Peripheral Blood Invariant Natural Killer T Cells of Pig-Tailed Macaques

In humans, invariant natural killer T (iNKT) cells represent a small but significant population of peripheral blood mononuclear cells (PBMCs) with a high degree of variability. In this study, pursuant to our goal of identifying an appropriate non-human primate model suitable for pre-clinical glycolipid testing, we evaluated the percentage and function of iNKT cells in the peripheral blood of pig-tailed macaques. First, using a human CD1d-tetramer loaded with α-GalCer (α-GalCer-CD1d-Tet), we found that α-GalCer-CD1d-Tet+ CD3+ iNKT cells make up 0.13% to 0.4% of pig-tailed macaque PBMCs, which are comparable to the percentage of iNKT cells found in human PBMCs. Second, we observed that a large proportion of Vα24+CD3+ cells are α-GalCer-CD1d-Tet+CD3+ iNKT cells, which primarily consist of either the CD4+ or CD8+ subpopulation. Third, we found that pig-tailed macaque iNKT cells produce IFN-γ in response to α-GalCer, as shown by ELISpot assay and intracellular cytokine staining (ICCS), as well as TNF-α, as shown by ICCS, indicating that these iNKT cells are fully functional. Interestingly, the majority of pig-tailed macaque iNKT cells that secrete IFN-γ are CD8+ iNKT cells. Based on these findings, we conclude that the pig-tailed macaques exhibit potential as a non-human animal model for the pre-clinical testing of iNKT-stimulating glycolipids.


Introduction
Natural killer T (NKT) cells are a unique subset of lymphoid cells that express both a T cell antigen receptor (TCR) and NK1.1 (NKR-P1 or CD161c), a C-lectin-type NK receptor [1,2]. A significant proportion of NKT cells express semi-invariant TCRs encoded by Va24 and Ja18 gene segments in humans and Va14 and Ja18 gene segments in mice, and these cells have been designated invariant NKT (iNKT) cells [3]. In humans, iNKT cells represent a small but significant proportion (0.01%-0.5%) of PBMCs with a high degree of variability [4,5]. Upon activation, iNKT cells rapidly secrete both Th1 and Th2 cytokines in vivo and induce a series of cellular activation events leading to the activation of innate immune cells, such as NK cells and dendritic cells (DCs), as well as the stimulation of adaptive immune cells, such as B and T cells [6][7][8][9][10][11][12]. In addition, upon stimulation, iNKT cells, like NK cells, display cytotoxic activity mediated by Fas, perforin, granzyme A/B, and granulysin [13,14]. iNKT cells have also been shown to display anti-tumor activity [15,16], mediate therapeutic effects against autoimmune diseases [17][18][19][20], and promote protection against certain infectious agents [21][22][23][24].
CD1d molecules and iNKT cells are conserved between mice and humans [25]. Accordingly, mouse models have been extensively used to study the biological activity of CD1d-binding, iNKT cell-stimulating glycolipids, and the phenotypes and functions of iNKT cells [1,26]. However, these studies have indicated substantial differences in the specificity, frequency, and function of CD1d and iNKT cells between the two species. Because of this, some studies have investigated the frequency, phenotype, and function of iNKT cells derived from non-human primates, including pig-tailed macaques, and found similar percentages and high variability of iNKT cells between monkeys and humans [27][28][29][30]. These studies have also indicated that the phenotypes and functions of monkey iNKT cells are significantly different among different macaque species [27][28][29][30]. Pig-tailed macaques have been used as animal models to study a number of human diseases, such as Chlamydia trachomatis [31][32][33] and HIV-1 infection [34,35]. In this study we sought to characterize in greater detail the base line frequency, specificity, and function of iNKT cells in pig-tailed macaques and address whether pig-tailed macaques could be used as an animal model for the pre-clinical testing of various iNKT cell-stimulating ligands.

