An ELF4 hypomorphic variant results in NK cell deficiency

Abstract

NK cell deficiencies (NKD) are a type of primary immune deficiency in which the major immunologic abnormality affects NK cell number, maturity, or function. Since NK cells contribute to immune defense against virally infected cells, patients with NKD experience higher susceptibility to chronic, recurrent, and fatal viral infections. An individual with recurrent viral infections and mild hypogammaglobulinemia was identified to have an X-linked damaging variant in the transcription factor gene ELF4. The variant does not decrease expression but disrupts ELF4 protein interactions and DNA binding, reducing transcriptional activation of target genes and selectively impairing ELF4 function. Corroborating previous murine models of ELF4 deficiency (Elf4-/-) and using a knockdown human NK cell line, we determined that ELF4 is necessary for normal NK cell development, terminal maturation, and function. Through characterization of the NK cells of the proband, expression of the proband's variant in Elf4-/- mouse hematopoietic precursor cells, and a human in vitro NK cell maturation model, we established this ELF4 variant as a potentially novel cause of NKD.

Keywords: Cell Biology; Immunology; Innate immunity; Monogenic diseases; NK cells.

Figure 1. Clinical phenotype and genotype of an individual with presumed NKD.

(A) Persistent herpes zoster lesions on proband crossing dermatomes showing trunk (left), arm, and neck (right). (B) Pedigree of the proband with X-linked inheritance. (C) Pedigree of a second family with ELF4 c.560C>A variant. Pedigrees show affected (black), carrier (black dot), unknown carrier (white dot), predicted carrier (gray dot), unknown (gray), and unaffected (white) individuals with their respective genotype: X* (c.560C>A ELF4 confirmed), X^u (unconfirmed genotype), and X (presumably wild-type ELF4). (D) Sanger sequencing of ELF4 in proband and his mother (arrow indicates nucleotide change c.560C>A. (E) Sanger sequencing of the genotyped individuals in the second family.

Figure 2. Reduced NK cell frequency is accompanied by impaired NK cell function and decreased perforin expression in the proband.

PBMCs from proband (red) and healthy control (blue) were used to assess NK cell phenotype. (A) NK cell frequency in the proband compared with healthy control and previously determined normal ranges (20) with FACS plots of NK cells defined as CD56⁺CD3^–. (B) CD56^bright NK cell frequency with histograms delineating CD56^bright and CD56^dim in the proband and healthy control (left) and compared with previously defined normal ranges and healthy control (right). (C) ⁵¹Cr release assay using PBMCs against K562 target cells with (solid line) and without (dashed line) IL-2 stimulation cell-mediated direct NK cell cytotoxicity. (D) CD8⁺ T cell cytotoxicity assay using in vitro expanded cytotoxic T lymphocytes against P815 target cells preincubated with anti-CD3. (E) Frequency of nascent (left) and processed (right) perforin in total NK cells and CD56^bright/CD56^dim subsets using perforin antibody clones BD48 and δG9, respectively. (F) Frequency of granzyme B in total NK cells and CD56^bright/CD56^dim subsets. Data represent mean ± SEM of ≥3 biological replicates (4 for NK cell cytotoxicity and 3 for T cell cytotoxicity, n ≥ 4 for all other); *P < 0.05, **P < 0.01, ****P < 0.0001; 2-tailed Student’s t test with Bonferroni’s correction for multiple comparisons and Wilcoxon matched pairs signed-rank test for specific lysis curves.

Figure 3. Reduced NK cell frequency is accompanied by impaired NK cell function and decreased perforin expression in a murine ELF4 BM chimera model.

(A) ELF4 BM chimera model experimental design. ELF4 wild-type or T187N-IRES-GFP constructs were expressed in C57BL/6 Elf4^–/– HSPCs and transplanted into recipient Elf4^+/+ or Elf4^–/– mice. (B) Percentage of ectopic expression of ELF4^WT (blue) and ELF4^T187N (red) and endogenous expression of Elf4^+/+ (purple) or Elf4^–/– (orange) from blood samples in murine NK cells identified as CD3^–NK1.1⁺. (C) NK cell maturation subsets from spleen samples identified by their CD11b and CD27 expression maturing as indicated by the arrow. Comparing control mice (black) and NK cells expressing the ELF4^WT (blue) and ELF4^T187N (red) constructs. (D) Perforin-positive splenic NK cells from ELF4^WT, ELF4^T187N, and Elf4^+/+ control samples from polyI:C-stimulated mice. (E) NK cell cytotoxicity against YAC-1 target cells measured via Cr⁵¹ release assay using sorted NK cells expressing Elf4^+/+ control, ELF4^WT, and ELF4^T187N from the spleens of polyI:C-stimulated chimeric mice. (n = 6: Elf4^+/+, 2: ELF4^WT, and 4: ELF4^T187N.) (F) LU calculated from the previous cytotoxicity assay. The data represent mean ± SEM of 3 independent experiments; each symbol represents an individual mouse with an n of ≥4 unless otherwise stated; *P < 0.05, ***P < 0.001; 2-tailed Student’s t test with Bonferroni’s correction for multiple comparisons.

Figure 4. Expression of ELF4 in human NK cell precursors reflects its role in maturation.

