 mDCs/ml) (Fig. 3B). However, no significant difference (Mann–Whitney U test) between naive and infected animals was noted, probably due to the broad distribution of mDCs and small sample sizes of each group. In general, the numbers of mDCs began to increase by day 4 or 5 and peaked around day 8; relative increases for individual animals ranged from 1.5- to 12-fold, with no differences between naive and infected animals (Fig. 3B). The relative increases in mDCs in SIVmac239-infected were higher than those in either SHIV-89.6P-infected or naive animals, resulting in slightly greater numbers of circulating mDCs when the numbers peaked.

FIG. 3.
Influence of FLT3-L on DC subsets in macaques. (A) Using flow cytometry, mDCs and pDCs were identified among blood mononuclear cells as negative for Lin markers (CD3, CD8 or CD16, CD14, and CD20), but positive for HLA-DR and either CD11c or CD123, respectively. ...

Consistent with previous observations, on day 0 the mean numbers of blood pDCs were lower in animals infected with SIVmac239 (3405 pDCs/ml) and, to a lesser extent, SHIV-89.6P (6,242 pDCs/ml), compared to naive macaques (12,220 pDCs/ml) (Fig. 3C).32,33 Again, these differences were not statistically different (Mann–Whitney U test). After FLT3-L treatment, pDCs increased as much as 10-fold in all groups, but the total numbers remained lower in infected compared to naive animals. Furthermore, the peak numbers of pDCs in both infected cohorts were greater than the normal range of 4000 to 17,000 pDCs/ml of blood previously reported for SIV-naive pigtailed macaques.33 Although the numbers of pDCs in all naive macaques peaked on day 8, in five of seven infected animals, the peak occurred on day 12. Numbers of circulating mDCs and pDCs did not correlate with serum levels of FLT3-L (data not shown).

Immune activation following administration of FLT3-L

Initially, expression of activation markers on blood DCs during or after administration of FLT3-L was not examined. Later, cryopreserved PBMCs, obtained either before treatment or on either day 8 or 12 (depending on availability), were used to compare expression of CD40, CD80, CD86, and HLA-DR. No significant changes in expression (MFI) of CD40, CD80, or CD86 were observed on either mDCs or pDCs, but in most animals, CD40 on mDCs was elevated moderately and changes in MFIs approached significance (Fig. 4). In contrast, after FLT3-L treatment HLA-DR expression on both DC subsets was upregulated significantly, with MFIs increasing as much as 3-fold.

FIG. 4.
Expression of activation markers on FLT3-L-mobilized DCs. Using four-color flow cytometry and cryopreserved PBMCs, cell-surface expression (MFIs) of the activation/costimulatory molecules CD40, CD80, and CD86 was measured on CD11c+HLA-DR+Lin ...

Monocytes and mDCs produce IL-12 and also secrete TNF-α; these cytokines synergize with pDC-secreted IFN-α to induce NK cell activation and promote a strong Th1 response.5861 However, during HIV infection, secretion of IL-12 and IFN-α is impaired. Exogenous FLT3-L resulted in transient increases in absolute numbers of circulating monocytes, mDCs, and pDCs, and also was associated with higher cell surface expression of HLA-DR; therefore, we determined whether these changes coincided with increased concentrations of DC- and monocyte-specific cytokines. On day 0, IL-12 was detected in serum from all animals, but the mean values were more than 2-fold higher in naive compared to both SIVmac239- and SHIV-89.6P-infected animals (Fig. 5A). Moderate increases in serum IL-12 occurred between days 8 to 12, with the largest increase (>4-fold) in macaque 97P045. Before the first FLT3-L injection, only two macaques had detectable serum IFN-α (Table 2). In macaques 97P009, CT19, AP1P, and AV1C, IFN-α was measured as early as day 4, but generally after day 10. On day 0, all serum samples were negative for TNF-α, but in one naive and three of four SHIV-89.6P-infected macaques, TNF-α was detected on one or more occasions during the 21 days of FLT3-L treatment and observation. Concentrations of TNF-α increased to greater than 27 pg/ml in 97P045 and 97P009, which was unusual because serum TNF-α typically is undetectable in naive pigtailed macaques and in those infected with various SIV/SHIV strains.62

FIG. 5.
Immune system activation in association with FLT3-L administration to macaques. (A) Serum concentrations of IL-12 were quantified by ELISA. Vertical dashed lines indicate the last day FLT3-L was administered. (B) The frequencies of CD4+CD3+ and CD8+CD3 ...
Table 2.
Serum Concentrations of IFN-α and TNF-α in Macaquesa

Both mDCs and pDCs activate T and NK cells, either by direct interactions or indirectly through cytokines, such as IFN-α, TNF-α, and IL-12, suggesting that the larger numbers of circulating DCs and higher serum cytokine concentrations might be associated with generalized activation and proliferation of both T and NK cells.9,5860 However, no consistent changes were observed in the numbers of CD3+CD4+ T cells and any increases were probably influenced by fluctuations in the absolute numbers of total lymphocytes that occurred in response to FLT3-L (Fig. 2B). Also, there were no significant changes in the total numbers of CD3+CD8+ T cells and NK cells (defined as CD16+CD8+CD3 or NKG2A+CD8+CD3) in either the infected or naive animals (data not shown). As a measure of T cell activation, we determined the frequencies of cells expressing the IL-2 receptor (CD25), which can be used either as a marker of T cell activation and proliferation or to identify Tregs.6366 In most animals, increases in the frequencies of CD4+ and CD8+ T cells expressing CD25 were observed: mean days of maximum expression on CD4+ and CD8+ T cells were 11.4 and 7.9 days, respectively (Fig. 5B). The frequencies of NK and CD8+ T cells expressing another activation marker, CD69, also significantly increased; the mean times of maximum CD69 expression on CD8+ T cells were 10.4 days and on NK cells were 11.2 days. The frequencies of CD69+ NK cells were always greater than those of CD8+CD69+ T cells, indicative of a more activated cell phenotype and consistent with previous observations for pigtailed macaques (Q. Wei and P.N. Fultz, unpublished data).62 Increases in both serum cytokine concentrations and activation states of T and NK cells suggested that FLT3-L administration induced peripheral immune system activation that coincided with maturation and increased numbers of circulating mDCs and pDCs.

