Rhesus Macaque Blood Research Articles

The effects of human and rhesus macaque blood meal sources on mosquito reproduction and adult survival under laboratory conditions

Mass rearing of mosquitoes as required to fulfil research studies is a technically challenging endeavor. Blood meal source has been recognized as a key consideration in mass rearing of mosquitoes that affects colony health and fecundity. Four species of laboratory-colonized mosquitoes from the Department of Entomology, US Army Medical Directorate – Armed Forces Research Institute of Medical Sciences (USAMD-AFRIMS); Anopheles cracens, An. dirus, An. minimus and An. sawadwongporni were fed blood meals from human and rhesus macaque sources using an artificial membrane feeder. The effects of different blood meal sources were evaluated concerning blood-feeding, survival and reproduction (fecundity and hatching rates). Adult survival was monitored at days 7, 14 and 21 post blood-feeding. Although the mosquitoes fed on human blood exhibited higher rates of engorgement, there were no significant differences in blood-feeding rates in An. cracens (P = 0.08) and An. dirus (P = 0.91) between rhesus macaque and human blood sources. Twenty-one days post-feeding, no significant differences were observed in the survival rates of mosquitoes fed on human versus rhesus macaque blood. Except for An. dirus, which had better survival rates with human blood (97.5%) than after feeding on rhesus macaque blood (95.4%). All mosquito species fed on human blood produced significantly more eggs when compared to those fed on rhesus macaque blood. However, there was no statistical difference in hatching rates between blood sources, except for An. dirus, which had better hatching rates with human blood. These results indicate that human and rhesus macaque blood may be a viable alternative for maintaining Anopheles mosquitoes in colony.

Urinary cytokine measurements do not reflect surgery-induced inflammation in rhesus macaques.

Measurement of the health and disease status of free-ranging primates is often limited by a lack of available biomarkers of immune activation and inflammation that can be applied noninvasively via the measurement of urine or fecal samples. Here, we evaluate the potential usefulness of noninvasive urinary measurements of a number of cytokines, chemokines, and other markers of inflammation and infection. We took advantage of surgery-associated inflammation in seven captive rhesus macaques, collecting urine samples before and after the medical interventions. We measured these urine samples for 33 different markers of inflammation and immune activation that are known to be responsive to inflammation and infection in rhesus macaque blood samples, via the Luminex platform. We also measured all samples for concentrations of the soluble urokinase plasminogen activator receptor (suPAR),which we had validated in a prior study as an effective biomarker of inflammation. Despite urine samples being collected in captivity under ideal conditions (clean, no contamination with feces or soil, frozen quickly), 13/33 biomarkers measured via Luminex were found at concentrations below detection limits in >50% of samples. Of the remaining 20 markers, only 2 showed significant increases in response to surgery-IL18 and MPO (myeloperoxidase). However, suPAR measurements of the same samples show a consistent marked increase in response to surgery that is absent from the patterns of IL18 and MPO measurement. Given that our samples were collected under conditions that are greatly preferable to those usually encountered in the field, urinary cytokine measurements via the Luminex platform seem overall unpromising for primate field studies.

Generation of macrophages with altered viral sensitivity from genome-edited rhesus macaque iPSCs to model human disease

Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro. Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models.

T Cells Specific for a Mycobacterial Glycolipid Expand after Intravenous Bacillus Calmette-Guérin Vaccination.

Intradermal vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) protects infants from disseminated tuberculosis, and i.v. BCG protects nonhuman primates (NHP) against pulmonary and extrapulmonary tuberculosis. In humans and NHP, protection is thought to be mediated by T cells, which typically recognize bacterial peptide Ags bound to MHC proteins. However, during vertebrate evolution, T cells acquired the capacity to recognize lipid Ags bound to CD1a, CD1b, and CD1c proteins expressed on APCs. It is unknown whether BCG induces T cell immunity to mycobacterial lipids and whether CD1-restricted T cells are resident in the lung. In this study, we developed and validated Macaca mulatta (Mamu) CD1b and CD1c tetramers to probe ex vivo phenotypes and functions of T cells specific for glucose monomycolate (GMM), an immunodominant mycobacterial lipid Ag. We discovered that CD1b and CD1c present GMM to T cells in both humans and NHP. We show that GMM-specific T cells are expanded in rhesus macaque blood 4 wk after i.v. BCG, which has been shown to protect NHP with near-sterilizing efficacy upon M. tuberculosis challenge. After vaccination, these T cells are detected at high frequency within bronchoalveolar fluid and express CD69 and CD103, markers associated with resident memory T cells. Thus, our data expand the repertoire of T cells known to be induced by whole cell mycobacterial vaccines, such as BCG, and show that lipid Ag-specific T cells are resident in the lungs, where they may contribute to protective immunity.

