Accumulation of functionally immature myeloid dendritic cells in lymph nodes of rhesus macaques with acute pathogenic simian immunodeficiency virus infection.

Myeloid dendritic cells (mDC) are key mediators of innate and adaptive immunity to virus infection, but the impact of HIV infection on the mDC response, particularly early in acute infection, is ill-defined. We studied acute pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques to address this question. The mDC in blood and bone marrow were depleted within 12 days of intravenous infection with SIVmac251, associated with a marked proliferative response. In lymph nodes, mDC were apoptotic, activated and proliferating, despite normal mDC numbers, reflecting a regenerative response that compensated for mDC loss. Blood mDC had increased expression of MHC class II, CCR7 and CD40, whereas in lymph nodes these markers were significantly decreased, indicating that acute infection induced maturation of mDC in blood but resulted in accumulation of immature mDC in lymph nodes. Following SIV infection, lymph node mDC had an increased capacity to secrete tumour necrosis factor-α upon engagement with a Toll-like receptor 7/8 ligand that mimics exposure to viral RNA, and this was inversely correlated with MHC class II and CCR7 expression. Lymph node mDC had an increased ability to capture and cleave soluble antigen, confirming their functionally immature state. These data indicate that acute SIV infection results in increased mDC turnover, leading to accumulation in lymph nodes of immature mDC with an increased responsiveness to virus stimulation.

TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection.

Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activate DC and the expression patterns of TLR3 protein in DC subsets have not been established. In this article, we show that cross-priming CD8α(+) and CD103(+) DC subsets express much greater levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1(+) endosomes on stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of ∼90 bp was sufficient to induce CD86, IL-12p40, IFN-ß, TNF-α, and IL-6 expression, and to mature DC into APC that cross-presented exogenous Ags to CD8(+) T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of Ag-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.

In acute pathogenic SIV infection plasmacytoid dendritic cells are depleted from blood and lymph nodes despite mobilization.

BACKGROUND: Plasmacytoid dendritic cells (pDC) are depleted from blood of individuals with HIV infection associated with progression to disease. It has been postulated but not proven that pDC accumulate in lymph nodes and induce sustained immune activation characteristic of disease. METHODS: The dynamics of the pDC response to acute pathogenic SIV infection of rhesus macaques were studied using methods to track recently divided cells. RESULTS: pDC were lost from blood and lymph nodes in acute SIV infection despite rapid mobilization and recruitment. pDC had a low frequency of infection, were uniformly activated and had increased levels of apoptosis, while maintaining normal function. CONCLUSIONS: pDC mobilization into blood and lymph nodes in acute SIV infection does not keep pace with excessive pDC loss through activation and apoptosis. The depletion of pDC from lymphoid tissues in acutely infected rhesus macaques does not support a pathogenic role for pDC in disease.

Early myeloid dendritic cell dysregulation is predictive of disease progression in simian immunodeficiency virus infection.

Myeloid dendritic cells (mDC) are lost from blood in individuals with human immunodeficiency virus (HIV) infection but the mechanism for this loss and its relationship to disease progression are not known. We studied the mDC response in blood and lymph nodes of simian immunodeficiency virus (SIV)-infected rhesus macaques with different disease outcomes. Early changes in blood mDC number were inversely correlated with virus load and reflective of eventual disease outcome, as animals with stable infection that remained disease-free for more than one year had average increases in blood mDC of 200% over preinfection levels at virus set-point, whereas animals that progressed rapidly to AIDS had significant loss of mDC at this time. Short term antiretroviral therapy (ART) transiently reversed mDC loss in progressor animals, whereas discontinuation of ART resulted in a 3.5-fold increase in mDC over preinfection levels only in stable animals, approaching 10-fold in some cases. Progressive SIV infection was associated with increased CCR7 expression on blood mDC and an 8-fold increase in expression of CCL19 mRNA in lymph nodes, consistent with increased mDC recruitment. Paradoxically, lymph node mDC did not accumulate in progressive infection but rather died from caspase-8-dependent apoptosis that was reduced by ART, indicating that increased recruitment is offset by increased death. Lymph node mDC from both stable and progressor animals remained responsive to exogenous stimulation with a TLR7/8 agonist. These data suggest that mDC are mobilized in SIV infection but that an increase in the CCR7-CCL19 chemokine axis associated with high virus burden in progressive infection promotes exodus of activated mDC from blood into lymph nodes where they die from apoptosis. We suggest that inflamed lymph nodes serve as a sink for mDC through recruitment, activation and death that contributes to AIDS pathogenesis.