Effects of FLT3-L on DCs in BM and secondary lymphoid tissues

To ascertain the effects of exogenous FLT3-L on DCs in macaque BM and peripheral LNs (PLNs), multiple single-cell suspensions of these tissues were evaluated. Before the study, the range of mDCs in BM was broad, but there was no apparent difference between numbers of mDCs in naive and infected animals, possibly related to the limited sample size (Fig. 6A). Similarly, the range of pDCs was highly variable, with some indication that pDCs in untreated animals might be lower in infected compared to naive macaques. In contrast to changes observed in blood, in the three animals for which day 4 biopsies were performed, the absolute numbers of both mDCs and pDCs in BM increased 8.5- and 2.5-fold, respectively. In most BM biopsies done after day 4, the numbers of mDCs and pDCs were consistently higher than those in the biopsies done pre-FLT3-L; e.g., in 98P016 mDCs and pDCs increased approximately 45- and 7-fold, respectively.

FIG. 6.
Augmentation of DC subsets in BM (A) and PLN (B) of macaques by administration of FLT3-L. The absolute numbers of DCs per milliliter of BM aspirate were calculated by multiplying the percentages of CD11c+HLA-DR+Lin mDCs and CD123+HLA-DR+Lin ...

In these cohorts of naive and infected animals there were no differences in the initial numbers of either DC subset in PLNs (Fig. 6B). In the three macaques for which PLN biopsies were performed on day 4, no changes in either mDCs or pDCs were observed; however, mDCs and pDCs in most animals increased during days 8 to 12 and then stabilized or decreased. The largest increase in mDCs was 35-fold for macaque 98P012 and in pDCs, 11-fold for macaque CT19.

mDCs and pDCs were enumerated in mesenteric LNs (MLNs), spleens, lungs, and jejunums collected at necropsy (Table 3). While the numbers of DCs in each of these tissues varied considerably, mDCs typically were more prevalent than pDCs, consistent with observations in blood, BM, and PLNs. Lung and spleen tissues were particularly enriched for mDCs, while jejunums appeared to contain the lowest numbers (Fig. 7). Since MC fractions from jejunums were eluted over glass wool columns to remove epithelial cells and mDCs exhibit some adherent properties (R.K. Reeves, unpublished), the prevalence of these cells in jejunums might be underestimated. Furthermore, although DCs were not enumerated in MLNs, spleens, lungs, and jejunums of FLT3-L-naive animals, we previously reported pDC frequencies in MLNs and spleens of SIV/SHIV-infected and uninfected macaques that did not receive FLT3-L.33 The median numbers of pDCs/106 MLN-MCs were 300, 600, and 400 in SIVmac239- and SHIV-89.6P-infected and uninfected animals, respectively. The numbers of pDCs in MLNs of FLT3-L-treated macaques in the current study were consistently greater (up to 15-fold, Table 3), regardless of viral status. Interestingly, the numbers of pDCs in spleens of FLT3-L-treated virus-naive and SHIV-89.6P-infected macaques were comparable or only slightly greater than the numbers of pDCs in the corresponding FLT3-L-naive macaques previously studied.33 All FLT3-L-treated SIVmac239-infected animals had greater numbers than the median of approximately 600 pDCs in untreated, similarly infected macaques; e.g., 99PO32 had almost 5000 pDCs/106 splenic MCs. These results suggested that expansion of DCs in tissues other than blood, BM, and PLNs also occurs in response to high levels of FLT3-L; however, a caveat to the tissue data (Table 3) is that MLNs, spleens, lungs, and jejunums from FLT3-L-treated macaques were taken on day 12 or later, which could be after the time of peak DC expansion.

FIG. 7.
Detection of mDCs (A) and pDCs (B) in macaque jejunum and lung. DC subsets were identified by flow cytometry among mononuclear cells (gated as in Fig. 3A, panel 1) in single-cell suspensions of various tissues (Table 3). Representative examples of FACScan ...
Table 3.
DC Subsets in Peripheral Tissues of Macaques at Time of Necropsya

Discussion

Strategies to treat HIV disease and its associated hematologic abnormalities are often tested using macaque models. In this study we inoculated uninfected and SIV- or SHIV-infected pigtailed macaques with the hematopoietic cytokine FLT3-L and evaluated its effects on (1) hematologic parameters, (2) SIVmac239 and SHIV-89.6P viremia and disease progression, and (3) immune cell activation. Previous studies in which humans and nonhuman primates were inoculated with FLT3-L did not quantify serum concentrations and most did not evaluate animals infected with a lentivirus. Normal serum concentrations of FLT3-L in pigtailed macaques were similar to levels found in two studies of healthy humans, which reported medians of 69 and 72 pg of FLT3-L/ml,38,40 but they were lower than a mean of 132 pg of FLT3-L/ml reported for a cohort of 72 cynomolgus macaques (M. fascicularis).67 In our study, serum FLT3-L increased to more than 1000 pg/ml in some animals, but quickly returned to baseline levels, suggesting rapid receptor-mediated uptake and/or metabolism of the exogenous cytokine. Our findings, however, indicate that peak levels of FLT3-L in macaques were similar to those observed in humans and macaques with chemotherapeutic- or radiation-induced aplasia where hematopoiesis is upregulated due to pancytopenia.41,44,67

