A Novel Biallelic LCK Variant Resulting in Profound T-Cell Immune Deficiency and Review of the Literature

Abstract

Lymphocyte-specific protein tyrosine kinase (LCK) is an SRC-family kinase critical for initiation and propagation of T-cell antigen receptor (TCR) signaling through phosphorylation of TCR-associated CD3 chains and recruited downstream molecules. Until now, only one case of profound T-cell immune deficiency with complete LCK deficiency [1] caused by a biallelic missense mutation (c.1022T>C, p.L341P) and three cases of incomplete LCK deficiency [2] caused by a biallelic splice site mutation (c.188-2A>G) have been described. Additionally, deregulated LCK expression has been associated with genetically undefined immune deficiencies and hematological malignancies. Here, we describe the second case of complete LCK deficiency in a 6-month-old girl born to consanguineous parents presenting with profound T-cell immune deficiency. Whole exome sequencing (WES) revealed a novel pathogenic biallelic missense mutation in LCK (c.1393T>C, p.C465R), which led to the absence of LCK protein expression and phosphorylation, and a consecutive decrease in proximal TCR signaling. Loss of conventional CD4⁺ and CD8⁺ αβT-cells and homeostatic T-cell expansion was accompanied by increased γδT-cell and Treg percentages. Surface CD4 and CD8 co-receptor expression was reduced in the patient T-cells, while the heterozygous mother had impaired CD4 and CD8 surface expression to a lesser extent. We conclude that complete LCK deficiency is characterized by profound T-cell immune deficiency, reduced CD4 and CD8 surface expression, and a characteristic TCR signaling disorder. CD4 and CD8 surface expression may be of value for early detection of mono- and/or biallelic LCK deficiency.

Keywords: CD4 and CD8 co-receptor expression; LCK deficiency; TCR signaling; inborn errors of immunity; profound T-cell immune deficiency.

Fig. 1

A novel biallelic missense variant in the LCK kinase domain. A Pedigree of the consanguineous family. The patient is indicated with an arrow. B Chest X-ray of the patient upon admission to Erciyes University Hospital in Kayseri. C Sanger sequencing of the index patient and indicated family members. Consensus amino acid and DNA sequence indicate on top, variant sequence below. D CADD vs. minor allele frequency (MAF)-plot of homozygous variants in LCK present in the gnomAD database and variants LCK C465R and LCK L341P [1]. Created with the PopViz webserver application [38]. The CADD cut-off was calculated with a 95% confidence interval. E Scheme of LCK protein: SH, Src homology region; UD, unique domain; KD, kinase domain. Numbers below indicate starting/ending aa residues of the individual domains. Arrows localize the variants L341P [1] and C465R. F (Right) Ribbon diagram of the X-ray structure of the kinase domain of human LCK in an open conformation (PDB ID: 3LCK) based on [17]. α-helices in purple, β-sheets in yellow, unstructured regions in grey. Residues Y394 and C465 are highlighted. (Left) Detailed view of the local structure surrounding C465. Carbon atoms depicted in green, nitrogen in blue, oxygen in red, and sulfur in yellow. Residues C465, P466, and Y469 are highlighted, and dashed lines indicate hydrogen bonds. Image created with Mol*Viewer [39] and RCSB PDB [40]. G Logo plot of LCK sequence conservation across the species indicated on the left. Created with Jalview [41]

Fig. 2

Reduced protein expression and TCR signaling due to LCK C465R. A LCK and GAPDH immunoblots of whole cell lysates of patient or healthy control (HC) T-cell blasts unstimulated or stimulated with anti-CD3 (2 min), anti-CD3/CD28 (2 min), PMA/ionomycin (P/I, 5 min) or treated with the selective LCK-inhibitor A770041 (A77, 10 min). Orange arrowheads indicate LCK bands. B pZAP70, pSFK (pY416), pERK1/2 or GAPDH immunoblots, conditions as in A. Red arrowheads indicate pLCK bands, black arrowheads pFYN-T. Numbers below pERK1/2 blot indicate fold change pERK1/2 band intensity (HC unstimulated =1), intensity normalized to GAPDH loading (numbers below GAPDH blot; GAPDH signal intensity normalized to HC unstimulated) C LCK, HA-tag and GAPDH immunoblots of whole cell lysates of Jurkat E6.1, J.CaM1.6, and stable transduced cells line expressing either LCK WT or LCK C465R with or without on C-terminal HA-tag or transduced with pCDH containing only the expression cassette for the extracellular domain of the low-affinity nerve growth factor receptor (LNGFR) (J.CaM EV). D Overlay of Ca²⁺-flux measurement in the indicated cell lines. Experiment representative of 3 independent experiments. E pZAP70, pSFK (pY416), pERK1/2 immunoblots in J.CaM1.6 cells transduced with pLJM1 containing LCK WT or LCK C465R and stimulated with either anti-CD3 (2, 5 or 15 min), anti-CD3/CD28 (2 or 5 min), PMA/ionomycin (5 min) or left untreated

Fig. 3

Immune phenotype of patient, mother, and two healthy controls (HC) by flow cytometry. Markers used for staining are indicated on the arrows next to the contour plots, gated populations above. For gating strategy, see Fig. S4. A (upper row) CD4 vs. CD8 expression on CD3⁺ lymphocytes, CD4/CD8 ratio in red; (lower row) αβ and γδT-cell frequency in CD3⁺ lymphocytes. B CD4⁺ T-cell differentiation (upper row) and CD57 expression (lower row). C CD8⁺ T-cell differentiation (upper row) and CD57 expression (lower row). D CD4⁺ CD45RO⁺ CCR6^- T helper cell phenotype. F Treg cells