Eosinophil Purification from Peripheral Blood of Rhesus Monkeys.

Eosinophils are granulocytes involved mainly in allergic inflammation and parasitic responses and constitute 1-5% of the circulating leukocytes in human healthy subjects. New immunotherapies targeting eosinophils have been developed and evaluated recently, and the availability of animal models that could mimic human eosinophil responses is important to consider. Differences in eosinophil biology and pathogenesis between humans and murine models have limited their utility in some settings. Isolation of viable eosinophils from rhesus macaque blood suitable for ex vivo and in vitro experimentation could provide a valuable tool for the study of eosinophil-targeted therapies and for the exploration of eosinophilic associated responses. Here, a new technique for the isolation of human eosinophils from rhesus macaque blood by negative selection from whole blood is described.

Phenotyping of Myeloid-Derived Suppressor Cells in Nonhuman Primates.

While myeloid-derived suppressor cells (MDSCs) in humans and mice have been intensively investigated, there is limited knowledge of these cells in nonhuman primates (NHPs). NHPs serve as critical models for late-stage testing of several biomedical inventions before proceeding with clinical trials and it is therefore important to fully understand their immune compartments and similarities with humans. Here, using antihuman cross-reactive antibodies, we provide flow cytometric analysis protocols for identification of MDSCs in the blood of rhesus macaques, one of the major NHP species as experimental models. Discrepancies and similarities between rhesus and human MDSCs are discussed.

Mucosal-associated invariant T (MAIT) cells provide B-cell help in vaccinated and subsequently SIV-infected Rhesus Macaques

Mucosal-associated invariant T (MAIT) cells help combat opportunistic infections. Thus, MAIT cells are of interest in HIV/SIV vaccination and infection. We investigated MAIT cell dynamics and function in rhesus macaque blood and bronchoalveolar lavage (BAL) following mucosal adenovirus (Ad)-SIV recombinant priming, intramuscular SIV envelope boosting and infection following repeated low-dose intravaginal SIV exposures. Increased frequencies of blood MAIT cells over the course of vaccination were observed, which were maintained even 12-weeks post-SIV infection. BAL MAIT cells only increased after the first Ad immunization. Vaccination increased MAIT cell levels in blood and BAL expressing the antiviral cytokine IFN-γ and TNF-α and the proliferation marker Ki67. Upon T cell-specific α-CD3, α-CD28 stimulation, MAIT cells showed a greater capacity to secrete cytokines/chemokines associated with help for B cell activation, migration and regulation compared to CD3+MR1− cells. Culture of MAIT cell supernatants with B cells led to greater tissue like memory B cell frequencies. MAIT cell frequencies in blood and BAL correlated with SIV-specific antibody levels in rectal secretions and with SIV-specific tissue resident memory B cells. Overall, SIV vaccination influenced MAIT cell frequency and functionality. The potential for MAIT cells to provide help to B cells was evident during both vaccination and infection.

Identification of novel rhesus macaque microRNAs from naïve whole blood.

MicroRNAs (miRNAs) are emerging as novel molecular tools for diagnosing and treating diseases. Rhesus monkeys (Macaca mulatta) are the most widely used nonhuman primate species for biomedical studies, yet only 912 mature miRNAs have been identified in this species compared to 2654 in humans and 1978 in mice. The aim of this project was to help bridge that gap in knowledge by evaluating circulating miRNA in naïve rhesus monkeys and comparing results with currently available databases in different species in order to identify novel, mature miRNAs. Total RNA was isolated from whole blood of ten healthy, adult rhesus macaques. After performing next generation sequencing(NGS), 475 novel, mature miRNAs were identified in rhesus macaques for the first time; of those, 423 were identified for the first time in any species. The most abundantly expressed novel rhesus macaque miRNA, hsa-miR-744-5p, has previously been described in humans. Database assessment of hsa-miR-744-5p potential gene targets showed that while the gene targets showed > 90% sequence similarity between rhesus and humans, many did not share the same consensus sequences. The identification of 475 novel miRNAs in the blood of rhesus macaque reflects the complexity and variety of miRNAs across species. Further NGS studies are needed to reveal novel miRNA that will inform on species-, tissue-, and condition-specific miRNAs.

