Species-specific differences in the expression and regulation of α4ß7 integrin in various nonhuman primates.

Among nonhuman primates, SIV-infected Asian pigtailed macaques (PM) are relatively more susceptible to infection and disease progression than SIV-infected rhesus macaques (RM). In addition, SIV-infected African natural hosts such as the sooty mangabeys (SM) are resistant to disease. The mechanisms associated with such species-related variable clinical outcomes remain ill-defined but hold the potential to provide insights into the underlying mechanisms surrounding HIV pathogenesis. Recent findings indicate that the expression of the heterodimeric gut homing integrin α4ß7 can influence both susceptibility and disease progression in RM. It was reasoned that differences in the frequencies/surface densities of α4ß7-expressing lymphocytes might contribute to the differences in the clinical outcome of SIV infection among NHPs. In this article, we report that CD4(+) T cells from PM constitutively express significantly higher levels of α4ß7 than RM or SM. Retinoic acid, a key regulator of α4ß7 expression, was paradoxically found at higher levels in the plasma of SM versus RM or PM. We also observed pairing of ß7 with αE (αEß7) on CD4(+) T cells in the peripheral blood of SM, but not PM or RM. Finally, the differential mean density of expression of α4ß7 in RM versus SM versus PM was predominantly dictated by species-specific sequence differences at the level of the ß7 promoters, as determined by in vitro reporter/promoter construct transfection studies. We propose that differences in the regulation and expression of α4ß7 may explain, in part, the differences in susceptibility and SIV disease progression in these NHP models.

In vivo administration of a JAK3 inhibitor during acute SIV infection leads to significant increases in viral load during chronic infection.

The studies reported herein are the first to document the effect of the in vivo administration of a JAK3 inhibitor for defining the potential role of NK cells during acute SIV infection of a group of 15 rhesus macaques (RM). An additional group of 16 MHC/KIR typed RM was included as controls. The previously optimized in vivo dose regimen (20 mg/kg daily for 35 days) led to a marked depletion of each of the major NK cell subsets both in the blood and gastro-intestinal tissues (GIT) during acute infection. While such depletion had no detectable effects on plasma viral loads during acute infection, there was a significant sustained increase in plasma viral loads during chronic infection. While the potential mechanisms that lead to such increased plasma viral loads during chronic infection remain unclear, several correlates were documented. Thus, during acute infection, the administration of the JAK3 inhibitor besides depleting all NK cell subsets also decreased some CD8⁺ T cells and inhibited the mobilization of the plasmacytoid dendritic cells in the blood and their localization to the GIT. Of interest is the finding that the administration of the JAK3 inhibitor during acute infection also resulted in the sustained maintenance during chronic infection of a high number of naïve and central memory CD4⁺ T cells, increases in B cells in the blood, but decreases in the frequencies and function of NKG2a⁺ NK cells within the GIT and blood, respectively. These data identify a unique role for JAK3 inhibitor sensitive cells, that includes NK cells during acute infection that in concert lead to high viral loads in SIV infected RM during chronic infection without affecting detectable changes in antiviral humoral/cellular responses. Identifying the precise mechanisms by which JAK3 sensitive cells exert their influence is critical with important implications for vaccine design against lentiviruses.

Relationships between IL-17(+) subsets, Tregs and pDCs that distinguish among SIV infected elite controllers, low, medium and high viral load rhesus macaques.

Comprehensive studies of the frequencies and absolute numbers of the various cell lineages that synthesize IL-17 in the blood and corresponding gastrointestinal (GI) tissues, their correlation with CD4(+) Tregs, CD8(+) Tregs, total and IFN-α synthesizing plasmacytoid dendritic cells (pDC) relative to plasma viral load in SIV infection has been lacking. The unique availability of SIV infected rhesus macaques (RM) classified as Elite Controllers (EC), and those with Low, Intermediate and High Viral Loads (HVL) provided a unique opportunity to address this issue. Results of these studies showed that EC demonstrated a remarkable ability to reverse changes that are induced acutely by SIV in the various cell lineages. Highlights of the differences between EC and HVL RM within Gastro-intestinal tissues (GIT) was the maintenance and/or increases in the levels of IL-17 synthesizing CD4, CD8, and NK cells and pDCs associated with slight decreases in the levels of CD4(+) Tregs and IFN-α synthesizing pDCs in EC as compared with decreases in the levels of IL-17 synthesizing CD4, CD8 and NK cells associated with increases in pDCs and IFN-α synthesizing pDCs in HVL monkeys. A previously underappreciated role for CD8(+) Tregs was also noted with a moderate increase in ECs but further increases of CD8(+) Tregs with increasing VL in viremic monkeys. Positive correlations between plasma VL and decreases in the levels of Th17, Tc17, NK-17, CD4(+) Tregs and increases in the levels of CD8(+) Tregs, total and IFN-α synthesizing pDCs were also noted. This study also identified 2 additional IL-17(+) subsets in GIT as CD3(-/)CD8(+)/NKG2a(-) and CD3(+)/CD8(+)/NKG2a(+) subsets. Studies also suggest a limited role for IFN-α synthesizing pDCs in chronic immune activation despite persistent up-regulation of ISGs. Finally, elevated persistent innate immune responses appear associated with poor prognosis. These findings provide an initial foundation for markers important to follow for vaccine design.

