Non-human Primate Disease Models Research Articles

Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases

Microglial activation plays central roles in neuroinflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18 kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells depends on the transcription factor AP1 and is unique to a subset of rodent species within the Muroidea superfamily. Finally, we identify LCP2 and TFEC as potential markers of microglial activation in humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO-PET signals in humans reflect the density of inflammatory cells rather than activation state.

Development of a 40+ color full spectrum flow cytometry panel for in-depth immunophenotyping of chief cell subsets in non-human primate peripheral blood mononuclear cells

Abstract Leveraging full-spectrum technology, we designed and optimized a 40+ color flow cytometry panel for the analysis of non-human primate peripheral blood mononuclear cells (PBMCs). Non-human primate models of disease allow for the closest approximation of human outcomes to vaccination, disease, and treatment due to similarities in physiological host responses, which increase predictive accuracy. This first panel of its kind achieves comprehensive profiling of immune subpopulations by the inclusion of surface, intracellular, and cytokine markers representative of differentiation, activation, functionality, and proliferation to deeply interrogate the immune response to infectious disease and vaccination. The optimized panel is additionally appropriate for the discovery of new immune phenotypes important for protection against pathogens. After initial optimization for use with cynomolgus macaque (Macaca fascicularis) and rhesus macaque (Macaca mulatta) PBMCs, the panel was further adapted for use with African green monkey (Chlorocebus aethiops) PBMCs to allow for cross-comparison between three frequently implemented non-human primate models of infectious disease. This study was supported by the US Army Medical Research Acquisition Activity contract number W81XWH1910027 to TWG and Department of Health and Human Services, National Institutes of Health grant number UC7AI094660 for BSL-4 operations support of the Galveston National Laboratory.

Phosphoramidate Prodrugs Continue to Deliver: The Journey of Remdesivir (GS-5734) from the Liver to Peripheral Blood Mononuclear Cells.

Remdesivir (GS-5734) is a monophenol, 2-ethylbutylalanine phosphoramidate prodrug of GS-441524 that is FDA-approved for the treatment of patients hospitalized for COVID-19. Despite showing strong, broad-spectrum antiviral activity in preclinical models, the clinical efficacy of remdesivir is mixed. This work highlights the pharmacodynamic discordance of remdesivir between humans and non-human primates, thereby demonstrating that non-human primate disease models overestimate the therapeutic efficacy of phosphoramidate prodrugs.

Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.

PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys.

Mutations of PTEN-induced kinase I (PINK1) cause early-onset Parkinson’s disease (PD) with selective neurodegeneration in humans. However, current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients. This suggests that generating PINK1 disease models in non-human primates (NHPs) that are close to humans is essential to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both of which can reduce off-target effects without compromising on-target editing, are two optimized strategies in the CRISPR/Cas9 system for establishing disease animal models. Here, we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target the PINK1 gene. We achieved precise and efficient gene editing of the target site in three newborn cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts led to a reduction in mRNA. However, western blotting and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in wild-type fibroblasts. We further reprogramed mutant fibroblasts into induced pluripotent stem cells (iPSCs), which showed similar ability to differentiate into dopamine (DA) neurons. Taken together, our results showed that co-injection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enabled the generation of human disease models in NHPs and target editing by pair truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact protein expression.

A DNA-based vaccine protects against Crimean-Congo haemorrhagic fever virus disease in a Cynomolgus macaque model.

There is currently no specific prophylaxis or vaccine against Crimean-Congo hemorrhagic fever virus (CCHFV). Crimean-Congo hemorrhagic fever (CCHF) is a severe febrile-illness transmitted by Hyalomma ticks in endemic areas, handling of infected livestock or care of infected patients. We report here the successful protection against CCHFV-mediated disease in a non-human primate disease model. Cynomolgus macaques were vaccinated with a DNA-based vaccine using in vivo electroporation-assisted delivery. The vaccine contained two plasmids encoding the glycoprotein precursor (GPC) and the nucleoprotein (NP) of CCHFV. Animals received three vaccinations and we recorded potent antibody and T-cell responses after vaccination. While all sham-vaccinated animals developed viremia, high tissue viral loads and CCHF-induced disease, the NP + GPC vaccinated animals were significantly protected. In conclusion, this is the first evidence of a vaccine that can protect against CCHFV-induced disease in a non-human primate model. This supports clinical development of the vaccine to protect groups at risk for contracting the infection.