Plasmodium 18S rRNA of intravenously administered sporozoites does not persist in peripheral blood

BackgroundPlasmodium 18S rRNA is a biomarker used to monitor blood-stage infections in malaria clinical trials. Plasmodium sporozoites also express this biomarker, and there is conflicting evidence about how long sporozoite-derived 18S rRNA persists in peripheral blood. If present in blood for an extended timeframe, sporozoite-derived 18S rRNA could complicate use as a blood-stage biomarker.MethodsBlood samples from Plasmodium yoelii infected mice were tested for Plasmodium 18S rRNA and their coding genes (rDNA) using sensitive quantitative reverse transcription PCR and quantitative PCR assays, respectively. Blood and tissues from Plasmodium falciparum sporozoite (PfSPZ)-infected rhesus macaques were similarly tested.ResultsIn mice, when P. yoelii sporozoite inoculation and blood collection were performed at the same site (tail vein), low level rDNA positivity persisted for 2 days post-infection. Compared to intact parasites with high rRNA-to-rDNA ratios, this low level positivity was accompanied by no increase in rRNA-to-rDNA, indicating detection of residual, non-viable parasite rDNA. When P. yoelii sporozoites were administered via the retro-orbital vein and blood sampled by cardiac puncture, neither P. yoelii 18S rRNA nor rDNA were detected 24 h post-infection. Similarly, there was no P. falciparum 18S rRNA detected in blood of rhesus macaques 3 days after intravenous injection with extremely high doses of PfSPZ. Plasmodium 18S rRNA in the rhesus livers increased by approximately 101-fold from 3 to 6 days post infection, indicating liver-stage proliferation.ConclusionsBeyond the first few hours after injection, sporozoite-derived Plasmodium 18S rRNA was not detected in peripheral blood. Diagnostics based on 18S rRNA are unlikely to be confounded by sporozoite inocula in human clinical trials.

Targeting the rhesus macaque TRIM5α gene to enhance the susceptibility of CD4+ T cells to HIV-1 infection.

The host range of human immunodeficiency virus type 1 (HIV-1) is extremely narrow, which has hampered the establishment of non-human primate models for HIV-1 infection. The species-specific innate immune factor tripartite motif 5 alpha (TRIM5α) is a key molecule that confers potent resistance against HIV-1 infection. In this study, we targeted the TRIM5α gene of rhesus macaques (rhTRIM5α) using the transcription activator-like effector nuclease (TALEN) to study the effect on HIV-1 infection. CD4+ T cells were separated from the peripheral blood of rhesus macaques by magnetic cell sorting, and the positive rate was greater than 99%. TALEN plasmids targeting rhTRIM5α were constructed and introduced into CD4+ T cells by electroporation, with a transfection efficiency of approximately 25%. The genome of the targeted cells was extracted, and the target efficiency was analyzed by T7E1 enzyme digestion. After sorting the positive transductants, the TALENs induced rhTRIM5α mutations at a rate of more than 40%. The ability of the HIV-1 virus to infect the targeted cells was demonstrated by ELISA. The results showed that targeting rhTRIM5α enhanced the susceptibility to HIV-1 infection. This finding will pave the way for further establishment of a new rhesus macaque model for HIV-1 studies.

Collection of hematopoietic CD34 stem cells in rhesus macaques using Spectra Optia.

Nonhuman primates, particularly rhesus macaques, are ideal preclinical large animal models to investigate organ tolerance induction protocols using donor hematopoietic stem cells (HSCs) to induce chimerism. Their relatively small size poses some challenges for the safe and effective collection of peripheral blood HSCs through apheresis procedures. We describe our experiences using the Spectra Optia apheresis unit to successfully obtain HSCs from mobilized peripheral blood of rhesus macaques. Mobilization of peripheral blood HSCs was induced using granulocyte stimulating factor (G-CSF) and Mozobil. The Spectra Optia unit was used in 18 apheresis procedures in 13 animals (4.9-10 kg). Animal health was carefully monitored during and after the procedure. Changes in peripheral blood cells before, during and after procedure were determined by complete blood count and flow cytometry. The automatic settings of the Spectra Optia unit were applied successfully to the procedures on the rhesus macaque. All animals tolerated the procedure well with no mortality. Mobilization of HSCs were most consistently achieved using 50 μg/kg of G-CSF for 5 days and a single dose of Mozobil on the 5th day, followed by collection of cells 3 h after Mozobil injection. The final apheresis product contained an average of 23 billion total nucleated cells with 47% granulocytes, 3,871 million total CD3 cells and 77 million CD34 cells which resulted in an average of 10 million CD34+ cells/kg of donor weight. Apheresis of peripheral blood mobilized HSCs in rhesus macaques using Spectra Optia is a safe and effective procedure.

