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Abstract
The Tec kinases ITK (interleukin-2-inducible T-cell kinase) and RLK (resting lymphocyte kinase) are critical components of the proximal TCR/CD3 signal transduction machinery, and data in mice suggest that ITK negatively modulates regulatory T cell (TREG) differentiation. However, whether Tec kinases modulate TREG development and/or function in human T cells remains unknown. Using a novel self-delivery siRNA platform (sdRNA), we found that ITK knockdown in human primary naïve peripheral blood CD4 T cells increased Foxp3+ expression under both TREG and T helper priming conditions. TREG differentiated under ITK knockdown conditions exhibited enhanced expression of the co-inhibitory receptor PD-1 and were suppressive in a T cell proliferation assay. ITK knockdown decreased IL-17A production in T cells primed under Th17 conditions and promoted Th1 differentiation. Lastly, a dual ITK/RLK Tec kinase inhibitor did not induce Foxp3 in CD4 T cells, but conversely abrogated Foxp3 expression induced by ITK knockdown. Our data suggest that targeting ITK in human T cells may be an effective approach to boost TREG in the context of autoimmune diseases, but concomitant inhibition of other Tec family kinases may negate this effect.
Highlights
Interleukin-2-inducible T-cell kinase (ITK) is a member of the TEC kinase family of non-receptor tyrosine kinases and mediates T cell signaling downstream of TCR activation [1]
To assess the effect of ITK on human TREG differentiation, we used a recently developed selfdelivered siRNA technology which circumvents the low efficiency of conventional gene knockdown methods in resting primary human T cells
Knockdown of ITK resulted in a 3-8-fold increase in the frequency of FoxP3+ T cells and a 2-5-fold increase in FoxP3 MFI under TREG, Th17 and Th1 polarizing conditions relative to non-targeting sdRNA control (NTC) (Figure 1B, C)
Summary
Interleukin-2-inducible T-cell kinase (ITK) is a member of the TEC kinase family of non-receptor tyrosine kinases and mediates T cell signaling downstream of TCR activation [1]. ITK is an important switch for Th1 and Th2 mediated immunity, and murine ITK deficiency results in reduced differentiation and effector cytokine production from Th1, Th2, and Th17 polarized CD4+ T cells while bolstering TREG development [6,7,8,9]. While ITK is required for IL-17a production in human T cell lines [14] and it regulates Th17 and TREG differentiation in mice [6], its role in human TREG differentiation is not defined. We investigated the roles of ITK in human Foxp3+ TREG differentiation and function using self-delivered siRNA (sdRNA) technology optimized to modulate ITK expression in resting primary human T cells. Th17 differentiation in primary human T cells and highlights the need for more specific pharmacologic targeting of TEC kinases in inflammatory and autoimmune disease
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