Higher serum concentrations of FLT3-L coincided with higher numbers of monocytes, but alterations in the numbers of circulating lymphocytes were inconsistent. This finding agrees with that of Papayannopoulou et al.68 who showed that in a cohort of FLT3-L-treated naive pigtailed macaques, myeloid progenitors were mobilized at a greater frequency than lymphoid progenitor cells. Coates et al.,47 however, reported a significantly higher frequency of lymphocytes in rhesus macaques given FLT3-L and, although the absolute numbers of circulating lymphocytes were not shown, this disparity might reflect minor differences in the responsiveness to FLT3-L of pigtailed compared to rhesus macaques. We observed a transient decrease in platelets, a result not unexpected since it was reported that when added to cell cultures, FLT3-L inhibited megakaryocyte differentiation from CD34+ progenitors.69,70 Furthermore, increased serum levels of FLT3-L in cynomolgus macaques that developed radiation-induced aplasia were associated with a decline in platelets, and FLT3-L treatment counteracted the effects of thrombopoietin-induced restoration of platelet counts in myelosuppressed rhesus macaques.67,71 Although the decrease in platelets that we observed was minimal and resolved within about 4 days after the last FLT3-L injection, it is possible that therapeutic administration of FLT3-L during advanced lentiviral disease, when thrombocytopenia is common, might elicit complications.

FLT3-L is a requisite cytokine for development of progenitor cells of lymphoid and myeloid lineages in BM and, also, for expanding mDCs and pDCs derived from these progenitor cells in peripheral lymphoid tissues.36,4346,72,73 We report here expansion of mDCs and pDCs not only in naive but also in infected macaques that have reduced DC numbers, similar to HIV-1-infected patients.25,28,3133 Compared to previous studies in which mDCs in SIV-naive rhesus macaques were amplified more than 50-fold,47,48 we observed variable increases of up to 45-fold in mDCs. Since the magnitude of expansion of mDCs in naive and infected macaques did not differ, the generally higher fold increases in other studies cannot be attributed to infection status, but might reflect either differences in rhesus versus pigtailed macaques or natural variation among outbred animals. Also, it is possible that our evaluations were not done at optimum times since Teleshova et al.48 found the peak of DC expansion to be 10 days after initiation of the 7-day regimen, a day on which we did not examine most animals. While the magnitude and kinetics of monocyte and mDC expansion did not differ significantly between naive and infected macaques, the peak numbers of pDCs generally occurred later in infected (day 12) than in naive animals (day 8). The initial numbers of pDCs in blood from SIVmac239- and SHIV-89.6P-infected, compared to naive, animals were lower, suggesting that immediate pDC precursors might also be lower, which might delay increases of pDCs in blood. Although viral replication was generally not detected in these animals, it is possible newly activated, more mature pDCs, which are susceptible to lentivirus infection,33 might have become infected and destroyed, thus obscuring migration of these cells in blood. It should be noted, however, that tissue pDCs have short half-lives and must be replenished continually.74 Thus, maintenance of higher numbers of pDCs in LNs during HIV/SIV infections implies continual migration of these cells from BM to blood to LNs, which would be facilitated by persistent inflammatory conditions.

Compared to uninfected persons, activation marker expression on DCs is generally higher during chronic HIV-1 infections,31 but we found no such distinction in CD40, CD80, and CD86 expression when comparing cells from untreated naive and infected macaques. These differences might be related to the aviremic status of most of the macaques, a condition that might be associated with minimal immune system activation, or simply to differences in virus–host interactions in humans versus macaques. Also it is possible that the limited number of animals in our cohorts did not exhibit the full range of expression of these cell surface proteins. FLT3-L treatment of macaques, however, was associated with increases in HLA-DR expression on both mDCs and pDCs; CD80, CD86, and CD40 appeared to be upregulated in some animals. Using the same FLT3-L regimen, Teleshova et al.48 reported significant upregulation of CD80 and CD86 on mDCs and pDCs in rhesus macaques. In FLT3-L-treated melanoma and renal cancer patients, CD86 and HLA-DR, but not CD80, were upregulated on DCs.75 DCs mobilized in the lungs of FLT3-L-treated mice expressed higher levels of CD40, CD86, and HLA-DR than DCs in placebo-treated controls.76 Combined with our data, these observations strongly indicate that, in general, blood DCs not only increase in number during FLT3-L treatment, but also can be activated.

Coinciding with increases in circulating mDCs and pDCs were transiently higher serum concentrations of IL-12 and, in some macaques, of IFN-α and TNF-α. Consistent with our results, FLT3-L treatment of melanoma patients elicited higher serum levels of TNF-α (up to 100 pg/ml),77 and isolated DCs from both macaques and mice produced higher amounts of IFN-α and IL-12 ex vivo.78 Similarly, in vivo administration of FLT3-L resulted in higher amounts of IL-12 that accumulated in medium when macaque PBMCs were cultured ex vivo and compared to cultures from untreated animals.48 The results with IFN-α were in contrast to those regarding IL-12. In both SIV-infected macaques and HIV-infected persons, pDCs were activated and produced large amounts of IFN-α in vivo, but these cells were refractory to stimulation in vitro and, consequently, produced lower amounts of this cytokine when cultured.79,80 While the overall conclusion is that administration of FLT3-L to macaques enhances cytokine production, the identity of specific cell types and the tissue compartments in which they reside is unclear.