Enhanced innate CD161+ T cell responses in mucosal tissues of nonhuman primates

Abstract Innate T cells recognize non-peptide antigens presented by non-classical MHC molecules, such as CD1 and MR1. The three major populations within the innate T cell group, namely, γδ T cells, MAIT cells, and NKT cells express high levels of the C-type lectin molecule CD161. To understand the significance of CD161 expressing innate T cells in mucosal immunity, we investigated the phenotype and functions of these cells in mucosal tissues, lymph nodes and blood of rhesus macaques. The frequency of CD161+ T cells was higher in the lungs and colonic mucosa (9.4%±3.8 and 6.2%±1.9) in comparison to peripheral blood (2.9%±1) and mesenteric lymph nodes (0.5%±0.3). Regardless of the tissue compartment, 30 to 80% of the CD161+ T cells comprised of γδ T cells and TCR Vα 7.2+ MAIT cells in approximately equal proportions. However, only 1 to 6% consisted of CD1d tetramer+ NKT cells. Moreover, the mucosal tissue CD161+ T cells displayed a highly activated phenotype based on CD69 and HLA-DR expression in comparison to blood and lymph node cells. HMBPP stimulated γδ T cells from lungs and colonic mucosa produced significantly higher amounts of IFN-γ, Perforin and IL-17 than those in blood. Similarly, E.coli stimulated MAIT cells from mucosal tissues produced significantly higher amounts of IFN-γ and IL-17 than those in blood. The enhanced Th1 and Th17 type effector functions of mucosal innate T cells along with their cytotoxic potential suggests that they can mount early immune responses to mucosal pathogens like HIV and Mtb prior to the development of adaptive immune responses and thereby play a role in the initial control of pathogen burden/dissemination.

Differentiation Kinetics of Blood Monocytes and Dendritic Cells in Macaques: Insights to Understanding Human Myeloid Cell Development.

Monocyte and dendritic cell (DC) development was evaluated using in vivo BrdU pulse-chase analyses in rhesus macaques, and phenotype analyses of these cells in blood also were assessed by immunostaining and flow cytometry for comparisons among rhesus, cynomolgus, and pigtail macaques, as well as African green monkeys and humans. The nonhuman primate species and humans have three subsets of monocytes, CD14(+)CD16(-), CD14(+)CD16(+), and CD14(-)CD16(+) cells, which correspond to classical, intermediate, and nonclassical monocytes, respectively. In addition, there exist presently two subsets of DC, BDCA-1(+) myeloid DC and CD123(+) plasmacytoid DC, that were first confirmed in rhesus macaque blood. Following BrdU inoculation, labeled cells first appeared in CD14(+)CD16(-) monocytes, then in CD14(+)CD16(+) cells, and finally in CD14(-)CD16(+) cells, thus defining different stages of monocyte maturation. A fraction of the classical CD14(+)CD16(-) monocytes gradually expressed CD16(+) to become CD16(+)CD14(+) cells and subsequently matured into the nonclassical CD14(-)CD16(+) cell subset. The differentiation kinetics of BDCA-1(+) myeloid DC and CD123(+) plasmacytoid DC were distinct from the monocyte subsets, indicating differences in their myeloid cell origins. Results from studies utilizing nonhuman primates provide valuable information about the turnover, kinetics, and maturation of the different subsets of monocytes and DC using approaches that cannot readily be performed in humans and support further analyses to continue examining the unique myeloid cell origins that may be applied to address disease pathogenesis mechanisms and intervention strategies in humans.

Rapid Development of gp120-Focused Neutralizing B Cell Responses during Acute Simian Immunodeficiency Virus Infection of African Green Monkeys.

