Animal models are an integral part of the drug development and evaluation process. However, they are unsurprisingly imperfect reflections of humans, and the extent and nature of many immunological differences are unknown. With the rise of targeted and biological therapeutics, it is increasingly important that we understand the molecular differences in the immunological behavior of humans and model organisms. However, very few antibodies are raised against non-human primate antigens, and databases of cross-reactivity between species are incomplete. Thus, we screened 332 antibodies in five immune cell populations in blood from humans and four non-human primate species generating a comprehensive cross-reactivity catalog that includes cell type-specificity. We used this catalog to create large mass cytometry universal cross-species phenotyping and signaling panels for humans, along with three of the model organisms most similar to humans: rhesus and cynomolgus macaques and African green monkeys; and one of the mammalian models most widely used in drug development: C57BL/6 mice. As a proof-of-principle, we measured immune cell signaling responses across all five species to an array of 15 stimuli using mass cytometry. We found numerous instances of different cellular phenotypes and immune signaling events occurring within and between species, and detailed three examples (double-positive T cell frequency and signaling; granulocyte response to Bacillus anthracis antigen; and B cell subsets). We also explore the correlation of herpes simian B virus serostatus on the immune profile. Antibody panels and the full dataset generated are available online as a resource to enable future studies comparing immune responses across species during the evaluation of therapeutics.

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IntroductionNon-human primates (NHPs) are critical components of drug development because of their similarity to humans. Many key immunology assays, such as flow cytometry, Western blots, immunohistochemistry and immunofluorescence microscopy, make use of antibodies to demarcate specific cell types and quantify signaling moieties. Very few antibodies are raised against non-human primate antigens; instead, researchers typically use anti-human antibodies that are cross-reactive with the non-human primate species that they are studying. To help researchers find antibodies for NHP research, the National Institutes of Health supports a highly valuable database of the cross-reactivity of commercially available antibodies with 13 NHP species (http://www.nhpreagents.org). The database is derived from manufacturer and investigator reports, and typically provides a simple yes/no statement about whether a clone stains a species, with occasional comments about staining intensity or specificity. While an invaluable resource, the database is limited in its coverage. For example, prior to this study, only 28 CD markers had been evaluated in African green monkeys.Additionally, with few exceptions, the database lacks information about the cell types bound by cross-reactive antibodies, and there are many known instances of antibody clones binding different cell types in different species. For example, granulocyte and monocyte marker expression is known to be substantially different in humans than in non-human primates. Anti-human CD33 clone AC104.3E3 was reported in the NIH database and manufacturer's datasheet as cross-reactive with rhesus and cynomolgus macaques, but our lab and others determined that in those species, it prominently stains granulocytes (1, 2), while in humans it stains monocytes and classical dendritic cells. As another example, the Fcγ receptor CD16 is found on granulocytes in humans and sooty mangabeys, but not in macaques or baboons (3, 4), which will likely confound animal studies evaluating therapeutic antibodies, which may bind, transduce signals through and mediate internalization via this Fcγ receptor. Yet another example is CD56, which is expressed on monocytes in macaques (5), but is a canonical NK cell marker in humans. Thus, researchers must confirm that each clone they use is staining the cell population of interest through literature review or experimental verification.Here we present an expansion of both the breadth and depth of primate cross-reactivity data. We screened 332 monoclonal antibodies in blood from two individuals of each of four NHP species: rhesus macaque (Macaca mulatta), cynomolgus macaque (Macaca fascicularis), African green monkey (Chlorcebus aethiops) and olive/ yellow baboon (Papio hamadryas anubis x Papio hamadryas cynocephalus hybrid); and found more than 120 clones that stained one or more populations in each species. Furthermore, we included counter-stain antibodies that allowed us to determine staining specificity in at least five major immune cell populations. Data from ...

Assessing the health and competence of the immune system is central to evaluating vaccination responses, autoimmune conditions, cancer prognosis, and treatment. With an increasing number of studies examining immune dysregulation, there is a growing need for a curated reference of variation in immune parameters in healthy individuals. We used mass cytometry (CyTOF) to profile blood from 86 humans in response to 15 ex vivo immune stimuli. We present reference ranges for cell-specific immune markers and highlight differences that appear across sex and age. We identified modules of immune features that suggest there exists an underlying structure to the immune system based on signaling pathway responses across cell types. We observed increased MAPK signaling in inflammatory pathways in innate immune cells and greater overall coordination of immune cell responses in females. In contrast, males exhibited stronger pSTAT1 and pTBK1 responses. These reference data are publicly available as a resource for immune profiling studies.