Generation of a Hutchinson\u2013Gilford progeria syndrome monkey model by base editing

Many human genetic diseases, including Hutchinson-Gilford progeria syndrome (HGPS), are caused by single point mutations. HGPS is a rare disorder that causes premature aging and is usually caused by a de novo point mutation in the LMNA gene. Base editors (BEs) composed of a cytidine deaminase fused to CRISPR/Cas9 nickase are highly efficient at inducing C to T base conversions in a programmable manner and can be used to generate animal disease models with single amino-acid substitutions. Here, we generated the first HGPS monkey model by delivering a BE mRNA and guide RNA (gRNA) targeting the LMNA gene via microinjection into monkey zygotes. Five out of six newborn monkeys carried the mutation specifically at the target site. HGPS monkeys expressed the toxic form of lamin A, progerin, and recapitulated the typical HGPS phenotypes including growth retardation, bone alterations, and vascular abnormalities. Thus, this monkey model genetically and clinically mimics HGPS in humans, demonstrating that the BE system can efficiently and accurately generate patient-specific disease models in non-human primates.

Behavioral shaping of rhesus macaques using the Cambridge neuropsychological automated testing battery

BackgroundThe Cambridge neuropsychological test automated battery (CANTAB) is a set of computerized visuospatial tests used to probe cognition in humans. The non-human primate (NHP) version of the battery is a valuable translational research tool to quantify cognitive changes in NHP models of disease by allowing direct comparison with performance data from human patient populations. One limitation is the long training times required for NHPs to reach appropriate levels of task performance, which is prohibitive for high throughput experimental designs. New methodWe report a new training regimen to teach NHPs a subset of CANTAB cognitive tasks using a method of successive approximations (shaping), where rewarded behaviors progressively approximate the goal behavior, and sequential task learning is used to build upon previously learned rules. Using this refined method, we taught 9 adult rhesus macaques to perform three tasks: the self-ordered spatial search (SOSS), delayed match-to-sample (DMTS), and paired associative learning (PAL) tasks. Results and comparison with existing methodsNHPs learned all three cognitive tasks in approximately 130 training sessions, roughly 200 sessions faster than previously published training times. NHPs were able to perform each task to a stable level of performance (>80 % correct) enabling their use in future cognitive experiments. ConclusionsOur approach of behavioral shaping reduced the time to train NHPs to performance criteria on SOSS, DMTS, and PAL tasks. This allows efficient use of the NHP-adapted CANTAB to compare cognitive changes in NHP models of neurological disease with those observed in human patient populations.

Zika virus: out of sight but not out of the mind. Viral persistence and host responses within the central and peripheral nervous systems

Following the rapid spread of Zika virus infection through the Caribbean and Americas, model systems of Zika virus infection have been established to determine disease pathology and potential health impacts for those infected with this neurotropic flavivirus. NIBSC has established both Old World (rhesus and cynomolgus macaques) and New World (red-bellied tamarins) non-human primate disease models of human Zika virus infection. Animals were infected sub-cutaneously with the Caribbean Zika isolate PRVABC59 and pairs terminated during primary viremia (3 dpc) or post clearance of plasma viremia (42 and 101 dpc). FFPE sections from multiple tissues, including brain and peripheral nerves were analysed for Zika virus RNA (RNAscope), viral proteins and host responses (immunohistochemistry). All animals became infected and rapidly cleared high levels of peripheral viremia. Zika virus RNA was detected in multiple tissues from 3 dpc with low levels of viral RNA and viral NS1 protein remaining detectable through to 101 dpc. Viral RNA was associated with both glial and neuronal cells throughout the brain and schwann cell nuclei in peripheral nerves. Neuroinflammation (astrogliosis, microgliosis, peri-vascular cuffing) and increased levels of CD3+, CD8+ or CD45+ cells were present from 3 dpc through to 101 dpc. Disruption of MAP2 neuronal dendrite staining and peripheral nerve myelin basic protein staining was also observed. The continued detection of Zika virus within tissues 3 months post infection and in the absence of a detectable peripheral viremia raises questions regarding potential long term effects of this virus and associated inflammatory responses within the nervous systems.

Role of surface charge in determining the biological effects of CdSe/ZnS quantum dots.

