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 1

In vitro activated murine NK cells do not upregulate PD-1.

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In vitro activated murine NK cells do not upregulate PD-1. (A) Represent...
(A) Representative NK1.1 and CD3 gating shows NK and T cell populations from resting C57BL/6 mice. (B and C) Untreated NK cells (CD3–NK1.1+) and T cells (CD3+NK1.1–) show low CD69 expression and minimal PD-1 expression. (D) ALAK cells were prepared by culturing splenocytes in 1000 IU/mL rhIL-2 for 7 days. Parent gating shows the enriched NK cell population. With cytokine activation, (E) NK cells show high CD69 expression, but still lack PD-1 expression, (F) while T cells exhibit moderate CD69 expression and low PD-1 expression. (G) Culture with T cell mitogen ConA leads to robust PD-1 expression on T cells, while PD-1 expression on NK cells remains minimal. (H) Schematic for RNA-Seq analysis of resting versus IL-2–stimulated sorted NK cells from WT splenocytes. (I) Despite marked upregulation of activation and proliferation markers, no expression of PD-1 is observed. (J) Rag2–/– splenocytes were used to isolate pure NK cells. (K) NK cells are highly activated by CD69 expression, but PD-1 remains minimal. (L) Comparing WT ALAK cells and Rag2–/– ALAK cells to unstimulated WT splenocytes, mRNA expression of PD-1 was minimal on Rag2–/– NK cells, while expression of granzyme B (Gzmb) is upregulated. Data show mean ± SD for n = 3–4/group and are representative of 3 independent experiments. **P < 0.01; ***P < 0.001, 1-way ANOVA compared with unstimulated WT splenocytes.