Rapid influx and death of plasmacytoid dendritic cells in lymph nodes mediate depletion in acute simian immunodeficiency virus infection.

Plasmacytoid dendritic cells (pDC) are essential innate immune system cells that are lost from the circulation in human immunodeficiency virus (HIV)-infected individuals associated with CD4(+) T cell decline and disease progression. pDC depletion is thought to be caused by migration to tissues or cell death, although few studies have addressed this directly. We used precise methods of enumeration and in vivo labeling with 5-bromo-2'-deoxyuridine to track recently divided pDC in blood and tissue compartments of monkeys with acute pathogenic simian immunodeficiency virus (SIV) infection. We show that pDC are lost from blood and peripheral lymph nodes within 14 days of infection, despite a normal frequency of pDC in bone marrow. Paradoxically, pDC loss masked a highly dynamic response characterized by rapid pDC mobilization into blood and a 10- to 20-fold increase in recruitment to lymph nodes relative to uninfected animals. Within lymph nodes, pDC had increased levels of apoptosis and necrosis, were uniformly activated, and were infected at frequencies similar to CD4(+) T cells. Nevertheless, remaining pDC had essentially normal functional responses to stimulation through Toll-like receptor 7, with half of lymph node pDC producing both TNF-alpha and IFN-alpha. These findings reveal that cell migration and death both contribute to pDC depletion in acute SIV infection. We propose that the rapid recruitment of pDC to inflamed lymph nodes in lentivirus infection has a pathologic consequence, bringing cells into close contact with virus, virus-infected cells, and pro-apoptotic factors leading to pDC death.

Surface phenotype and rapid quantification of blood dendritic cell subsets in the rhesus macaque.

BACKGROUND: The study of dendritic cell (DC) biology in the rhesus macaque is becoming increasingly important but is limited by incomplete characterization and the lack of a rapid assay to quantify cells. METHODS: We characterized the surface phenotype of myeloid (mDC) and plasmacytoid DC (pDC) subsets in healthy rhesus macaque blood and developed a flow cytometry-based assay for absolute DC determinations. RESULTS: Rhesus CD11c(+) mDC were CD16(+) CD11b(+) CD56(lo) CD8(-) CD1c(-) whereas CD123(+) pDC lacked expression of these markers. Precise DC determinations were performed using a rapid two-step assay combining the analysis of whole blood and peripheral blood leukocytes (PBL). CONCLUSIONS: Antibodies to CD11b, CD56 and CD16 must be omitted from the lineage antibody cocktail to prevent inadvertent gating-out of DC when analyzing rhesus blood. The combined whole-blood/PBL quantification assay will be invaluable for the rapid and repeated monitoring of blood DC counts in this species.

Studies of plasmacytoid dendritic cell dynamics in simian immunodeficiency virus infection of nonhuman primates provide insights into HIV pathogenesis.

Plasmacytoid dendritic cells (pDC) play a key role in antiviral immunity through their immense capacity to produce type I interferons (IFN) and other cytokines and through induction of antigen-specific T cell responses. Several reports have documented decreased numbers and reduced function of pDC in the circulation of HIV patients associated with progression to disease, indicating that pDC are likely to be important in control of HIV infection. The mechanism of pDC loss has not been determined and is difficult to address in natural infection of humans. As highlighted in this brief review, the study of pDC dynamics in simian immunodeficiency virus (SIV) infection of nonhuman primates paves the way to understanding the complex biology of this important innate system cell in HIV and other viral infections.

Parallel loss of myeloid and plasmacytoid dendritic cells from blood and lymphoid tissue in simian AIDS.