The growing potential of quantum dots (QDs) in biomedical applications has provoked the urgent need to thoroughly address their interaction with biological systems. However, only limited studies have been performed to explore the effects of surface charge on the biological behaviors of QDs. In the present study, three commercially available QDs with different surface coatings were used to systematically evaluate the effects of surface charge on the cellular uptake, cytotoxicity, and in vivo biodistribution of QDs. Our results demonstrated that charged QDs entered both cancer cells and macrophages more efficiently than neutral ones, while negative QDs internalized mostly. Upon entry into cells, QDs were localized in different subcellular compartments (eg, cytoplasm and lysosomes) depending on the surface charge. Interestingly, inconsistent with the result of internalization, positive QDs but not negative QDs exhibited severe cytotoxicity, which was likely due to their disruption of cell membrane integrity, and production of reactive oxygen species. Biodistribution studies demonstrated that negative and neutral QDs preferentially distributed in the liver and the spleen, whereas positive QDs mainly deposited in the kidney with obvious uptake in the brain. In general, surface charge plays crucial roles in determining the biological interactions of QDs.

Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430

Filoviruses are emerging pathogens and causative agents of viral haemorrhagic fever. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, exceeding 90% (ref. 1). Licensed therapeutic or vaccine products are not available to treat filovirus diseases. Candidate therapeutics previously shown to be efficacious in non-human primate disease models are based on virus-specific designs and have limited broad-spectrum antiviral potential. Here we show that BCX4430, a novel synthetic adenosine analogue, inhibits infection of distinct filoviruses in human cells. Biochemical, reporter-based and primer-extension assays indicate that BCX4430 inhibits viral RNA polymerase function, acting as a non-obligate RNA chain terminator. Post-exposure intramuscular administration of BCX4430 protects against Ebola virus and Marburg virus disease in rodent models. Most importantly, BCX4430 completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 hours after infection. In addition, BCX4430 exhibits broad-spectrum antiviral activity against numerous viruses, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. This is the first report, to our knowledge, of non-human primate protection from filovirus disease by a synthetic drug-like small molecule. We provide additional pharmacological characterizations supporting the potential development of BCX4430 as a countermeasure against human filovirus diseases and other viral diseases representing major public health threats.

Large-scale polymorphism discovery in macaque G-protein coupled receptors

BackgroundG-protein coupled receptors (GPCRs) play an inordinately large role in human health. Variation in the genes that encode these receptors is associated with numerous disorders across the entire spectrum of disease. GPCRs also represent the single largest class of drug targets and associated pharmacogenetic effects are modulated, in part, by polymorphisms. Recently, non-human primate models have been developed focusing on naturally-occurring, functionally-parallel polymorphisms in candidate genes. This work aims to extend those studies broadly across the roughly 377 non-olfactory GPCRs. Initial efforts include resequencing 44 Indian-origin rhesus macaques (Macaca mulatta), 20 Chinese-origin rhesus macaques, and 32 cynomolgus macaques (M. fascicularis).ResultsUsing the Agilent target enrichment system, capture baits were designed for GPCRs off the human and rhesus exonic sequence. Using next generation sequencing technologies, nearly 25,000 SNPs were identified in coding sequences including over 14,000 non-synonymous and more than 9,500 synonymous protein-coding SNPs. As expected, regions showing the least evolutionary constraint show greater rates of polymorphism and greater numbers of higher frequency polymorphisms. While the vast majority of these SNPs are singletons, roughly 1,750 non-synonymous and 2,900 synonymous SNPs were found in multiple individuals.ConclusionsIn all three populations, polymorphism and divergence is highly concentrated in N-terminal and C-terminal domains and the third intracellular loop region of GPCRs, regions critical to ligand-binding and signaling. SNP frequencies in macaques follow a similar pattern of divergence from humans and new polymorphisms in primates have been identified that may parallel those seen in humans, helping to establish better non-human primate models of disease.

Old world monkeys and new age science: the evolution of nonhuman primate systems virology.

Nonhuman primate (NHP) biomedical models are critical to our understanding of human health and disease, yet we are still in the early stages of developing sufficient tools to support primate genomic research that allow us to better understand the basis of phenotypic traits in NHP models of disease. A mere 7 years ago, the limited NHP transcriptome profiling that was being performed was done using complementary DNA arrays based on human genome sequences, and the lack of NHP genomic information and immunologic reagents precluded the use of NHPs in functional genomic studies. Since then, significant strides have been made in developing genomics capabilities for NHP research, from the rhesus macaque genome sequencing project to the construction of the first macaque-specific high-density oligonucleotide microarray, paving the way for further resource development and additional primate sequencing projects. Complete published draft genome sequences are now available for the chimpanzee ( Chimpanzee Sequencing Analysis Consortium 2005), bonobo ( Prufer et al. 2012), gorilla ( Scally et al. 2012), and baboon ( Ensembl.org 2013), along with the recently completed draft genomes for the cynomolgus macaque and Chinese rhesus macaque. Against this backdrop of both expanding sequence data and the early application of sequence-derived DNA microarrays tools, we will contextualize the development of these community resources and their application to infectious disease research through a literature review of NHP models of acquired immune deficiency syndrome and models of respiratory virus infection. In particular, we will review the use of -omics approaches in studies of simian immunodeficiency virus and respiratory virus pathogenesis and vaccine development, emphasizing the acute and innate responses and the relationship of these to the course of disease and to the evolution of adaptive immunity.

