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Immunophenotyping Panel Development using Flow Cytometry in Non-human Primates Toxicology Studies

Posted on: October 28, 2020

Development of Cross- Reactive Immunophenotyping Panel for Leukocyte Subset Identification in Cynomolgus Monkey and Human Tissues

Animal models play an important role in both the scientific discovery and in the preclinical toxicology safety assessment process for drug/therapeutic development.  Due to relative genetic similarities to humans, non-human primates (NHP) are used as model systems within this process. Non-human primate species in research include rhesus macaques, crab-eating macaques, stump-tailed macaques, pig-tailed macaques, squirrel monkeys, owl monkeys, baboons, chimpanzees, marmosets and spider monkeys, with rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) monkeys being the most widely used species. The similarity between the human and the non-human primate immune systems coupled with non-human primates susceptibility to varying human pathogens makes them a valuable animal model for studying immune responses to various infectious diseases, as well as assessment of vaccine-induced immunity. As more novel therapeutics are being developed and tested, non-human primate (NHP) may be the most relevant species for evaluating pre-clinical safety endpoints that may lack cross-reactivity in other species. Immuno-toxicological endpoints are routinely used in pre-clinical safety studies and immunophenotyping by flow cytometry is often utilized to better understand what immune mechanisms may be involved in functional endpoints [1].

Immunophenotyping Panel Development

Because of the genetic proximity between humans and non-human primates and the fact that there are very few antibodies developed against non-human primate antigens, researchers generally use anti-human antibodies that are found to be cross reactive with non-human primates. However, it should be noted that there can be key differences that exist between the two species that can limit the use of anti-human antibodies in non-human primate immunologic assays including flow cytometry [2]. The National Institute of Health has developed a database (https://www.nhpreagents.org/) of some commercially available antibodies that show cross reactivity between human and NHP targets, but the list is not complete. Furthermore, the database only indicates the presence or absence of staining and does not distinguish if different populations are stained in NHP’s than in humans. There are known instances of antibody clones staining different cell types in the different species. Granulocytic and monocytic marker expression is known to be different in humans versus non-human primates. Yet another example is CD56, which is restricted to NK cell expression in humans but is primarily expressed on monocytes and a mDC subset population in primates [3] [4]. CD8 is expressed on B lymphocytes in rhesus monkeys but not in humans [2]. It is important for researchers to confirm that the clones that they are using are truly staining the cell population of interest through either careful literature review or by experimental verification. 

Figure 1 is an example of a study that was undertaken to develop a cross-reactive immunophenotyping panel for analysis of leukocyte subsets within cynomolgus PBMCs using flow cytometry. The panel was designed to examine total leukocytes, T cell subsets, NK and NKT cells and B cells. A series of commercially available human monoclonal antibodies that were known to cross react with NHP mononuclear cells were used for flow cytometry analysis [5].

Fig. 1.  Gating strategy for phenotypic analyses of PBMC from a cynomolgus macaque. Lymphocytes and monocytes were first gated based on forward scatter (FCS) versus side scatter (SSC). Then CD3 +, CD4 +, CD8 +, (CD4 + CD8 +), and CD20+, and NK and NKT subpopulations were positively identified from the lymphocyte subset. The specificity of staining for the various markers was determined using isotype control antibody staining for each combination of markers.

Limitations of NHP Models for Toxicology Studies

There are critical differences between the Fc regions of human and NHP models that could have major implications on clinical toxicology endpoints particularly in relation to IgG-based therapeutics. Humans FcγRIIIA (CD16A) is expressed on monocytes, macrophages and NK cells whereas the FcγRIIIB (CD16B) isoform is expressed on neutrophils, eosinophils and other cells. This is different in NHPs, there is only one CD16 gene, that shows higher homology to the human CD16A, and is only expressed in NK cells and monocytes. This is critical because the mechanism of action of IgG-based therapeutic drugs involves the binding to low-affinity CD16. The concept that IgG/CD16 interactions in NHP model those in humans is largely unproven and so there should be careful consideration of the use of NHPs for the evaluation of these types of drug candidates.  

In spite of efforts to characterize CD biomarkers on non-human primate species, there is still a shortage of validated reagents. It is critical to understand the cross-reactivity of antibodies to CD markers in non-human species. Antigens may be characterized into 3 groups based on monoclonal antibody binding characteristics and cellular biomarker distribution. Firstly, there are CD markers for which both monoclonal antibody binding profiles and cellular distribution patterns are consistent with humans. Then there are CD markers for which monoclonal antibodies bind to the same antigen, but the cellular expression of these antigens is inconsistent with human cells. And lastly, there are CD markers for which monoclonal antibody staining is extremely dim or non-existence on non-human leukocytes compared with human leukocytes.

Cell Type/Species Human Macaque
Regulatory T cell CD3+, CD4+, CD25+, CD127-, FoxP23+ CD3+, CD4+, CD25+, FoxP23+
Naive T cell CD3+, CD45RA+, CCR7+, CD95- CD3+, CD45RA+, CCR7+, CD95-
Central Memory T cell CD3+, CD45RA+, CCR7+ CD3+, CD45RA-, CCR7+
Effector Memory T cell CD3+, CD45RA-, CCR7- CD3+, CD45RA-, CCR7-
Terminally Differentiated Effector Memory CD3+, CD45RA+, CCR7- CD3+, CD45RA+, CCR7-


Table 1 T-cell Immunophenotyping Panel of Human versus Macaques.

