Germline Mutations within the IL2-Inducible T Cell Kinase Impede T Cell Differentiation or Survival, Cause Protein Destabilisation, Loss of Membrane Recruitment and Lead to Severe EBV Lymphoproliferation
Authors: Linka Rene Martin , Huck Kirsten , Krux Frank , Stepenski Polina , Synaeve Cindy , Vettenranta Kim , Deenen Rene , Müller-Fleckenstein Ingrid , Linka Yvonne , Ginzel Sebastian , Halenius Anne , Köhrer Karl , Fleckenstein Bernhard , Laws Hans Jürgen , Hartig Roland , Schraven Burkhart , Latour Sylvain , Borkhardt Arndt 
Published: October 2019

Abstract
Abstract 280
Recently we described mutations in the autosomal encoded IL2-inducible T cell kinase (ITK) as reason for insufficient immune response to EBV in two girls (Huck et al., JCI 2009). After initial discovery of ITK as cause for this novel primary immunodeficiency disease, we established a clinical database within the framework of all pediatric hospitals in Germany (ESPED). In addition, we collected patient samples from 38 EBV-associated lymphoproliferations, 33 genetically undefined autoimmune lymphoproliferative syndromes (ALPS) or suspicion of a congenital hemophagocytic syndrome (40 patients), respectively. We sequenced the ITK locus in those children and identified so far 7 patients with ITK mutations in the pleckstrin homology (PH) domain (ITKR29H), in the SH2 domain (ITKR335W) or the kinase domain (ITKD500T,F501L,D503-620 or ITKD578-620). Patients came from Morocco, Turkey, India or Israel. Immunophenotyping revealed that all patients showed dramatically reduced naive CD4+ T cells (CD45RA) during the course of the disease and a characteristic lack of invariant NKT (Valpha24;Valpha18) cells, which has also been demonstrated for X-linked lymphoproliferative diseases (Purtillo syndrome) caused by SH2D1A or XIAP deficiency. ITK is a crucial part of the T cell receptor (TCR) mediated signaling cascade, which has been shown to critically influence calcium signalling in mouse T cells. Thus, we assayed Ca-response in Herpes saimiri transformed patient T cells harbouring the R335W mutation. We observed a nearly complete absence of Ca mobilisation from its intracellular storage pools after TCR-activation by CD3 antibody. Both heterozygous parents showed a reduced, but still detectable calcium flux which reached approximately 50% of the value found in healthy control cells. Western blot analyses showed significant reduction or even absence of ITK expression for both R29H and R335W mutants. To determine the exact protein half-life, we performed S35 pulse chase analyses in HEK 293 cells, stably expressing HA-ITK or the respective mutants. Our wild type ITK constructs showed a half-life of 125 min, whereas either mutant exhibited a destabilising effect leading to a 32% reduction for the half-life of ITKR29H (85 min) or 12% reduction for the half-life of ITKR335W (110 min), respectively.
As one of the patients (R29H) harboured a homozygous defect in the PH domain of the enzyme, which is believed to be necessary for the membrane recruitment of ITK, we performed in vivo colocalisation studies in HEK 293 cells. Confocal microscopy of living cells, stably co-expressing EYFP-ITK and ECFP-ITKR29H from a polycistronic expression construct, confirmed the lack of membrane binding for the R29H mutant.
Finally, for further evaluation of our ITK deficiency, we subjected these cell lines carrying the homozygous ITKR335W to Affymetrix gene expression analyses. Among the differentially regulated genes, in unstimulated CD4+ cells, 46 matched corresponding gene expression analyses in ITK knockout mice (Blomberg KE et al., BMC Genomics 2009). Among these were, for example, CD55 and CAMK4, the calmodulin-dependent protein kinase IV (both downregulated) or Granzyme B (GZMB) as well as the nuclear factor of activated T cells (NFATC1), either upregulated.
In conclusion, mutations within ITK are widely distributed over the entire protein and include missense and nonsense mutations. ITK mutations can cause loss of membrane recruitment, protein destabilisation and probably loss of kinase activity. This situation is reminiscent to BTK mutations in Brutons disease. We recommend analysis of the ITK locus in all cases with EBV-associated lymphoproliferation.

Disclosures:
No relevant conflicts of interest to declare.