(A–C) Isolated CD34⁺ precursors were cultured with stromal cells for 4 weeks to generate mature NK cells in vitro, with the lines in the bar graphs (bottom) connecting experimental repeats. (A) NK cell frequency from healthy control and proband samples. (B) Frequency of the mature population of the NK cell gate identified as CD16⁺. (C) Additional analysis of the same in vitro differentiation data (as shown in A and B) identifying the frequency of the NK cell developmental stages as Lin^– stage 1 CD34⁺CD117^–, stage 2 CD34⁺CD117⁺, stage 3 CD34^–CD117⁺CD94^–, stage 4 CD34^–CD117⁺CD94⁺CD56⁺CD16^–, and stage 5 CD34^–CD117⁺CD94⁺CD56⁺CD16⁺. (D and E) ELF4 expression analyzed by flow cytometry in NK cell precursors from human peripheral blood (D) or tonsil (E) analyzed as per Supplemental Figure 3A. ELF4 MFI of the positive cells with representative histograms. (n = 5.) Box plots show the interquartile range (box), median (line), and minimum and maximum (whiskers). Data represent mean ± SEM of ≥3 biological replicates unless N otherwise specified; *P < 0.05, **P < 0.01, 2-tailed Student’s t test (A–C) and ANOVA with multiple comparisons (D and E).

Figure 5. ELF4 regulates perforin expression in human NK cell lines.

Doxycycline-inducible ELF4 shRNA or scramble control were expressed in NK92 and YTS human NK cell lines. (A and C) Representative Western blot (left) of samples without or with doxycycline treatment and parental cells with graph of ELF4 expression normalized to loading control and subsequently normalized to respective doxycycline-treated negative control (right) in NK92 (A) and YTS (C) cells. (B and D) ELF4 expression (MFI) measured by flow cytometry and normalized to respective doxycycline-treated negative control in NK92 (B) and YTS (D) cells. (E and F) Nascent (left) and processed (middle) perforin and granzyme B (right) MFI measured by FACS and normalized to respective doxycycline-treated negative control in NK92 (E) and YTS (F). (G and H) 48 hours after treatment with CMA, nascent (left) and processed (middle) perforin and granzyme B (right) MFI normalized to respective doxycycline-treated negative control in NK92 (G) and YTS (H) cells. Data represent mean ± SEM ≥3 biological replicates (n ≥ 4 scr, ≥9 ELF4); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 2-tailed Student’s t test.

Figure 6. Normal ELF4 quantity and localization in the presence of c.C560A p.T187N.

(A) Western blot of proband and healthy participant BLCLs (top) and quantification (bottom) of ELF4 expression normalized to tubulin loading control. (B) ELF4 MFI with a representative histogram (top) and quantification (bottom) assessed using imaging flow cytometry, with IgG control. (C) ELF4 MFI in different stages of cell cycle within representative histograms for healthy control and proband (top) and quantification (bottom), with IgG control. (D) ELF4 protein domains. P, phosphorylation sites predicted (gray) and experimentally confirmed (magenta); S, SUMOylation; Ub, ubiquitination; NLS, nuclear localization signal with T187N variant location (inset). (E) Proportion of ELF4 localizing to the nucleus in BLCLs assessed by imaging flow cytometry (left) with representative histograms (middle) and images (right) of bright-field, nucleus, and ELF4, and an overlay of ELF4/nuclear dye (scale bar: 7 μm). (F) Nuclear localization of ELF4 determined in different stages of the cell cycle. (G) Western blot of ELF4 wild-type and T187N variant overexpression in HEK293T in the nuclear and cytoplasmic fractions (left), with quantification (right), normalized to Lamin B1 and tubulin loading control for the respective fractions and further normalized to wild-type control. The data represent mean ± SEM of ≥3 independent experiments; images represent experiments repeated at least 3 times; 1-way ANOVA with multiple comparisons (A–C, E, and F), 2-tailed Student’s t test (G).

Figure 7. ELF4 functional changes induced by p.T187N.

(A) I-TASSER prediction of ELF4 structure showing the backbone with a cluster predicted to form a binding pocket encircled and DNA binding residues with red arrow pointing at aa 187. (B) UCSF Chimera software analysis from predicted models of hydrogen bonds (green lines) with listed aa residue interactions of ELF4^T187 (blue, left) and ELF4^N187 (pink, right). (C) LIGPLOT software analysis from predicted models of Van der Waals interactions (pink) and hydrogen bonds, showing predicted interactions with aa within the same chain or proximal (across the black line) based on protein folding for ELF4^T187 and ELF4^N187. (D) Relative fluorescence units of luciferase promoter reporter (PRF1 and MDM2) for ELF4 T187N overexpression normalized to the WT control. (E) Independent repeats of ChIP quantitative PCR for overexpressed WT and T187N ELF4 bound to promoters (NT, nontransfected). (F) Salt-titrated extraction assay of chromatin-bound ELF4. Western blot run on a gel including the total cell lysate and the cytoplasmic and nuclear soluble fractions and a second gel including the chromatin-bound salt extraction. Showing the proteins detached with the increasing concentrations of salt from the chromatin-bound fraction after sequential extraction of the cytoplasmic, nuclear soluble, and chromatin-bound fractions (top, representative blot) with red lines indicating the significant difference in extraction between the WT and T187N ELF4 at lower salt concentrations. ELF4 expression normalized to loading control and subsequently to the wild-type control (bottom) across 3 independent experiments. The data represent mean ± SEM of ≥3 independent experiments. Western blot is representative of experiment repeated at least 3 times; *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed paired Student’s t test and Wilcoxon matched pairs signed-rank test.