An Effort to Use Human-Based Exome Capture Methods to Analyze Chimpanzee and Macaque Exomes

Non-human primates have emerged as an important resource for the study of human disease and evolution. The characterization of genomic variation between and within non-human primate species could advance the development of genetically defined non-human primate disease models. However, non-human primate specific reagents that would expedite such research, such as exon-capture tools, are lacking. We evaluated the efficiency of using a human exome capture design for the selective enrichment of exonic regions of non-human primates. We compared the exon sequence recovery in nine chimpanzees, two crab-eating macaques and eight Japanese macaques. Over 91% of the target regions were captured in the non-human primate samples, although the specificity of the capture decreased as evolutionary divergence from humans increased. Both intra-specific and inter-specific DNA variants were identified; Sanger-based resequencing validated 85.4% of 41 randomly selected SNPs. Among the short indels identified, a majority (54.6%–77.3%) of the variants resulted in a change of 3 base pairs, consistent with expectations for a selection against frame shift mutations. Taken together, these findings indicate that use of a human design exon-capture array can provide efficient enrichment of non-human primate gene regions. Accordingly, use of the human exon-capture methods provides an attractive, cost-effective approach for the comparative analysis of non-human primate genomes, including gene-based DNA variant discovery.

ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody

Tumor necrosis factor (TNF) signals through two membrane receptors, TNFR1 and TNFR2, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB, produced in CHO cells, showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1-70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.

Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28

Although embryonic stem (ES) cell–like induced pluripotent stem (iPS) cells have potential therapeutic applications in humans, they are also useful for creating genetically modified human disease models in nonhuman primates. In this study, we generated common marmoset iPS cells from fetal liver cells via the retrovirus-mediated introduction of six human transcription factors: Oct-3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28. Four to five weeks after introduction, several colonies resembling marmoset ES cells were observed and picked for further expansion in ES cell medium. Eight cell lines were established, and validation analyses of the marmoset iPS cells followed. We detected the expression of ES cell–specific surface markers. Reverse transcription-PCR showed that these iPS cells expressed endogenous Oct-3/4, Sox2, Klf4, c-Myc, Nanog and Lin28 genes, whereas all of the transgenes were silenced. Karyotype analysis showed that two of three iPS cell lines retained a normal karyotype after a 2-month culture. Both embryoid body and teratoma formation showed that marmoset iPS cells had the developmental potential to give rise to differentiated derivatives of all three primary germ layers. In summary, we generated marmoset iPS cells via the transduction of six transcription factors; this provides a powerful preclinical model for studies in regenerative medicine.

RNAa Is Conserved in Mammalian Cells

BackgroundRNA activation (RNAa) is a newly discovered mechanism of gene activation triggered by small double-stranded RNAs termed ‘small activating RNAs’ (saRNAs). Thus far, RNAa has only been demonstrated in human cells and is unclear whether it is conserved in other mammals.Methodology/Principal FindingsIn the present study, we evaluated RNAa in cells derived from four mammalian species including nonhuman primates (African green monkey and chimpanzee), mouse, and rat. Previously, we identified saRNAs leading to the activation of E-cadherin, p21, and VEGF in human cells. As the targeted sequences are highly conserved in primates, transfection of each human saRNA into African green monkey (COS1) and chimpanzee (WES) cells also resulted in induction of the intended gene. Additional saRNAs targeting clinically relevant genes including p53, PAR4, WT1, RB1, p27, NKX3-1, VDR, IL2, and pS2 were also designed and transfected into COS1 and WES cells. Of the nine genes, p53, PAR4, WT1, and NKX3-1 were induced by their corresponding saRNAs. We further extended our analysis of RNAa into rodent cell types. We identified two saRNAs that induced the expression of mouse Cyclin B1 in NIH/3T3 and TRAMP C1 cells, which led to increased phosphorylation of histone H3, a downstream marker for chromosome condensation and entry into mitosis. We also identified two saRNAs that activated the expression of CXCR4 in primary rat adipose–derived stem cells.Conclusions/SignificanceThis study demonstrates that RNAa exists in mammalian species other than human. Our findings also suggest that nonhuman primate disease models may have clinical applicability for validating RNAa-based drugs.