The loss of myeloid (mDC) and plasmacytoid dendritic cells (pDC) from the blood of HIV-infected individuals is associated with progressive disease. It has been proposed that DC loss is due to increased recruitment to lymph nodes, although this has not been directly tested. Similarly as in HIV-infected humans, we found that lineage-negative (Lin(-)) HLA-DR(+)CD11c(+)CD123(-) mDC and Lin(-)HLA-DR(+)CD11c(-)CD123(+) pDC were lost from the blood of SIV-infected rhesus macaques with AIDS. In the peripheral lymph nodes of SIV-naive monkeys the majority of mDC were mature cells derived from skin that expressed high levels of HLA-DR, CD83, costimulatory molecules, and the Langerhans cell marker CD1a, whereas pDC expressed low levels of HLA-DR and CD40 and lacked costimulatory molecules, similar to pDC in blood. Surprisingly, both DC subsets were depleted from peripheral and mesenteric lymph nodes and spleens in monkeys with AIDS, although the activation status of the remaining DC subsets was similar to that of DC in health. In peripheral and mesenteric lymph nodes from animals with AIDS there was an accumulation of Lin(-)HLA-DR(moderate)CD11c(-)CD123(-) cells that resembled monocytoid cells but failed to acquire a DC phenotype upon culture, suggesting they were not DC precursors. mDC and pDC from the lymphoid tissues of monkeys with AIDS were prone to spontaneous death in culture, indicating that apoptosis may be a mechanism for their loss in disease. These findings demonstrate that DC are lost from rather than recruited to lymphoid tissue in advanced SIV infection, suggesting that systemic DC depletion plays a direct role in the pathophysiology of AIDS.

Broad cellular immunity with robust memory responses to simian immunodeficiency virus following serial vaccination with adenovirus 5- and 35-based vectors.

Adenovirus serotype 35 (Ad35) is a promising vaccine platform for human immunodeficiency virus (HIV) infection and emerging infectious diseases as it is uncommon in humans worldwide and is distinct from Ad5, the major vaccine serotype for which many individuals have pre-existing immunity. The immunogenicity of a first-generation, replication-competent Ad35-based vaccine was tested in the simian immunodeficiency virus (SIV) rhesus macaque model by evaluating its capacity to boost immunity generated by Ad5-based vectors. A series of four immunizations with replication-defective Ad5 vectors expressing SIVmac239 gag induced high-frequency responses mediated by both CD8(+) and CD4(+) T cells directed against several epitopes. Ad5-specific neutralizing antibody responses that did not neutralize Ad35 were rapidly induced but waned over time. Subsequent immunization with Ad5-based vectors was minimally effective, whereas immunization with Ad35-based vectors generated a strong increase in the frequency of Gag-specific T cells with specificities that were unchanged. While this boosting response was relatively transient, challenge with the distinct pathogenic isolate SIV/DeltaB670 generated robust and selective recall responses to Gag with similar specificities as induced by vaccination that were elevated for 25 weeks relative to controls. Vaccination had measurable albeit minor effects on virus load. Unexpectedly, regional hypervariability within the Gag sequence of SIV/DeltaB670 was associated with mutation of the conserved CD8(+) T-cell epitope CM9 without concurrent flanking mutations and in the absence of immune pressure. These findings support the further development of Ad35 as a vaccine vector, and promote vaccine regimens that utilize serial administration of heterologous adenoviruses.

Understanding and exploiting dendritic cells in human immunodeficiency virus infection using the nonhuman primate model.

Dendritic cells (DC) are pivotal cells in the innate immune system. Recent interest in the role of DC in human immunodeficiency virus (HIV) pathogenesis has increased with the finding that both myeloid (mDC) and plasmacytoid DC (pDC) are lost from blood during infection, associated with progression to disease. DC are also being studied intensively for their capacity to stimulate robust virus-specific immunity as vaccines. Here we discuss our work in these contrasting fields of DC biology using the rhesus macaque nonhuman primate model. We focus on studies of DC dynamics in lymphoid tissues during pathogenic simian immunodeficiency virus (SIV) infection, DC trafficking in health and disease, DC-based vaccination and the use of autologous virus as antigen for stimulation of virus-specific T cells.