FLT3-L treatment of macaques induced higher frequencies of circulating CD25+CD4+ T cells, which are likely to be activated CD4+ T cells, with the possibility that some might be Tregs. Although evaluation of a more definitive Treg marker, such as FoxP3, would confirm the latter idea, mDCs and pDCs are known to induce Treg development by mechanisms that can involve IFN-α, TNF-α, and indoleamine 2,3-dioxygenase.11,8185 Specifically, during acute SIV infection of cynomolgus macaques, Malleret et al.85 found that pDCs and type I IFNs played important roles in eliciting an immunosuppressive state. Furthermore, IFN-α/β produced by pDCs following HIV infection can modulate proliferation and depletion of effector CD4+ T cells, also contributing to immunosuppression. Since Tregs can influence HIV/SIV disease progression by limiting virus-specific immune responses,65,66,8588 an increase in this population potentially might be detrimental for FLT3-L-based immunotherapies for HIV. However, Tregs, in general, are only a minor proportion of any CD4+CD25+ population.89 Our data also indicated that FLT3-L administration upregulated activation states, but not numbers, of NK cells, which contrasts with some murine studies in which FLT3-L treatment led to significant increases in NK cells concurrent with DC mobilization.90 The increased CD69 expression on NK cells that we observed likely is linked to higher serum IL-12, which activates NK cells.91 Whether activation enhanced the lytic activity of NK cells, which would be beneficial in lentivirus infections where NK cell numbers and functions can be impaired,9294 was not evaluated.

FLT3-L promotes cellular activation and proliferation; therefore, a major concern when treating lentivirus-infected individuals with this cytokine is the possibility of inducing virus replication. Transient increases in viremia were detected only in three animals, similar to Bucur et al.95 who found no increases in viral burdens in a group of SIVmac239-infected rhesus macaques treated with FLT3-L. Also, Koopman et al.49 observed no changes in viremia in a cohort of SIV-infected macaques treated with the related FLT3 agonist, ProGP-1. However, in our study, vRNA in plasma from 98P016 increased almost 6-fold after 15 days, suggesting that FLT3-L potentially might augment viral replication in animals with measurable viremia. Studies using larger numbers of macaques with higher levels of viremia at the start of the study are needed to address this possibility.

With the exception of Thomson's group, who also evaluated LNs, livers, and kidneys in rhesus macaques,47,96 previous studies showing FLT3-L-induced mobilization of DCs in humans and macaques evaluated only blood. Other studies utilized rhesus or cynomolgus macaques whereas we used pigtailed macaques—three distinct species—that are known to differ physiologically and in their pathological responses to some SIV strains. FLT3-L is expressed ubiquitously in tissues, while the FLT3 receptor is limited to CD34+ progenitor cells, including those for DCs36,37; therefore, initial DC activation should occur where CD34+ cells are concentrated. As in humans, we verified this site to be macaque BM and both mDC and pDC activation and mobilization in BM appeared to precede that in blood.35 In support of this finding, there was a significant correlation between serum levels of FLT3-L and DC numbers in BM (mDCs, R = 0.5630, p = 0.006; pDCs, R = 0.5991, p = 0.003), but not in blood. Although BM damage occurs in HIV, SIV, and SHIV infections,9799 it was not obvious that DC activation in BM was impaired in infected animals. Our detection of significant increases in both mDCs and pDCs in the PLNs of all animals is similar to that in previous surveys of macaques. It is interesting that, as in blood, DC numbers did not increase in PLNs on day 4, which is highly suggestive that DCs originated in the BM and then migrated through the blood to LNs. Because we detected low frequencies of CD34+ cells in both blood and LNs (not shown), we cannot exclude the possibility that some newly generated DCs were derived from localized progenitor cells and did not originate directly from BM precursors. Consistent with this possibility, Waskow et al.73 recently showed that DC precursors transit from BM to spleen and LNs where their division is controlled by FLT3-L.

Thus, based on our preliminary results in macaques chronically infected with SIV or SHIV and those of others showing increased numbers of different leukocyte subsets in blood and tissues following administration of FLT3-L to macaques,4749 additional studies are required to establish the potential efficacy of FLT3-L therapy in HIV-infected persons. These future studies should be a comprehensive evaluation of the positive and negative effects of FLT3-L administration to SIV-infected macaques that are nonprogressors with low viral loads and to those exhibiting signs of disease progression.

Acknowledgments

The authors thank M.L. Spell and the UAB Center for AIDS Research Flow Cytometry core, supported by NIH Grant P30 AI027767, for flow cytometric analyses; Dr. Robert Baker and Deidra Isbell for performing biopsies; and Drs. Mike Piatak and Jeff Lifson for determining plasma virion RNA concentrations. Amgen Corp. (Thousand Oaks, CA) generously provided FLT3-L. This work was supported by grants from the National Institutes of Health: UO1 AI028147; three animals were supported by NIAID RO1 AI049784, as part of a collaborative study with Dr. James Hoxie (to be reported elsewhere), who provided the SIVmac239 mutant viruses for the original study. R.K.R. was supported by an NIH Basic Mechanisms of Virology training grant, T32 AI007150-29.

Disclosure Statement

No competing financial interests exist.

