Minimal PD-1 expression in mouse and human NK cells under diverse conditions
Sean J. Judge, … , Robert J. Canter, William J. Murphy
Sean J. Judge, … , Robert J. Canter, William J. Murphy
Published March 5, 2020
Citation Information: J Clin Invest. 2020;130(6):3051-3068. https://doi.org/10.1172/JCI133353.
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Research Article Immunology

Minimal PD-1 expression in mouse and human NK cells under diverse conditions

Abstract

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Authors

Sean J. Judge, Cordelia Dunai, Ethan G. Aguilar, Sarah C. Vick, Ian R. Sturgill, Lam T. Khuat, Kevin M. Stoffel, Jonathan Van Dyke, Dan L. Longo, Morgan A. Darrow, Stephen K. Anderson, Bruce R. Blazar, Arta M. Monjazeb, Jonathan S. Serody, Robert J. Canter, William J. Murphy

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Figure 3

In vivo acute MCMV infection leads to upregulation of TIGIT, not PD-1, on highly activated NK cells.

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In vivo acute MCMV infection leads to upregulation of TIGIT, not PD-1, o...
(A) WT C57BL/6 and Rag2–/– mice were infected with 2 × 104 PFU MCMV. (B) At 72 hours, viral titers were elevated in the liver. (C) Representative parent gating from spleens of mice shows NK (CD3–NK1.1+) and T (CD3+NK1.1–) cell populations. NK cells were further gated on NKp46+ and compared with those of uninfected mice. MCMV-infected mice had significant upregulation of (D) CD69 and (E) Thy1.2 on NK cells in the spleen and liver at 3 days after infection. (F) PD-1 expression on NK cells in WT mice remained minimal, while (G) TIGIT was upregulated. (H) Though minimal (1%–2% increase), the measured increase in PD-1 was statistically significant, while TIGIT was upregulated by 15% to 25% under identical conditions. (I) Parent gating of splenocytes from acute MCMV infection in Rag2–/– mice shows marked upregulation of CD69, while (J) PD-1 was minimal and TIGIT upregulation significant. (K) PD-1 mRNA expression on MCMV-infected Rag2–/– splenocytes was minimal compared with uninfected WT splenocytes, while both infected groups exhibited marked upregulation of granzyme B. (L) At day 7 after infection, (M) PD-1 expression on T cells was upregulated, while NK expression of PD-1 remained minimal. (N) Summary data of PD-1 expression on NK and T cells at 5 to 7 days after infection. Data are shown as mean ± SD for n = 3 mice/group and are representative of 3 independent experiments (B, D, E, H, and K). Data are shown as mean ± SD for n = 2–3 mice/group, showing results from 4 independent experiments using 1 to 2 × 104 PFU MCMV (N). **P < 0.01; ***P < 0.001; ****P < 0.0001, unpaired Student’s t test (D, E, and H) and 1-way ANOVA compared with uninfected splenocytes (K).