The initial phases of acute human immunodeficiency virus type 1 (HIV-1) infection may be critical for development of effective envelope (Env)-specific antibodies capable of impeding the establishment of the latent pool of HIV-1-infected CD4(+) T cells, preventing virus-induced immune hyperactivation to limit disease progression and blocking vertical virus transmission. However, the initial systemic HIV-1 Env-specific antibody response targets gp41 epitopes and fails to control acute-phase viremia. African-origin, natural simian immunodeficiency virus (SIV) hosts do not typically progress to AIDS and rarely postnatally transmit virus to their infants, despite high milk viral loads. Conversely, SIV-infected rhesus macaques (RMs), Asian-origin nonnatural SIV hosts, sustain pathogenic SIV infections and exhibit higher rates of postnatal virus transmission. In this study, of acute SIV infection, we compared the initial systemic Env-specific B cell responses of AGMs and RMs in order to probe potential factors influencing the lack of disease progression observed in AGMs. AGMs developed higher-magnitude plasma gp120-specific IgA and IgG responses than RMs, whereas RMs developed more robust gp140-directed IgG responses. These gp120-focused antibody responses were accompanied by rapid autologous neutralizing responses during acute SIV infection in AGMs compared to RMs. Moreover, acute SIV infection elicited a higher number of circulating Env-specific memory B cells in peripheral blood of AGMs than in the blood of RMs. These findings indicate that AGMs have initial systemic Env-specific B cell responses to SIV infection distinct from those of a nonnatural SIV host, resulting in more functional SIV-specific humoral responses, which may be involved in impairing pathogenic disease progression and minimizing postnatal transmission. Due to the worldwide prevalence of HIV-1 infections, development of a vaccine to prevent infection or limit the viral reservoir remains an important goal. HIV-1-infected humans, as well as SIV-infected nonnatural SIV hosts, develop pathogenic infections and readily transmit the virus to their infants. Conversely, natural SIV hosts do not develop pathogenic infections and rarely transmit the virus to their infants. The immunologic factors contributing to these favorable outcomes in natural SIV hosts could prove invaluable for directing HIV-1 vaccine and therapy design. This study identified distinctions in the specificity and function of the initial systemic SIV envelope-specific B cell response that developed during acute SIV infection in natural and nonnatural SIV host species. Identification of distinct acute B cell responses in natural SIV hosts may inform vaccine strategies seeking to elicit similar responses prior to or during the initial phases of acute HIV-1 infection.

Imidazoquinoline Toll-like receptor 8 agonists activate human newborn monocytes and dendritic cells through adenosine-refractory and caspase-1–dependent pathways

Newborns have frequent infections and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant but might confer a T(H)2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes demonstrate impaired T(H)1-polarizing responses to many TLR agonists caused by plasma adenosine acting through cyclic AMP. TLR8 agonists, including imidazoquinolines (IMQs), such as the small synthetic 3M-002, induce adult-level TNF from neonatal monocytes, but the scope and mechanisms of IMQ-induced activation of neonatal monocytes and monocyte-derived dendritic cells (MoDCs) have not been reported. We sought to characterize IMQ-induced activation of neonatal monocytes and MoDCs. Neonatal cord and adult peripheral blood monocytes and MoDCs were cultured in autologous plasma; levels of alum- and TLR agonist-induced cytokines and costimulatory molecules were measured. TLR8 and inflammasome function were assayed by using small interfering RNA and Western blotting/caspase-1 inhibitory peptide, respectively. The ontogeny of TLR8 agonist-induced cytokine responses was defined in rhesus macaque whole blood exvivo. IMQs were more potent and effective than alum at inducing TNF and IL-1β from monocytes. 3M-002 induced robust TLR pathway transcriptome activation and T(H)1-polarizing cytokine production in neonatal and adult monocytes and MoDCs, signaling through TLR8 in an adenosine/cyclic AMP-refractory manner. Newborn MoDCs displayed impaired LPS/ATP-induced caspase-1-mediated IL-1β production but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8 IMQs induced robust TNF and IL-1β in whole blood of rhesus macaques at birth and infancy. IMQ TLR8 agonists engage adenosine-refractory TLR8 and inflammasome pathways to induce robust monocyte and MoDC activation and represent promising neonatal adjuvants.