References

1. Kohrgruber N. Halanek N. Groger M, et al. Survival, maturation, and function of CD11c- and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines. J Immunol. 1999;163:3250–3259. [PubMed]
2. O'Doherty U. Peng M. Gezelter S, et al. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology. 1994;82:487–493. [PubMed]
3. Hoeffel G. Ripoche AC. Matheoud D, et al. Antigen crosspresentation by human plasmacytoid dendritic cells. Immunity. 2007;27:481–492. [PubMed]
4. Iwasaki A. Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol. 2004;5:987–995. [PubMed]
5. Reis e Sousa C. Toll-like receptors and dendritic cells: For whom the bug tolls. Semin Immunol. 2004;16:27–34. [PubMed]
6. Siegal FP. Kadowaki N. Shodell M, et al. The nature of the principal type 1 interferon-producing cells in human blood. Science. 1999;284:1835–1837. [PubMed]
7. Fonteneau JF. Larsson M. Beignon AS, 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. [PMC free article] [PubMed]
8. Lou Y. Liu C. Kim GJ. Liu YJ. Hwu P. Wang G. Plasmacytoid dendritic cells synergize with myeloid dendritic cells in the induction of antigen-specific antitumor immune responses. J Immunol. 2007;178:1534–1541. [PubMed]
9. Gerosa F. Gobbi A. Zorzi P, et al. The reciprocal interaction of NK cells with plasmacytoid or myeloid dendritic cells profoundly affects innate resistance functions. J Immunol. 2005;174:727–734. [PubMed]
10. MacDonald KPA. Rowe V. Clouston AD, et al. Cytokine expanded myeloid precursors function as regulatory antigen-presenting cells and promote tolerance through IL-10-producing regulatory T cells. J Immunol. 2005;174:1841–1850. [PubMed]
11. Kawamura K. Kadowaki N. Kitawaki T. Uchiyama T. Virus-stimulated plasmacytoid dendritic cells induce CD4+ cytotoxic regulatory T cells. Blood. 2006;107:1031–1038. [PubMed]
12. Blom B. Ho S. Antonenko S. Liu YJ. Generation of interferon-α-producing predendritic cell (pre-DC)2 from human CD34+ hematopoietic stem cells. J Exp Med. 2000;192:1785–1795. [PMC free article] [PubMed]
13. Szabolcs P. Park KD. Reese M. Marti L. Broadwater G. Kurtzberg J. Absolute values of dendritic cell subsets in bone marrow, cord blood, and peripheral blood enumerated by a novel method. Stem Cells. 2003;21:296–303. [PubMed]
14. Li W. Liu YJ. Development of dendritic-cell lineages. Immunity. 2007;26:741–750. [PubMed]
15. Pinchuk LM. Grouard-Vogel G. Magaletti DM. Doty RT. Andrews RG. Clark EA. Isolation and characterization of macaque dendritic cells from CD34+ bone marrow progenitors. Cell Immunol. 1999;196:34–40. [PubMed]
16. Hartmann E. Graefe H. Hopert A, et al. Analysis of plasmacytoid and myeloid dendritic cells in nasal epithelium. Clin Vaccine Immunol. 2006;13:1278–1286. [PMC free article] [PubMed]
17. Pashenkov M. Teleshova N. Kouwenhoven M, et al. Recruitment of dendritic cells to the cerebrospinal fluid in bacterial neuroinfections. J Neuroimmunol. 2002;122:106–116. [PubMed]
18. Zhao X. Deak E. Soderberg K, et al. Vaginal submucosal dendritic cells, but not Langerhans cells, induce protective Th1 responses to herpes simplex virus-2. J Exp Med. 2003;197:153–162. [PMC free article] [PubMed]
19. Middel P. Raddatz D. Gunawan B. Haller F. Radzun HJ. Increased number of mature dendritic cells in Crohn's disease: Evidence for a chemokine mediated retention mechanism. Gut. 2006;55:220–227. [PMC free article] [PubMed]
20. Vandenabeele S. Hochrein H. Mavaddat N. Winkel K. Shortman K. Human thymus contains 2 distinct dendritic cell populations. Blood. 2001;97:1733–1741. [PubMed]
21. Summers KL. Hock BD. McKenzie JL. Hart DNJ. Phenotypic characterization of five dendritic cell subsets in human tonsils. Am J Pathol. 2001;159:285–295. [PubMed]
22. Masten BJ. Olson GK. Tarleton CA, et al. Characterization of myeloid and plasmacytoid dendritic cells in human lung. J Immunol. 2006;177:7784–7793. [PubMed]
23. Tanis W. Mancham S. Binda R, et al. Human hepatic lymph nodes contain normal numbers of mature myeloid dendritic cells but few plasmacytoid dendritic cells. Clin Immunol. 2004;110:81–88. [PubMed]
24. Woltman AM. de Fijter JW. Zuidwijk K, et al. Quantification of dendritic cell subsets in human renal tissue under normal and pathological conditions. Kidney Int. 2007;71:1001–1008. [PubMed]
25. Donaghy H. Pozniak A. Gazzard B, 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. [PubMed]
26. Goutagny N. Vieux C. Decullier E, et al. Quantification and functional analysis of plasmacytoid dendritic cells in patients with chronic hepatitis C infection. J Infect Dis. 2004;189:1646–1655. [PubMed]
27. Duan XZ. Wang M. Li HW. Zhuang H. Xu D. Wang FS. Decreased frequency and function of circulating plasmacytoid dendritic cells (pDC) in hepatitis B virus infected humans. J Clin Immunol. 2004;24:637–646. [PubMed]
