Human NK cell deficiency as a result of biallelic mutations in MCM10
Emily M. Mace, … , Anja K. Bielinsky, Jordan S. Orange
Emily M. Mace, … , Anja K. Bielinsky, Jordan S. Orange
Published August 31, 2020
Citation Information: J Clin Invest. 2020;130(10):5272-5286. https://doi.org/10.1172/JCI134966.
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Research Article Immunology

Human NK cell deficiency as a result of biallelic mutations in MCM10

Abstract

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage–response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.

Authors

Emily M. Mace, Silke Paust, Matilde I. Conte, Ryan M. Baxley, Megan M. Schmit, Sagar L. Patil, Nicole C. Guilz, Malini Mukherjee, Ashley E. Pezzi, Jolanta Chmielowiec, Swetha Tatineni, Ivan K. Chinn, Zeynep Coban Akdemir, Shalini N. Jhangiani, Donna M. Muzny, Asbjørg Stray-Pedersen, Rachel E. Bradley, Mo Moody, Philip P. Connor, Adrian G. Heaps, Colin Steward, Pinaki P. Banerjee, Richard A. Gibbs, Malgorzata Borowiak, James R. Lupski, Stephen Jolles, Anja K. Bielinsky, Jordan S. Orange

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Figure 7

Increased replication stress in an MCM10-KD cell line.

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Increased replication stress in an MCM10-KD cell line. WT or MCM10-KD NK...
WT or MCM10-KD NK92 cells were fixed, permeabilized, and incubated with anti-γH2AX Alexa Fluor 647. (A) Images were acquired by confocal microscopy. Scale bar: 5 μm. (B) The frequency of cells per field of view that were positive for γH2AX by microscopy was scored by manual counting. Shown are the means from 3 independent replicates performed on different days and normalized to WT NK92. NS, 1-sample t test and Wilcoxon’s test. Data are represented as mean ± 95% CI. (C) The number of γH2AX foci per cell were determined by manual counting of microscopy images. Shown are the means from 3 independent replicates performed on different days and normalized to WT NK92. n = 18–60 cells per condition. **P < 0.01, 1-sample t test and Wilcoxon’s test. Data are represented as mean ± 95% CI. (D) WT NK92 or NK92 MCM10-KD were irradiated with 2 Gy and allowed to recover for 24 hours before fixing and immunostaining for γH2AX. Images were acquired by confocal microscopy, and foci were enumerated by manual counting. Data shown are means from 3 technical replicates performed on different days normalized to WT NK92. n = 10–29 cells per condition. Data are represented as mean ± 95% CI. *P < 0.05, 1-sample t test and Wilcoxon’s test. (E) Cytotoxic function of WT NK92 or MCM10-KD cells against K562 targets was performed by 51Cr release assay. Representative data shown from 3 technical replicates performed on different days. E:T ratio, effector/target ratio. (F) CD56 expression on NK92 (solid line) or MCM10-KD (dashed line) NK92 cells was measured by FACS analysis. Data are representative of 3 technical replicates performed on different days.