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.
View: Text | PDF
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

×

Figure 5

In vitro activation of human PBMCs leads to PD-1 expression on T cells, not NK cells.

Options: View larger image (or click on image) Download as PowerPoint
In vitro activation of human PBMCs leads to PD-1 expression on T cells, ...
(A) Representative gating shows NK and T cell populations isolated from human PBMCs after 72 hours in media. (B) Resting CD3–CD56+ NK cells lack expression of activation marker CD69 and are negative for PD-1, (C) while resting CD3+CD56– T cells also lack expression of CD69, but show baseline expression of PD-1. (D) After culture in ConA for 72 hours, NK cells show increased CD56 expression, (E) with upregulation of CD69 and continued absence of PD-1. (F) After ConA stimulation, T cells exhibit increased CD69 expression and robust PD-1 expression. (G) After culture in 1000 IU/mL rhIL-2 for 72 hours, (H) NK cells upregulate CD69 with minimal PD-1 expression. (I) In contrast, CD3+ T cells exhibit moderate increases in both CD69 and PD-1. (J) After culture in 100 ng/mL rhIL-15 for 72 hours, (K) NK cells upregulate CD69 with minimal PD-1 expression. (L) Similarly to what occurs with IL-2 stimulation, CD3+ T cells exhibit moderate increases in both CD69 and PD-1 after IL-15 stimulation. (M) Summary data show significantly increased expression of CD69 on NK and T cells with either ConA, IL-2, or IL-15 compared with media alone. (N) Under identical conditions, PD-1 was significantly upregulated on ConA-stimulated T cells with no change in PD-1 expression on NK cells under any conditions. Data are shown as mean ± SD for n = 4–6 unique human donors. ***P < 0.001; ****P < 0.0001, 1-way ANOVA compared with cell-specific media only controls.