Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation
Celia C. Cubitt, … , Jacqueline E. Payton, Todd A. Fehniger
Celia C. Cubitt, … , Jacqueline E. Payton, Todd A. Fehniger
Published May 28, 2024
Citation Information: J Clin Invest. 2024;134(15):e173602. https://doi.org/10.1172/JCI173602.
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Research Article Immunology

Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation

Abstract

The surface receptor CD8α is present on 20%–80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses in patients with leukemia in prior studies, thus, we hypothesized that CD8α may affect critical NK cell functions. Here, we discovered that CD8α– NK cells had improved control of leukemia in xenograft models compared with CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, we found that CD8α expression was induced on approximately 30% of previously CD8α– NK cells following IL-15 stimulation. These induced CD8α+ (iCD8α+) NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity compared with those that sustained existing CD8α expression (sustained CD8α+) or those that remained CD8α– (persistent CD8α–). These iCD8α+ cells originated from an IL-15Rβhi NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identified human NK cell capacity for IL-15–induced proliferation and metabolism in a time-dependent fashion, and its presence had a suppressive effect on NK cell–activating receptors.

Authors

Celia C. Cubitt, Pamela Wong, Hannah K. Dorando, Jennifer A. Foltz, Jennifer Tran, Lynne Marsala, Nancy D. Marin, Mark Foster, Timothy Schappe, Hijab Fatima, Michelle Becker-Hapak, Alice Y. Zhou, Kimberly Hwang, Miriam T. Jacobs, David A. Russler-Germain, Emily M. Mace, Melissa M. Berrien-Elliott, Jacqueline E. Payton, Todd A. Fehniger

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Figure 9

CD8A KO enhances cytokine secretion and degranulation following NKp30 stimulation.

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CD8A KO enhances cytokine secretion and degranulation following NKp30 st...
(A–C) Primary human NK cells were electroporated with Cas9 mRNA and sgRNA targeting CD8A or a control gRNA (TRAC) and cultured in vitro in 1 ng/mL IL-15 for 6 days. NK cells were stimulated with plate-bound antibodies (10 μg/mL) targeting NKp30 or mouse IgG1 isotype control antibody for 6 hours, with GolgiPlug/Stop for the last 5 hours. The percentage of NK cells positive for expression of (A) CD107a, (B) IFN-γ, or (C) TNF is shown. n = 13 donors and 7 independent experiments. (D–F) Control or CD8-KO cells were labeled with 50 nM CFSE, mixed together at a 1:1 ratio, and then labeled with the UV-excitable, Ca2+-sensing dye Indo-1. A mAb (5 μg/mL) targeting NKp30 alone or NKp30 (5 μg/mL) and KIR3DL1 (0.2 μg/mL) was added for 20 minutes at 4°C, cells were washed, and then cross-linking was induced at the indicated time point (black arrow) using goat anti–mouse IgG (10 μg/mL). Calcium flux was measured by flow cytometry. (E) Data are shown as the normalized ratio of Indo-violet over Indo-blue within control or CD8-KO cells as a function of time in cells from 1 representative donor. (F) Sum of the AUC of the normalized Indo-violet/Indo-blue ratio for all time points in control and CD8-KO cells. n = 3 donors and 3 independent experiments; donors were prescreened to ensure KIR3DL1 and CD8α expression of greater than 30%. Data represent the mean ± SEM. **P < 0.01 and ***P < 0.001, by (A–C) 2-way ANOVA with Holm-Šídák correction for multiple comparisons and (F–G) paired, 2-tailed Student’s t test.