28. Feldman S. Stein D. Amrute S, et al. Decreased interferon-α production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors. Clin Immunol. 2001;101:201–210. [PubMed]
29. Hishizawa M. Imada K. Kitawaki T. Ueda M. Kadowaki N. Uchiyama T. Depletion and impaired interferon-α-producing capacity of blood plasmacytoid dendritic cells in human T-cell leukaemia virus type 1-infected individuals. Br J Haematol. 2004;125:568–575. [PubMed]
30. Zhang Z. Xu D. Li Y, et al. Longitudinal alteration of circulating dendritic cell subsets and its correlation with steroid treatment in patients with severe acute respiratory syndrome. Clin Immunol. 2005;116:225–235. [PubMed]
31. 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. [PubMed]
32. Brown K. 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. [PubMed]
33. 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. [PMC free article] [PubMed]
34. 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. . doi:10.1371. [PMC free article] [PubMed]
35. Reeves RK. Fultz PN. Characterization of plasmacytoid dendritic cells in bone marrow of pig-tailed macaques. Clin Vaccine Immunol. 2008;15:35–41. [PMC free article] [PubMed]
36. Karsunky H. Merad M. Cozzio A. Weissman IL. Manz MG. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo. J Exp Med. 2003;198:305–313. [PMC free article] [PubMed]
37. D'Amico A. Wu L. The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hematopoietic precursors expressing Flt3. J Exp Med. 2003;198:293–303. [PMC free article] [PubMed]
38. Dettke M. Jurko S. Ruger BM, et al. Increased serum flt3-ligand in healthy donors undergoing granulocyte colony-stimulating factor-induced peripheral stem cell mobilization. J Hematother Stem Cell Res. 2001;10:317–320. [PubMed]
39. Gill MA. Blanco P. Arce E. Pascual V. Banchereau J. Palucka AK. Blood dendritic cells and DC-poietins in systemic lupus erythematosus. Hum Immunol. 2002;63:1172–1180. [PubMed]
40. Lyman SD. Seaberg M. Hanna R, et al. Plasma/serum levels of flt3 ligand are low in normal individuals and highly elevated in patients with Fanconi anemia and acquired aplastic anemia. Blood. 1995;86:4091–4096. [PubMed]
41. Wodnar-Filipowicz A. Lyman SD. Gratwohl A. Tichelli A. Speck B. Nissen C. Flt3 ligand level reflects hematopoietic progenitor cell function in aplastic anemia and chemotherapy-induced bone marrow aplasia. Blood. 1996;88:4493–4499. [PubMed]
42. Maraskovsky E. Brasel K. Teepe M, et al. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: Multiple dendritic cell subpopulations identified. J Exp Med. 1996;184:1953–1962. [PMC free article] [PubMed]
43. Shurin MR. Pandharipande PP. Zorina TD, et al. Flt3 ligand induces the generation of functionally active dendritic cells in mice. Cell Immunol. 1997;179:174–184. [PubMed]
44. Chklovskaia E. Nowbakht P. Nissen C. Gratwohl A. Bargetzi M. Wodnar-Filipowicz A. Reconstitution of dendritic and natural killer-cell subsets after allogeneic stem cell transplantation: Effects of endogenous flt3 ligand. Blood. 2004;103:3860–3868. [PubMed]
45. Pulendran B. Banchereau J. Burkeholder S, et al. Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo. J Immunol. 2000;165:566–572. [PubMed]
46. Maraskovsky E. Daro E. Roux E, et al. In vivo generation of human dendritic cell subsets by Flt3 ligand. Blood. 2000;96:878–884. [PubMed]
47. Coates PTH. Barratt-Boyes SM. Zhang L, et al. Dendritic cell subsets in blood and lymphoid tissue of rhesus monkeys and their mobilization with Flt3 ligand. Blood. 2003;102:2513–2521. [PubMed]
48. Teleshova N. Jones J. Kenney J, et al. Short-term Flt3L treatment effectively mobilizes functional macaque dendritic cells. J Leukoc Biol. 2004;75:1102–1110. [PubMed]
49. Koopman G. Niphuis H. Haaksma AGM, et al. Increase in plasmacytoid and myeloid dendritic cells by progenipoietin-1, a chimeric Flt-3 and G-CSF receptor agonist, in SIV-infected rhesus macaques. Hum Immunol. 2004;65:303–316. [PubMed]
50. Koopman G. Mortier D. Niphuis H, et al. Systemic mobilization of antigen presenting cells, with a chimeric Flt-3 and G-CSF receptor agonist, during immunization of Macaca mulatta with HIV-1 antigens is insufficient to modulate immune responses or vaccine efficacy. Vaccine. 2005;23:4195–4202. [PubMed]
51. Kwissa M. Amara RR. Robinson HL, et al. Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus. J Exp Med. 2007;204:2733–2746. [PMC free article] [PubMed]
52. Fultz PN. Stallworth JW. Porter D. Novak M. Anderson MJ. Morrow CD. Immunogenicity in pig-tailed macaques of poliovirus replicons expressing HIV-1 and SIV antigens and protection against SHIV-89.6P disease. Virology. 2003;315:425–437. [PubMed]
53. Wei Q. Stallworth JW. Vance PJ. Hoxie JA. Fultz PN. Simian immunodeficiency virus (SIV)/immunoglobulin G immune complexes in SIV-infected macaques block detection of CD16 but not cytolytic activity of natural killer cells. Clin Vaccine Immunol. 2006;13:768–778. [PMC free article] [PubMed]