Animals
Pig-tailed macaques (M. nemestrina) were used in this study. All animals were negative for simian immunodeficiency virus (SIV) and simian T-cell lymphotropic virus type 1 (STLV-1) by serology as well as simian type D retrovirus by serology and polymerase chain reaction (PCR). Peripheral blood was collected by venipuncture under anesthesia. All animals used in this study were housed and cared for according to the Guide for the Care and Use of Laboratory Animals at the Washington National Primate Research Center (WaNPRC), an institution accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The animal quarters are maintained at 75-78uF with controlled air humidity and quality. Commercial monkey chow was fed to the animals once daily, and drinking water was available at all times. Daily examinations and any medical care were provided by the WaNPRC veterinary staff in consultation with the clinical veterinarian. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington and conducted in compliance with the Public Health Services Policy on Humane Care and Use of Laboratory Animals (http://grants.nih.gov/ grants/olaw/references/PHSPolicyLabAnimals.pdf). The animals were kept under deep sedation with ketamine HCl at a dose of 10-15 mg/kg intramuscularly to alleviate any pain and discomfort during blood draws. An animal technician or veterinary technologist monitored the animals while under sedation.

Preparation of Peripheral Blood Mononuclear Cells (PBMCs)
PBMCs were isolated from buffy coats by Ficoll-Hypaque density gradient separation. Erythrocytes were removed by osmotic lysis in ACK lysing buffer (Life Technologies, Grand Island, NY), and the remaining nucleated cells were washed twice with RPMI supplemented with 10% fetal calf serum (FCS).

Antibodies, Glycolipid, and CD1d-tetramer
Anti-human antibodies known to cross-react with macaques were selected for this study.

Flow Cytometric Analysis
For cell surface staining, 1610 6 PBMCs were incubated for 20 min at 4uC in FACs staining buffer in the presence of the antibody of interest. After washing twice, labeled cells were subjected to multicolor FACScan flow cytometry on a BD LSRII (Becton Dickinson, Franklin Lakes, NJ) using forward and sidescatter characteristics to exclude dead cells. Anti-mouse-Ig or antirat compensation particle sets were used for compensation purposes (BD Biosciences). The data were analyzed using Flowjo software (Tree Star, Ashland, OR).

PBMCs Stimulation by a-GalCer
PBMCs were cultured in a 96-well U-bottom plate at 1610 6 cells/well in the presence of 5 mg/ml or 0.1 mg/ml of a-GalCer for 6 hours at 37uC followed by the addition of Brefeldin A (BioLegend) at 5 mg/ml for the last 4 hours of incubation. In a

Intracellular Cytokine Staining
For intracellular IFN-c and TNF-a staining, the PBMCs were stimulated with a-GalCer, as described above. After stimulation, the cells were incubated with anti-CD3, anti-CD4, and anti-CD8 antibodies, as well as the human CD1d tetramer loaded with a-GalCer for 20 min. The cells were then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer's instructions. The permeabilized cells were stained with PE-Cy7-labeled anti-TNF-a and APC-labeled anti-IFN-c antibodies for 30 min on ice in the dark. After washing twice, the cells were resuspended in staining buffer and analyzed by flow cytometric analysis. Acquisition and analysis were carried out by first gating for live cells by forward scatter (FSC) and side scatter (SSC) then subsequently gating for iNKT cells by positivity to CD3 + and a-GalCer-loaded CD1d tetramer + among the live cells. IFN-c + and TNF-a + cells were then further gated from the iNKT cells.

IFN-c ELISpot Assay
The total IFN-c producing cells among the a-GalCer stimulated PBMC cells were determined by an ELISpot assay using the monkey IFN-c ELISpot kit (Mabtech). Briefly, the Multiscreen HA ELISpot plate (Millipore, Billerica, MA) was first coated with anti-IFN-c antibodies. Next, the PBMC cells from pig-tailed macaques were added at 5610 5 cells/well and stimulated with a-GalCer at 0.1 mg/ml or 1 mg/ml for 24 hours at 37uC. In the negative control groups, the cells were cultured with 0.1% DMSO. After washing five times, the plate was incubated with biotinlabeled anti-IFN-c antibodies for 1 hour followed by incubation with avidin-HRP. Finally, the spots were developed with an AEC ELISpot substrate kit (BD Biosciences).