Rhesus Monkey Immature Monocyte-Derived Dendritic Cells Generate Alloantigen-Specific Regulatory T Cells From Circulating CD4+CD127−/lo T Cells

Generation of non-human primate regulatory T cells (Treg) with alloantigen (alloAg) specificity would allow their testing in preclinical transplant models. Low recovery of Treg from peripheral blood limits their potential utility. In small animals and humans, conventional myeloid dendritic cells (DC) have been shown to select or induce alloAg-specific Treg. We combined enrichment of rhesus macaque blood CD4 Treg based on IL-7Ralpha (CD127) expression with their stimulation in mixed leukocyte cultures with immature, allogeneic control or vitamin (Vit) D3/IL-10-conditioned monocyte-derived DC. After co-culture in IL-2 and IL-15 for up to 14 days, the ability of the resulting T cells to suppress alloreactive effector T-cell proliferation was assessed. CD4CD127 T cells represented approximately 7% of normal rhesus circulating CD4 T cells and were enriched for forkhead box P3 (Foxp3) cells. When stimulated with control allogeneic DC, they exhibited much inferior proliferative responses compared with bulk CD4 or CD4CD127 cells. This anergic state was reversed by exogenous IL-2 and IL-15. After 10 to 14 days culture of CD4CD127 T cells with immature allogeneic DC, particularly maturation-resistant VitD3/IL-10 DC, the frequency of Foxp3 T cells was increased. The cultured cells markedly inhibited CD4 effector T-cell proliferation in a dose-related and donor alloAg-specific manner. Stimulation of rhesus CD4CD127 T cells with immature and especially maturation-resistant allogeneic DC, generated highly-suppressive, alloAg-specific Treg. Without resorting to a more purified starting population, this approach may have therapeutic utility in clinically relevant transplant models.

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

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. 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. 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). 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.

CpG oligonucleotides induce strong humoral but only weak CD4 + T cell responses to protein antigens in rhesus macaques in vivo

Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for humoral immune responses but data on cellular immune responses in primates are scarce. Rhesus macaque blood contained similar numbers of plasmacytoid dendritic cells and B cells, the key sensors of CpG ODN, as human blood, and these cells were activated by CpG-A and CpG-B in vitro. In vivo, both ODNs induced equal plasma levels of interferon-inducible protein 10 and similarly enhanced antibody responses following i.m. injections of the ODNs, protein antigen, and aluminium hydroxide into rhesus macaques, whereas antigen-specific CD4 + T cell responses were only slightly increased by CpG ODN.

Characterization of an in vitro rhesus macaque blood–brain barrier

The blood–brain barrier (BBB) has been modeled in vitro in a number of species, including rat, cow and human. Coculture of multiple cell types is required for the correct expression of tight junction proteins by microvascular brain endothelial cells (MBEC). Markers of inflammation, especially MHC-II, and cell adhesion molecules, such as VCAM-1, are not expressed on the luminal surface of the barrier under resting conditions. The rhesus macaque model has been used to study early events of HIV-neuropathogenesis in vivo, but a suitable in vitro model has not been available for detailed mechanistic studies. Here we describe an in vitro rhesus macaque blood–brain barrier that utilizes autologous MBEC and astrocytes. We believe that this model is highly relevant for examining immunological events at the blood–brain barrier and demonstrate its potential usefulness for examining early events in AIDS neuropathogenesis.

Generation of monocyte-derived dendritic cells from precursors in rhesus macaque blood

While the dendritic cells (DCs) of mouse and man have been extensively studied, until recently those of non-human primates remained poorly characterized. We present a method for generating large numbers of DCs from precursors in rhesus macaque blood, based on techniques developed for human blood. For 7 days, a T cell-depleted population of mononuclear cells was cultured in 1% human plasma with GM-CSF and IL-4, both to initiate DC differentiation and to inhibit macrophage development. On day 7, 50% of the culture medium was replaced with a monocyte-conditioned medium (MCM), which is required for the final maturation of the DCs into potent stimulators of the allogeneic MLR. Between 0.5 and 1.0×10 6 DCs can be generated from 20 ml of rhesus macaque blood. We compared these cytokine-generated DCs to the adherent macrophages present in the same cultures. Cytokine-generated DCs were considerably more potent at stimulating allogeneic T cells than adherent macrophages. Furthermore, the DCs had a distinct morphology and phenotype, with long processes, high levels of p55, and a characteristic perinuclear collection of intracellular CD68. In contrast, adherent macrophages expressed very low levels of p55, and high diffuse levels of CD68. Macaque DCs generated by this method may be useful in vaccine development, and for studies of SIV pathogenesis.