The significance of non-human primates as preclinical models of human arthritic disease

Background: The broad immunological gap between inbred SPF-raised strains of mice and rats and the diverse rheumatoid arthritis (RA) patient population limits the predictive value of the existing disease models for clinical success of new therapies, in particular for those using highly specific biologicals. Objective: This review argues that because of their closer immunological and physiological proximity to patients, disease models in non-human primates (NHPs) may bridge this gap and help reduce the failure of many (± 80%) new therapies in clinical trials. In various research areas, NHPs are an accepted intermediate between disease models in rodents and the ultimate introduction for clinical use in patients. However, with the exception of transplantation, this is not the case for immune-mediated inflammatory disorders, such as RA, although useful preclinical models are being developed. Method: The validity and use of the rhesus monkey model of collagen-induced arthritis as a preclinical RA model is reviewed. The discussion comprises present genetic and immunological aspects, biomarkers, and an overview of published preclinical therapy evaluations. Conclusion: It is time to consider the use of NHPs with a greater evolutionary proximity to humans as models for preclinical evaluation of new human-specific drugs for arthritic disease.

Long versus short oligonucleotide microarrays for the study of gene expression in nonhuman primates

The high degree of sequence similarity between human and nonhuman primate (NHP) genomic DNA suggests that human genome sequence-based DNA microarrays may be used effectively to study gene expression in NHP disease models. In the present study, two distinct commercially available human genome microarray platforms, the Affymetrix HG U133A GeneChip ® System utilizing Human Genome U133A GeneChips ® and the Applied Biosystems Expression Array System utilizing the Human Genome Survey Microarray, were used to identify and characterize gene expression changes in the anterior cerebellum of a macaque monkey model of human alcoholism. The Affymetrix microarray consists of eleven short oligonucleotide probe sets for each gene while the Applied Biosystems Microarray uses a single long oligonucleotide per gene. Cross-mapping of probes revealed a total of 11,542 genes that are represented on both microarray platforms. Absolute measures of gene expression (“present calls”) from the cerebellum RNA samples were 65–70% (Applied Biosystems Expression Array System) and 27–30% (AffymetrixGeneChip ® System) among these common gene targets. Analysis of variance (ANOVA; p < 0.05; >1.2 fold change; detected on at least 50% of the arrays) indicated 932 and 515 differentially expressed genes for the Applied Biosystems and Affymetrix microarrays, respectively. Significance analysis of microarrays (SAM) identified 255 significant genes at 5% false discovery rate (FDR) for the Applied Biosystems data set and five significant genes at 60% FDR (minimum FDR) for the Affymetrix data set. TaqMan ® assay-based real-time PCR validation of a number of differentially-expressed genes yielded results that agreed well with the array data in the majority of comparisons. This study demonstrates that human sequence-based DNA arrays can be used effectively to detect differential gene expression in an NHP disease model and provides evidence that the use of this long oligonucleotide-based microarray platform may be more suitable for cross-species gene expression studies than a short oligonucleotide-based system.

Identification and simian immunodeficiency virus infection of CD1d-restricted macaque natural killer T cells.

Natural killer T (NKT) cells express a highly conserved T-cell receptor (TCR) and recognize glycolipids in the context of CD1d molecules. We recently demonstrated that CD4+ NKT cells are highly susceptible to human immunodeficiency virus type 1 (HIV-1) infection and are selectively depleted in HIV-infected individuals. Here, we identified macaque NKT cells using CD1d tetramers and human Valpha24 antibodies. Similar to human NKT cells, alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells activate and expand macaque NKT cells. Upon restimulation with alpha-GalCer-pulsed CD1d(+) cells, macaque NKT cells secreted high levels of cytokines, a characteristic of these T cells. Remarkably, the majority of resting and activated macaque NKT cells expressed CD8, and a smaller portion expressed CD4. Macaque NKT cells also expressed the HIV-1/simian immunodeficiency virus (SIV) coreceptor CCR5, and the CD4+ subset was susceptible to SIV infection. Identification of macaque NKT cells has major implications for delineating the role of these cells in nonhuman primate disease models of HIV as well as other pathological conditions, such as allograft rejection and autoimmunity.