Results and Discussion
Here, we aimed to characterize the properties of iNKT cells derived from pig-tailed macaques to determine whether the cells in this species exhibit similar properties to human iNKT cells. The goal of the study was to determine whether pig-tailed macaques represent an appropriate animal species for pre-clinical testing. We first determined the frequency of iNKT cells among PBMCs collected from pig-tailed macaques. To accomplish this, we identified iNKT cells by staining PBMCs with an a-GalCerloaded human CD1d-tetramer (a-GalCer-CD1d-Tet). As shown in Fig. 1, we detected a distinct population of PBMCs that react with a-GalCer-CD1d-Tet, but not with the unloaded human CD1d-tetramer. The percentage of these a-GalCer-CD1d-Tet + cells ranged from 0.13% to 0.4% of the total PBMCs (Fig. 2B). These results confirm those from a previously published study   [30], and indicate that the percentage of iNKT cells in the peripheral blood of pig-tailed macaques is comparable to what has been observed in the peripheral blood of humans. To determine the correlation between a-GalCer-CD1d-Tet + cells and Va24 + cells, we co-stained pig-tailed macaque PBMCs with a-GalCer-CD1d-Tet and anti-Va24 antibodies, as shown in Fig. 2A. We found that approximately two-thirds of the Va24 + cells were a-GalCer-CD1d-Tet + cells (Fig. 2B), and there was a strong positive linear correlation (p = 0.0187; R 2 = 0.9599) between the percentages of the two subpopulations (Fig. 2C).
In humans, the majority of a-GalCer-CD1d-Tet + iNKT cells have been shown to ''co-express'' an invariant Va24-Ja18 chain and a semi-invariant Vb11 chain [36][37][38]. Therefore, we sought to determine whether a-GalCer-CD1d-Tet + iNKT cells derived from pig-tailed macaques also co-express Va24 and Vb11. Unfortunately, the anti-human Vb11 antibody failed to cross-react with pig-tailed macaque iNKT cells, as has been shown with iNKT cells derived from other monkey species (Fig. 3) [27][28][29]. Furthermore, the 6B11 antibody, which is known to react with the CDR3 region of the Va24-Ja18 chain, failed to react with Va24 + cells derived from pig-tailed macaques (Fig. 3). Although the CDR3 region between the human and rhesus Vc24 chain is almost identical (98% homology) [39], it is possible that the amino-acid sequence of the pig-tailed macaque Va24 chain varies enough from the human Va24 chain to lack the 6B11 epitope. This issue requires clarification and will be resolved in a future study.
We next determined the CD4 and CD8 phenotypes of pig-tailed macaque iNKT cells (Fig. 4A) and found that iNKT cells primarily consisted of the CD4+ and CD8+ subpopulations. The few remaining cells were double negative (DN) (Fig. 4B). All CD8 + iNKT cells were also found to be CD8ab + (Fig. 4C). These results confirmed an earlier study showing that pig-tailed macaque iNKT cells consist of a significant percentage of a CD4 + subpopulation [30]. Furthermore, the CD4/CD8 distribution is somewhat different from iNKT cells derived from other monkey species, which are largely made up of CD8 + cells [27][28][29]. More importantly, this distribution pattern resembles human iNKT cells, except DN iNKT cells are more abundant in humans [4,5].
Regarding the functionality of iNKT cells, human iNKT cells activated by a-GalCer are known to secrete a myriad of cytokines, with CD8 + iNKT cells biased toward a Th1 phenotype, CD4 + iNKT cells predominantly secreting Th2 cytokines, and DN iNKT cells exhibiting an intermediate Th1/Th2 phenotype [40]. Nonhuman primate iNKT cells have been shown to display a similar function to human iNKT cells, but there are some differences among different species. For example, rhesus macaque iNKT cells secrete large amounts of TGF-b, IL-6, and IL-13, and modest levels of IFN-c, whereas IL-10 secretion was negligible and no detectable IL-4 was observed [41]. However, sooty mangabey iNKT cells have been shown to secrete virtually all cytokines tested, including IFN-c, TNF-a, IL-2, IL-13, and IL-10 [29]. In addition, their CD8 + NKT subpopulation produced a high amount of IFN-c and expressed significantly higher levels of granzyme B and perforin [42].
