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Genetic Deficiency and Biochemical Inhibition of ITK Affect Human Th17, Treg, and Innate Lymphoid Cells

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Abstract

Purpose

Interleukin-2-inducible T cell kinase (ITK) is an important mediator of T cell receptor signaling. Loss of function mutations in ITK results in hypogammaglobulinemia and CD4+ T cell loss in humans, and the patients often present with EBV-associated B cell lymphoproliferative syndrome. Itk-deficient mice show loss of T cell naivety, impaired cytolytic activity of CD8+ T cells, and defects in CD4+ T cell lineage choice decisions. In mice, Itk mutations were shown to affect Th17-Treg lineage choice in favor of the latter. In this study, we explored whether human ITK reciprocally regulates Th17-Treg balance as its murine ortholog.

Methods

Whole Exome Sequencing was used to identify the mutation. ITK-deficient peripheral blood lymphocytes were characterized by FACSAria III-based flow cytometric assays with respect to proliferation, apoptosis, cytokine production, and innate lymphoid cell (ILC) frequency. Sorted T cells from healthy donors were exposed to ibrutinib, an irreversible ITK inhibitor, to assess ITK’s contribution to Th17 and Treg cell generation and functions.

Results

In this study, we report a child with a novel ITK mutation who showed impaired CD3/CD28 induced proliferation in T cells. ITK-mutant cells were more apoptotic irrespective of TCR activation. More importantly, T cells produced less Th17-associated cytokines IL-17A, IL-22, and GM-CSF. Conversely, Th1-associated IFN-γ production was increased. An irreversible inhibitor of ITK, ibrutinib, blocked ex vivo Th17 generation and IL-17A production, conversely augmented FOXP3 expression only at low doses in Treg cultures. Finally, we analyzed peripheral ILC populations and observed a relative decrease in ILC2 and ILC3 frequency in our ITK-deficient patient.

Conclusions

To our knowledge, this is the first report showing that both genetic and chemical inhibition of ITK result in reduced Th17 generation and function in humans. We also report, for the first time, a reduction in ILC2 and ILC3 populations in an ITK-deficient human patient.

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Acknowledgments

We are grateful to our patient and his family for allowing us to study her disease and to the clinical staff taking care of her. We thank Dr. Mohamed Oukka, for providing us with IL-23RGFP mice, and Erciyes University Medical Biology and GENKOK administrative personnel.

Financial Support

This research was partially supported, in part, by the Erciyes University BAP grant, TOA-2016-6130; The Scientific and Technological Research Council of Turkey (TUBITAK) grants, 215S725 and 315S315 to AE; and by the German Academic Exchange Service (DAAD).

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Authors and Affiliations

  1. Faculty of Medicine, Department of Medical Biology, Genome and Stem Cell Center (GENKOK), Erciyes University, 38030, Melikgazi, Kayseri, Turkey

    Ahmet Eken, Fatma Zehra Okus, Serife Erdem & Halit Canatan

  2. Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology-Oncology, Erciyes University, 38030, Melikgazi, Kayseri, Turkey

    Murat Cansever, Sefika Akyol, Alper Ozcan, Musa Karakukcu, Ekrem Unal & Turkan Patiroglu

  3. Dr von Hauner Children’s Hospital, Department of Pediatrics, University Hospital, LMU Munich, Munich, Germany

    Ido Somekh, Yoko Mizoguchi, Natalia Zietara, Sebastian Hollizeck, Meino Rohlfs & Christoph Klein

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  1. Ahmet Eken
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Contributions

CK, IS, YM, SH, NZ, and MR performed NGS and analyses, identified the mutation, and read and revised the manuscript. AE, MC, HC, and EU conceived and supervised the study and wrote and edited the manuscript. TP, MK, AO, and SA cared the patient and provided samples, provided intellectual input, and read and revised the manuscript. FZO, SE, and AE performed the experiments.

Corresponding authors

Correspondence to Ahmet Eken or Ekrem Unal.

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Conflict of Interest

The authors declare that they have no conflict of interest.

Research Involving Human Participants

Informed consent for participation in this study was obtained in accordance with local regulations, with approval from the IRB. The experiments described here were performed in Turkey and in Germany. All the experiments were conducted in accordance with local regulations, and with the approval of the IRB for Erciyes University, Turkey, and were in line with the current guidelines of the Declaration of Helsinki. The ethics permit number is 2018/388.

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Written informed consent was obtained from the patients.

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Supplemental Fig.1

Gating strategy and exclusion of the doublets for the patient, mother and healthy control PBMCs was shown for representative samples. (PNG 1104 kb)

High Resolution Image (TIF 1630 kb)

Supplemental Fig.2

Real time qPCR results for LCK and THY1 (Normalized over 18S) for lymph node biopsies of ITK patient and control (top panel). FOXP3, IL17A, RORC and ITK gene expression is normalized to LCK (bottom panel). (PNG 175 kb)

High Resolution Image (TIF 605 kb)

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Eken, A., Cansever, M., Somekh, I. et al. Genetic Deficiency and Biochemical Inhibition of ITK Affect Human Th17, Treg, and Innate Lymphoid Cells. J Clin Immunol 39, 391–400 (2019). https://doi.org/10.1007/s10875-019-00632-5

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  • DOI: https://doi.org/10.1007/s10875-019-00632-5

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