Key Considerations for NHP Studies

There are some important considerations when designing panels for immunophenotyping clinical endpoints, including the effect of gender and age on subset profiles. In humans, peripheral blood leukocyte subsets vary between genders and with age; typically, females have higher total CD4+ T-cell and CD4+ memory cells and lower CD16+ NK cell counts than males, and both CD4+ and CD8+ memory T cells counts increase with age. However, in cynomolgus monkeys, the average cell counts of CD4+, CD8+, and B-cells were all higher in males than females, except for NK cells that were lower in males than females [1].  There are also reported differences between the immunophenotype profiles of cynomolgus monkeys from the island of Mauritius versus those from mainland China. 

Age is also an important consideration since cynomolgus monkeys have a much higher prevalence of CD4+/CD8+ (double positive) T-cells than humans. These cells exhibit a resting memory phenotype that increases proportionally with age, in parallel with CD28 expression. CD28-mediated T cells activation and cytokine release is markedly different between young and adult cynomolgus monkeys, and this should be a consideration for any toxicology study of immunomodulatory and T-cell targeting study. 

Another consideration for toxicology studies of drugs for T cell mediated diseases (e.g. bronchial asthma, rheumatoid arthritis and type I diabetes) is that human T-cells exhibit much stronger proliferation responses upon activation of the T cell receptor than most NHP models.

With the expansion of immunomodulatory monoclonal antibody-derived therapeutics, understanding the relative pharmacological effect and comparative immunology on both humans and non-human primate models is essential for safe drug development. Intrinsically, these studies have several considerations:

  1. The selection of the most pharmacologically- relevant non-human primate test species for the drug target, i.e. one that both expresses the drug target and evokes a similar pharmacological response to the drug.
  2. Should include a  comprehensive understanding of the limitation of this animal model system with respect to specific clinical end points. It may be necessary to combine in vivo studies with in vitro studies on human and NHP cell lines for comparison.
  3. Studies should be designed to enable the translation of the response in NHP to predict the risk of adverse immunotoxicology events in humans.
  4. Studies should be designed to enable the selection of safe starting doses for first in human clinical trials based on the minimum anticipated biological effect level determined in the NHP model system.

There are many factors to consider when translating toxicology studies in NHP to humans, making the process complex. Since a number of pharmacodynamic endpoints rely on immunophenotyping assays, particularly those pertaining to immunomodulating therapeutics, it is crucial to recognize the importance of cellular expression profiles and pharmacodynamic markers on both the selection of the test animal model and the correct toxicological assessment, in order to ensure that safe translation into human clinical trial studies.  

Learn More

At FlowMetric, our scientific team will collaborate with you to provide scientific expertise for the development of the appropriate immune profiling panel for use in your specific NHP model system for your toxicology and pre-clinical research needs.  Please contact us today to speak with one of our technical support team.

  1. J Immunotoxicol. 2013; (4): 361-372.An inter-laboratroy retrospective analysis of immunotoxicological endpoints in non-human priamtes: Flow Cytometry Immunophenotyping C. Krejsa  et.al.

  2. Immunology. 2005 Jun: 115(2): 206-214. Delineation of Multiple Subpopulations of Natural Killer Cells in Rhesus Macaque. R. Webster et. al.

  3. J. Med Primatol. 2009 Aug: 38 (4): 272-8. Surface Phenotype and Rapid Quantification of Blood Dendritic Cell Subsets in the Rhesus Macaque. K. Brown et. al.

  4. Biorxiv. 2019 Mar 14. Cell type specific monoclonal antibody cross-reactivity screening in non-human primates and development of comparative immunophenotyping panels for CyTOF. Z. Bjornson-Hooper et. al.

  5. J. Am Assoc Lab Anim Sci. 2019 Nov 1;58 (6):774-782. Effects of Transportation and Relocation on the Immunologic Measures in Cynomologus macaques. K. Shelton et. al.   https://pubmed.ncbi.nlm.nih.gov/31604484/


SR Authored by: Susan Reynolds

Susan Reynolds has worked at FlowMetric for over four years. She has over three decades of experience in the areas of flow cytometry and assay development.  Her work has focused on the therapeutic areas of immunology, immuno-oncology, cell therapy, and gene therapy. Sue holds a graduate degree in Immunology from the University of Maine and an executive education MBA from MIT Sloan School of Management.  Sue's career includes drug development research working within Pfizer and Sanofi and as a flow cytometry technical application specialist within Thermo Fisher Scientific and BD Biosciences. Sue was part of Becton-Dickinson’s Global HIV Health initiative working in Africa with the World Health Organization to advance training in flow cytometric applications for advancing vaccine development in the areas of HIV and malaria

JAB Authored by: Dr. Julie Bick



Dr. Julie Bick is a medicinal biochemist who has spent close to 7 years with FlowMetric Life Sciences. After receiving her doctorate in Biochemistry at Southampton University in the UK, she began her career as Associate Professor at Rutgers University, NJ, before moving to the west coast to perform biomedical research with Syngenta and Novartis at the Torrey Mesa Research Institute in San Diego. Dr. Bick specializes in biomedical engineering of cells and proteins in order to provide innovative therapeutic and diagnostic solutions. She brings to FlowMetric a clinical expertise across a wide range of therapeutic areas from autoimmunity to oncology and chronic inflammatory conditions, acquired over 25 years of research experience in academic, biotechnology and pharmaceutical laboratories. In leading FlowMetric Life Sciences’ innovation initiatives, Dr. Bick has been collaborating with BurstIQ to implement Block Chain solutions into the company’s Contract Research Organization division, with a focus on enhanced big data analytics and process control solutions in the regulated clinical environment. Dr. Bick is committed to working with local Community Colleges to support STEM programs for the next generation of scientists

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