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 1

Cross-species comparisons.

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Cross-species comparisons. (A) UMAP dimensional reduction of PBMCs from ...
(A) UMAP dimensional reduction of PBMCs from human and 3 NHP species demonstrating overlap of major populations. Species identity is color coded. (B) FlowSOM metaclusters (k = 17) projected onto UMAP plot from A. (C) Proportion of total live CD45+ PBMCs for each species represented by each metacluster from B. (D) Proportion of CD8+ T cells made comprised of central memory (CD28+CD95+) T cells, effector memory (CD28–CD95+) T cells, and naive (CD28+CD95–) T cells. Significance is shown in proportion among species. (E) Projections of central memory, effector memory, and naive CD8+ T cells onto UMAP dimensional reduction, with division by metaclustering as defined in B from 4 different species. (F) Proportion of CD4+ T cells made comprised of central memory T cells, effector memory T cells, and naive T cells. Significance is shown in proportion among species. (G) Projections of central memory, effector memory, and naive CD4+ T cells onto UMAP dimensional reduction, with division by metaclustering as defined in B from 4 different species. (H) Proportion of CD8+ T cells that are central memory plotted against human-equivalent age. (I) Proportion of CD8+ T cells that are effector memory plotted against human-equivalent age. (J) Proportion of CD4+ T cells that are naive plotted against human-equivalent age. The asterisks indicate significant differences among species as calculated by 2-way ANOVA with Šidák’s multiple comparison analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The number of green monkeys (GM), crab-eating macaques (CEM), humans, and rhesus monkeys (RhM) are 2, 3, 2, and 3, respectively, for A, C, and E–G and are 4, 3, 4, and 3, respectively, for D, F, and H–J. NHP, nonhuman primate; UMAP, Uniform Manifold Approximation and Projection.