Abstract

IL2-inducible T cell kinase (ITK) belongs to the Tec family of tyrosine kinases and is an important component of TCR-mediated signaling. ITK regulates T cell adhesion and migration, signaling and activation, Th17 polarization and Treg differentiation, which are all critical for immune responses in health and disease. Ibrutinib is a newly developed Bruton tyrosine kinase (BTK) inhibitor with outstanding clinical activity and tolerability in B-cell malignancies. Recently, it has been demonstrated that Ibrutinib as an irreversible inhibitor of both BTK and ITK that blocks downstream immune receptor activation. Given its critical role in T-cell signaling and activation, ITK is an appealing therapeutic target that can contribute to the pathogenesis of various immune disorders, which was our impetus for further studying Ibrutinib - a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility.

Herein, we report that Ibrutinib inhibits T cell adhesion, proliferation and immunological synapse formation. We found Ibrutinib treatment reduced the homotypic aggregation of primary mouse T cells mediated by PMA and CD3-antibody at 0.5µM, and completely blocked aggregation at 5µM concentration. We investigated whether Ibrutinib treatment can prevent mouse primary T cell proliferation upon activation in mixed lymphocyte reactions (MLR), and found the inhibition of T cell proliferation by Ibrutinib is dose-dependent. With the increase of Ibrutinib concentration from 0.5µM to 1µM and 5µM, the frequency of dividing cells were greatly decreased for both CD4+ and CD8+ T cells; the proliferation was completely blocked at about 1µM concentration with majority T cells were still alive. To further investigate the role of Ibrutinib in regulating TCR-induced activation, we examined the immune synapse formation in mouse CD8+ T cells using confocal microscope. We found that TCR was enriched proximal to the membrane and associated with the prominent cytoskeleton reorganization (FA) cluster in the after 30 min of TCR stimulation. In the presence of Ibrutinib (1µM), the predominant pattern was different with TCR and FA evenly distributed across the cell surface upon CD3-antibody simulation, similar as the control without any TCR stimulation. Thus, inhibition of ITK with Ibrutinib can prevents TCR signaling and activation.

Although ablation of ITK have been shown to subvert Th2 immunity, thereby potentiating Th1-based immune responses, more recent published data demonstrated ITK is a positive modulator of IL-17A production, with reduced percentages of IL17A-producing cells in ITK-deficient CD4+ T cells. In addition, ITK has been implicated in the development and suppressor function of Treg. ITK-deficient CD4+ T cells can differentiate more efficiently into Treg cells and exhibit increased FoxP3 induction in mice. To determine the effect of Ibrutinib treatment in Th17 and Treg differentiation, naïve CD4+ T cells (CD62Lhi CD44lo CD25neg CD4+) from C57BL/6 mice were cultured with irradiated splenic APC from BALB/C mice in the presence of polarizing reagents (Th17 or Treg) for 5 days, then restimulated and after which Foxp3-, IL-17- and IFNγ-producing cells were analyzed using intracellular staining. We found Ibrutinib treatment (1μM) decreased the Th17-producing cells from 23% (PBS control) to 14% under Th17 culture condition, but increased FoxP3-producing cells from 43% (PBS control) to 57% under Treg culture condition. Furthermore, mRNA expression of Th17 (IL-17 and RORgt) genes was significant decreased while Treg (FoxP3) gene was significant increased in the presence of Ibrutinib. This is consistent with recently published data demonstrating that ITK-mediated signaling regulates the balance between Th17 and Treg in vitro and in vivo. Thus, inhibition of ITK with Ibrutinib can influence Th17 and Treg cell differentiation.

In summary, our studies demonstrated that Ibrutinib treatment can: (1) inhibit primary mouse T cell aggregation and adhesion; (2) inhibit the proliferation of both CD4+ and CD8+ T cells; (3) prevent cytoskeleton reorganization, clustering and immune synapse formation mediated by TCR-activation and signaling; (4) decrease the number of Th17-producing cells and mRNA expression of Th17 (IL-17 and RORgt) genes under Th17 culture condition; (5) potentiate Treg differentiation and induces FoxP3 expression in vitro.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.