To investigate the function of pig-tailed macaque iNKT cells in this study, we first measured the percentage of a-GalCer-activated iNKT cells secreting IFN-c, TNF-a and IL-10, using an ICCS assay. As shown in Fig. 5, a significant percentage of pig-tailed macaque iNKT cells secreted TNF-a and/or IFN-c, whereas they failed to secrete a significant amount of IL-10 (data not shown). We next analyzed the percentages of CD4 + and CD8 + iNKT cell subpopulations among the total IFN-c-secreting iNKT cells. Although both CD4 + and CD8 + iNKT cells produced IFN-c after stimulation with a-GalCer, the percentage of IFN-c-secreting CD8 + iNKT cells was much higher than IFN-c-secreting CD4 + iNKT cells (Fig. 6). Furthermore, the IFN-c ELISpot assay showed that 100-400 per million PBMCs secreted IFN-c in response to 1 mg/ml of a-GalCer (Fig. 6C). This result corroborates our ICCS assay and indicates that a significant number of iNKT cells among PBMCs secrete IFN-c upon a-GalCer stimulation.
We then performed various correlation analyses and found a marginal correlation between the relative number of a-GalCeractivated cells secreting IFN-c among PBMCs, as determined by ELISpot assay, and the percentage of total iNKT cells among PBMCs, as determined by FACS analysis (R 2 = 0.7847, p = 0.0455) (Fig. 7A). However, the correlation became much stronger when we performed a correlation analysis between the relative number of a-GalCer-activated cells secreting IFN-c among PBMCs and the percentage of CD8 + iNKT cells among PBMCs (R 2 = 0.9576, p = 0.0135) (Fig. 7A). Interestingly, when we compared the relative number of IFN-c-secreting cells among PBMCs and the percentages of IFN-c-secreting CD8 + and CD4 + iNKT cells by ICCS assay, we found a strong correlation for the relative number of IFN-c-secreting cells among PBMCs with IFNc-secreting CD8 + iNKT cells (R 2 = 0.8965, p = 0.0146), but not with IFN-c + CD4 + iNKT cells (R 2 = 0.2559, p = 0.3866) (Fig. 7B). Thus, our current functional study demonstrates that the majority of pig-tailed macaque iNKT cells that secrete IFN-c consist of CD8 + iNKT cells, although CD4 + iNKT cells can also produce Th1 cytokines, including IFN-c and TNF-a.
We would like to emphasize that due to the lack of available antibodies that cross-react with pig-tailed macaque cells, we could only perform a limited study of the phenotype and function of pigtailed macaque iNKT cells. Despite this difficulty, however, our current study demonstrates that the percentage of iNKT cells present in the peripheral blood of pig-tailed macaques is comparable to the iNKT cells found in human peripheral blood. Furthermore, similar to humans, a large proportion of Va24 + CD3 + cells are a-GalCer-CD1d-Tet + CD3 + iNKT cells, and almost half of these express CD4 molecules.
Together, these results highlight the properties of pig-tailed macaque iNKT cells, which resemble human cells to some degree. In light of previous successful research studies using pig-tailed macaques for certain human diseases [31][32][33][34][35], our study provides further evidence supporting the use of pig-tailed macaques in the pre-clinical testing of various iNKT cell-stimulating ligands. In particular, they may be useful for evaluating therapeutic and prophylactic measures across a myriad of human diseases in the future.