Molecular and functional characterization of the first human gain of function variant In Lck
FUSARO M. 1,7, LOPES SOUZA C. 2,3, TANITA K. 2,3, SIMONIN M. 8, FOURNIER B. 2,3,4, NEVEN B. 3,4, LESOURNE R. 1, DUPRÉ L. 1,6, PICARD C. 2,3,4,5, SOUDAIS C. 2,3, LATOUR S. 2,3
1 Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), INSERM UMR1291, CNRS UMR5051, Toulouse, France; 2 Laboratory of Lymphocyte Activation and Susceptibility to EBV infection, Institut national de la santé et de la recherche médicale UMR 1163, Paris, France; 3 Paris Cité University, Imagine Institute, Paris, France; 4 Department of Pediatric Immunology, Hematology and Rheumatology, Necker-Enfants Malades Hospital, Assistance Publique - Hôpitaux de Paris, Paris, France; 5 Study Center for Primary Immunodeficiencies, Necker-Enfants Malades Hospital, Assistance Publique - Hôpitaux de Paris, Paris, France; 6 Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria; 7 Immunology Department Laboratory, Institut Fédératif de Biologie, Toulouse University Hospital, Toulouse, France; 8 Université Paris Cité, Institut Necker-Enfants Malades (INEM), Institut national de la santé et de la recherche médicale (Inserm) U1151, and Laboratory of Onco-Hematology, Assistance Publique-Hôpitaux de Paris, Hôpital Necker Enfants-Malades, Paris, France
T cell receptor (TCR) signaling is initiated by lymphocyte-specific protein tyrosine kinase (LCK) and is mandatory for T cell development, activation and function. Following newborn screening, we discovered a heterozygous in-frame deletion in LCK (F498del) in two siblings presenting severe CD8+ T cell lymphopenia and investigated its functional impact on T cell biology.
We assessed the functional effects of the LCK F498del allele on primary T cells and LCK-deficient Jurkat cells (Jcam1.6 cell line) reconstituted with LCK WT or LCK F498del. Additionally, we evaluated the immune phenotype of a heterozygous LCK F498del CRISPR Cas9 knock-in mouse model.
The deletion site is located in critical region for maintaining the inactive conformation of LCK. Immune phenotype revealed severe CD8+ T cell lymphopenia and very low mucosal-associated invariant T cells (MAIT) in both siblings. Interestingly, decreased expression of CD4 and CD8 co-receptors was observed, a feature also present in the only reported patient with complete LCK deficiency. We observed decreased reactivation induced cell death and abnormal Ca2+ mobilization upon weak TCR engagement in patients-derived T cells. Furthermore, Jcam1.6 cells transfected with LCK F498del showed increased Ca2+ mobilization, enhanced CD69 expression following TCR activation, and intensified phosphorylation of CD3zeta and ZAP70, two direct targets of LCK. Preliminary results obtained from heterozygous F498del mice exhibited a moderate thymopoiesis defect associated to an elevated CD4/CD8 ratio and a decreased expression of CD4 and CD8 co-receptors in periphery.
These findings indicate that LCK F498del functions as a gain-of-function variant, lowering the activation threshold for T cell signaling. Although the LCK F498del appears to impact T-cell differentiation and function, both patients are so far asymptomatic and healthy. Further study of heterozygous and homozygous F498del mice will help to better characterize the functional consequences of this variant in vivo and to increase our knowledge on LCK activity during thymopoiesis.