54. Lifson JD. Rossio JL. Piatak M Jr, et al. Role of CD8+ lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment. J Virol. 2001;75:10187–10199. [PMC free article] [PubMed]
55. Scaradavou A. HIV-related thrombocytopenia. Blood Rev. 2002;16:73–76. [PubMed]
56. Fultz PN. Vance PJ. Endres MJ, et al. In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail. J Virol. 2001;75:278–291. [PMC free article] [PubMed]
57. Wachtman LM. Tarwater PM. Queen SE. Adams RJ. Mankowski JL. Platelet decline: An early predictive hematologic marker of simian immunodeficiency virus central nervous system disease. J Neurovirol. 2006;12:25–33. [PubMed]
58. Romagnani C. Chiesa MD. Kohler S, et al. Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells. Eur J Immunol. 2005;35:2452–2458. [PubMed]
59. Marshall JD. Heeke DS. Abbate C. Yee P. Van Nest G. Induction of interferon-γ from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid dendritic cell-produced interferon-a and tumour necrosis factor-α Immunology. 2006;117:38–46. [PubMed]
60. Tomescu C. Chehimi J. Maino VC. Montaner LJ. NK cell lysis of HIV-1-infected autologous CD4 primary T cells: Requirement for IFN-mediated NK activation by plasmacytoid dendritic cells. J Immunol. 2007;179:2097–2104. [PubMed]
61. Chehimi J. Starr SE. Frank I, et al. Impaired interleukin 12 production in human immunodeficiency virus-infected patients. J Exp Med. 1994;179:1361–1366. [PMC free article] [PubMed]
62. McGinn TM. Wei Q. Stallworth JW. Fultz PN. Immune responses to HTLV-1(ACH) during acute infection of pig-tailed macaques. AIDS Res Hum Retroviruses. 2004;20:443–456. [PubMed]
63. Mahalingam M. Peakman M. Davies ET. Pozniak A. McManus TJ. Vergani D. T cell activation and disease severity in HIV infection. Clin Exp Immunol. 1993;93:337–343. [PubMed]
64. Giavedoni LD. Velasquillo MC. Parodi LM. Hubbard GB. Hodara VL. Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus. J Virol. 2000;74:1648–1657. [PMC free article] [PubMed]
65. Estes JD. Li Q. Reynolds MR, et al. Premature induction of an immunosuppressive regulatory T cell response during acute simian immunodeficiency virus infection. J Infect Dis. 2006;193:703–712. [PubMed]
66. Tsunemi S. Iwasaki T. Imado T, et al. Relationship of CD4+CD25+ regulatory T cells to immune status in HIV-infected patients. AIDS. 2005;19:879–886. [PubMed]
67. Bertho JM. Demarquay C. Frick J, et al. Level of Flt3-ligand in plasma: A possible new bio-indicator for radiation-induced aplasia. Int J Radiat Biol. 2001;77:703–712. [PubMed]
68. Papayannopoulou T. Nakamoto B. Andrews RG. Lyman SD. Lee MY. In vivo effects of Flt3/Flk2 ligand on mobilization of hematopoietic progenitors in primates and potent synergistic enhancement with granulocyte colony-stimulating factor. Blood. 1997;90:620–629. [PubMed]
69. Sigurjonsson OE. Gudmundsson KO. Haraldsdottir V. Rafnar T. Gudmundsson S. Flt3/Flk-2-ligand in synergy with thrombopoietin delays megakaryocyte development and increases the numbers of megakaryocyte progenitor cells in serum-free cultures initiated with CD34+ cells. J Hematother Stem Cell Res. 2002;11:389–400. [PubMed]
70. Case J. Hicks C. Trickett A. Kwan YL. Manoharan A. The expansion of megakaryocyte progenitors from CD34+-enriched mobilized peripheral blood stem cells is inhibited by Flt3-L. J Interferon Cytokine Res. 2006;26:76–82. [PubMed]
71. Hartong SC. Neelis KJ. Wagemaker G. Co-administration of Flt-3 ligand counteracts the actions of thrombopoietin in myelosuppressed rhesus monkeys. Br J Haematol. 2003;121:359–367. [PubMed]
72. Chen W. Antonenko S. Sederstrom JM, et al. Thrombopoietin cooperates with FLT3-ligand in the generation of plasmacytoid dendritic cell precursors from human hematopoietic progenitors. Blood. 2004;103:2547–2553. [PMC free article] [PubMed]
73. Waskow C. Liu K. Darasse-Jeze G, et al. The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues. Nat Immunol. 2008;9:676–683. [PMC free article] [PubMed]
74. Merad M. Manz MG. Dendritic cell homeostasis. Blood. 2009;113:3418–3427. [PubMed]
75. Marroquin CE. Westwood JA. Lapointe R, et al. Mobilization of dendritic cell precursors in patients with cancer by Flt3 ligand allows the generation of higher yields of cultured dendritic cells. J Immunother. 2002;25:278–288. [PMC free article] [PubMed]
76. Masten BJ. Olson GK. Kusewitt DF. Lipscomb MF. Flt3 ligand preferentially increases the number of functionally active myeloid dendritic cells in the lungs of mice. J Immunol. 2004;172:4077–4083. [PubMed]
77. Shackleton M. Davis ID. Hopkins W, et al. The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptide vaccination with adjuvant Flt3 ligand. Cancer Immun. 2004;4:9. [PubMed]