Depletion of CD4⁺ T cells abrogates post-peak decline of viremia in SIV-infected rhesus macaques.

CD4+ T cells play a central role in the immunopathogenesis of HIV/AIDS, and their depletion during chronic HIV infection is a hallmark of disease progression. However, the relative contribution of CD4+ T cells as mediators of antiviral immune responses and targets for virus replication is still unclear. Here, we have generated data in SIV-infected rhesus macaques (RMs) that suggest that CD4+ T cells are essential in establishing control of virus replication during acute infection. To directly assess the role of CD4+ T cells during primary SIV infection, we in vivo depleted these cells from RMs prior to infecting the primates with a pathogenic strain of SIV. Compared with undepleted animals, CD4+ lymphocyte-depleted RMs showed a similar peak of viremia, but did not manifest any post-peak decline of virus replication despite CD8+ T cell- and B cell-mediated SIV-specific immune responses comparable to those observed in control animals. Interestingly, depleted animals displayed rapid disease progression, which was associated with increased virus replication in non-T cells as well as the emergence of CD4-independent SIV-envelopes. Our results suggest that the antiviral CD4+ T cell response may play an important role in limiting SIV replication, which has implications for the design of HIV vaccines.

Blocking of α4ß7 gut-homing integrin during acute infection leads to decreased plasma and gastrointestinal tissue viral loads in simian immunodeficiency virus-infected rhesus macaques.

Intravenous administration of a novel recombinant rhesus mAb against the α4ß7 gut-homing integrin (mAb) into rhesus macaques just prior to and during acute SIV infection resulted in significant decrease in plasma and gastrointestinal (GI) tissue viral load and a marked reduction in GI tissue proviral DNA load as compared with control SIV-infected rhesus macaques. This mAb administration was associated with increases in peripheral blood naive and central memory CD4(+) T cells and maintenance of a high frequency of CCR5(+)CD4(+) T cells. Additionally, such mAb administration inhibited the mobilization of NK cells and plasmacytoid dendritic cells characteristically seen in the control animals during acute infection accompanied by the inhibition of the synthesis of MIP-3α by the gut tissues. These data in concert suggest that blocking of GI trafficking CD4(+) T cells and inhibiting the mobilization of cell lineages of the innate immune system may be a powerful new tool to protect GI tissues and modulate acute lentiviral infection.

Decreased NK cell frequency and function is associated with increased risk of KIR3DL allele polymorphism in simian immunodeficiency virus-infected rhesus macaques with high viral loads.

NK cells have been established as an important effector of innate immunity in a variety of viral infections. In HIV-1 infection in humans, alterations of NK cell function, frequency, and expression of various NK receptors have been reported to be associated with differential dynamics of disease progression. Expression of certain alleles of KIR3DL and KIR3DS receptors on NK cells was shown to correlate with levels of virus replication. In the SIV-infected rhesus macaque (RM) model of AIDS, several families of killer inhibitory Ig-related receptors (KIR receptors) corresponding to their human counterparts have been characterized, but only at the level of individual sequence variants. Here we define 14 different alleles of KIR3DL expressed among 38 SIV-infected RM, characterized by either high or low levels of SIV replication, by analyzing multiple sequences from individual animals and show an unequal distribution of certain alleles in these cohorts. High levels of SIV replication were associated with significant increases in KIR3DL mRNA levels in addition to decreases in both the frequency and function of NK cells in these animals. The higher frequency of inheritance of two KIR3DL alleles characterized by a single nucleotide polymorphism 159 H/Q was associated with RM that exhibited high plasma viral load. This data for the first time defines multiple alleles of KIR3DL in RM and shows an association between virus control, NK cell function and genetic polymorphisms of KIR receptors.

Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells.

Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcepsilonRIalpha(+) cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34(+) cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2-3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in ( approximately 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection.

Magnetic resonance imaging of activated proliferating rhesus macaque T cells labeled with superparamagnetic monocrystalline iron oxide nanoparticles.

Imaging of adoptively transferred cells in vivo by magnetic resonance imaging (MRI) could provide important information on disease-related patterns of lymphocyte homing in nonhuman primate models of AIDS. As a preliminary study to assess the feasibility of visualizing activated rhesus T cells by MRI, anti-CD3/CD28-expanded CD4+ T lymphocytes were labeled in vitro with monocrystalline iron oxide nanoparticles (MION). Intracellular incorporation of MION was determined by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrography (ICP-MS). Pretreatment with colchicine did not affect MION labeling, suggesting that cellular uptake of MION occurred by adsorptive pinocytosis or receptor-mediated endocytosis. TEM analysis revealed that MION were intracellularly compartmentalized exclusively in the cytoplasm and did not cause any measurable physiologic effects on T-cell function, including viability, proliferation, synthesis of select cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma), activation antigens (CD25 and CD69), adhesion molecules (alpha4beta7 and CD49d), and susceptibility to in vitro infection with simian immunodeficiency virus mac239. A sensitivity of 0.05% (1 MION-labeled T cell in 2000 unlabeled cells) could be achieved using T2-weighted gradient echo imaging. Furthermore, under these experimental conditions, the MRI signal did not decrease in proliferating MION-labeled CD4+ T cells over a period of 120 hours. These results indicate that intracellular labeling with MION can be a useful technique for noninvasively monitoring trafficking patterns of adoptively transferred leukocyte subsets in real-time by MRI in nonhuman primate models of AIDS.