Cellular immunophenotyping in human and primate tissues during healthy conditions and Ebola and Nipah infections
Andrew P. Platt, Bobbi Barr, Anthony Marketon, Rebecca Bernbaum, Deja F.P. Rivera, Vincent J. Munster, Daniel S. Chertow, Michael R. Holbrook, Scott M. Anthony, Bapi Pahar
Andrew P. Platt, Bobbi Barr, Anthony Marketon, Rebecca Bernbaum, Deja F.P. Rivera, Vincent J. Munster, Daniel S. Chertow, Michael R. Holbrook, Scott M. Anthony, Bapi Pahar
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Research Article Immunology Infectious disease

Cellular immunophenotyping in human and primate tissues during healthy conditions and Ebola and Nipah infections

Abstract

We developed a 29-color spectral cytometry panel to enhance nonhuman primate (NHP) models for cross-reactive immunophenotyping. This panel is suitable for biosafety level 4 (BSL-4) viruses and can be used with both human and NHP samples in BSL-2 research settings. Tissues from humans, rhesus monkeys (RhMs), crab-eating macaques (CEMs), and green monkeys (GMs) were stained with a 29-color immunophenotyping panel requiring only 2 clone substitutions. Comparable staining was observed for all samples. Unbiased analysis showed acceptable overlap in T cell phenotypes across samples, with differences in human and NHP B cells and granulocytes. In CEMs, most circulating CD8+ T cells were from effector memory cells, with significantly higher levels than in humans, RhMs, and GMs. Analysis of samples from various anatomical sites revealed distinct location-specific phenotypes. In Nipah virus–exposed animals, splenocytes showed a substantial increase in IgM+ B cells and a reduction in effector memory CD8+ T cells compared with unexposed controls. Lymph nodes from Ebola virus–exposed animals showed a loss of CXCR3+CD8+ T cells versus unexposed controls. This panel may guide the development of additional multicolor panels in preclinical and clinical settings and may increase understanding of the pathogenesis of diseases caused by emerging and reemerging viruses.

Authors

Andrew P. Platt, Bobbi Barr, Anthony Marketon, Rebecca Bernbaum, Deja F.P. Rivera, Vincent J. Munster, Daniel S. Chertow, Michael R. Holbrook, Scott M. Anthony, Bapi Pahar

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Figure 4

Comparison of Mes LN cell phenotypes in RhMs with and without EBOV exposure.

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Comparison of Mes LN cell phenotypes in RhMs with and without EBOV expos...
(A) UMAP dimension reduction of live CD45+ cells from Mes LN of EBOV-exposed versus unexposed animals (n =2). (B) The UMAP plot from A shows the FlowSOM metaclusters (k = 21) projected onto it. (C) Proportion of total cells from each group belonging to each metacluster are displayed using column graphs with means. (D) Expression of proliferation marker (Ki67), T cell exhaustion marker (PD-1), early activation marker (CD69), and late activation marker (HLA-DR) in Mes LN CD4+ T cells from EBOV-exposed versus unexposed RhMs. (E) Expression of Ki-67, PD-1, HLA-DR, and CD69 on CD8+ T cells in EBOV-exposed versus unexposed RhMs. (F) Representative dot plots of CD8+ T cells expressing CXCR3+ cells are shown for unexposed (left panel) and EBOV-exposed (right panel) samples. Circled areas represent CXCR3+CD8+ T cells. (G) The frequency of CXCR3+CD8+ T cells is presented for EBOV-exposed and unexposed RhMs in columns with means. (H) The proportion of CD20+ B cells, CD11b+ macrophages, and CD11b+CD11c+ DCs in Mes LN from EBOV-exposed versus unexposed RhMs. (I) The proportion of CD20+ B cells that are CD95+, CD38–, HLA-DR+, and IgM– in Mes LN from EBOV-exposed versus unexposed RhMs in columns with means (n =2). EBOV, Ebola virus; Mes LN, mesenteric lymph node; RhMs, rhesus monkeys; UMAP, Uniform Manifold Approximation and Projection.