78. Vollstedt S. O'Keeffe M. Odermatt B, et al. Treatment of neonatal mice with Flt3 ligand leads to changes in dendritic cell subpopulations associated with enhanced IL-12 and IFN-α production. Eur J Immunol. 2004;34:1849–1860. [PubMed]
79. Lehmann C. Harper JM. Taubert D, et al. Increased interferon alpha expression in circulating plasmacytoid dendritic cells in HIV-1-infected patients. J Acquir Immune Defic Syndr. 2008;48:522–530. [PubMed]
80. Tilton JC. Manion MM. Luskin MR, et al. Human Immunodeficiency virus viremia induces plasmacytoid dendritic cell activation in vivo and diminished alpha interferon production in vitro. J Virol. 2008;82:3997–4006. [PMC free article] [PubMed]
81. Moseman EA. Liang X. Dawson AJ, et al. Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. J Immunol. 2004;173:4433–4442. [PubMed]
82. Dhodapkar MV. Steinman RM. Antigen-bearing immature dendritic cells induce peptide-specific CD8+ regulatory T cells in vivo in humans. Blood. 2002;100:174–177. [PubMed]
83. Chen X. Baumel M. Mannel DN. Howard OM. Oppenheim JJ. Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. J Immunol. 2007;179:154–161. [PubMed]
84. Manches O. Munn D. Fallahi A, et al. HIV-activated human plasmacytoid DCs induce Tregs through an indoleamine 2,3-dioxygenase-dependent mechanism. J Clin Invest. 2008;118:3431–3439. [PMC free article] [PubMed]
85. Malleret B. Maneglier B. Karlsson I, et al. Primary infection with simian immunodeficiency virus: Plasmacytoid dendritic cell homing to lymph nodes, type I interferon, and immune suppression. Blood. 2008;112:4598–4608. [PubMed]
86. Karlsson I. Malleret B. Brochard P, et al. FoxP3+ CD25+ CD8+ T-cell induction during primary simian immunodeficiency virus infection in cynomolgus macaques correlates with low CD4+ T-cell activation and high viral load. J Virol. 2007;81:13444–13455. [PMC free article] [PubMed]
87. Sedaghat AR. German J. Teslovich TM, et al. Chronic CD4+ T-cell activation and depletion in human immunodeficiency virus type 1 infection: Type I interferon-mediated disruption of T-cell dynamics. J Virol. 2008;82:1870–1883. [PMC free article] [PubMed]
88. Nilsson J. Boasso A. Velilla PA, 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. [PubMed]
89. Piccirillo CA. Shevach EM. Naturally occurring CD4+ CD25+ immunoregulatory T cells: Central players in the arena of peripheral tolerance. Semin Immunol. 2004;16:81–88. [PubMed]
90. Shaw SG. Maung AA. Steptoe RJ. Thomson AW. Vujanovic NL. Expansion of functional NK cells in multiple tissue compartments of mice treated with Flt3-ligand: Implications for anti-cancer and anti-viral therapy. J Immunol. 1998;161:2817–2824. [PubMed]
91. Biron CA. Nguyen KB. Pien GC. Cousens LP. Salazar-Mather TP. Natural killer cells in antiviral defense: Function and regulation by innate cytokines. Annu Rev Immunol. 1999;17:189–220. [PubMed]
92. Hu PF. Hultin LE. Hultin P, et al. Natural killer cell immunodeficiency in HIV disease is manifest by profoundly decreased numbers of CD16+CD56+ cells and expansion of a population of CD16dimCD56- cells with low lytic activity. J Acquir Immune Defic Syndr. 1995;10:331–340. [PubMed]
93. Azzoni L. Rutstein RM. Chehimi J. Farabaugh MA. Nowmos A. Montaner LJ. Dendritic and natural killer cell subsets associated with stable or declining CD4+ cell counts in treated HIV-1-infected children. J Infect Dis. 2005;191:1451–1459. [PubMed]
94. Mansour I. Doinel C. Rouger P. CD16+ NK cells decrease in all stages of HIV infection through a selective depletion of the CD16+CD8+CD3- subset. AIDS Res Hum Retroviruses. 1990;6:1451–1457. [PubMed]
95. Bucur SZ. Lackey DA. Adams JW, et al. Hematologic and virologic effects of lineage-specific and non-lineage-specific recombinant human and rhesus cytokines in a cohort of SIVmac239-infected macaques. AIDS Res Hum Retroviruses. 1998;14:651–660. [PubMed]
96. Morelli AE. Coates PT. Shufesky WJ, et al. Growth factor-induced mobilization of dendritic cells in kidney and liver of rhesus macaques: Implications for transplantation. Transplantation. 2007;83:656–662. [PubMed]
97. Lee CI. Cowan MJ. Kohn DB. Tarantal AF. Simian immunodeficiency virus infection of hematopoietic stem cells and bone marrow stromal cells. J Acquir Immune Defic Syndr. 2004;36:553–561. [PubMed]
98. Thiebot H. Louache F. Vaslin B, et al. Early and persistent bone marrow hematopoiesis defect in simian/human immunodeficiency virus-infected macaques despite efficient reduction of viremia by highly active antiretroviral therapy during primary infection. J Virol. 2001;75:11594–11602. [PMC free article] [PubMed]
99. Folks TM. Kessler SW. Orenstein JM. Justement JS. Jaffe ES. Fauci AS. Infection and replication of HIV-1 in purified progenitor cells of normal human bone marrow. Science. 1988;242:919–922. [PubMed]

Articles from AIDS Research and Human Retroviruses are provided here courtesy of Mary Ann Liebert, Inc.