The Tec kinases Itk and Rlk regulate conventional versus innate T-cell development

Abstract

Tec family kinases are important components of antigen receptor signaling pathways in B cells, T cells, and mast cells. In T cells, three members of this family, inducible T-cell kinase (Itk), resting lymphocyte kinase (Rlk), and Tec, are expressed. In the absence of Itk and Rlk, T-cell receptor signaling is impaired, with defects in mitogen-activated protein kinase activation, Ca(2+) mobilization, and actin polymerization. During T-cell development in the thymus, no role has been found for these kinases in the CD4(+) versus CD8(+) T-cell lineage decision; however, several studies indicate that Itk and Rlk contribute to the signaling leading to positive and negative selection. In addition, we and others have recently described an important role for Itk and Rlk in the development of conventional as opposed to innate CD4(+) and CD8(+) T cells. Natural killer T and gammadelta T-cell populations are also altered in Itk- and Rlk/Itk-deficient mice. These findings strongly suggest that the strength of T-cell receptor signaling during development determines whether T cells mature into conventional versus innate lymphocyte lineages. This lineage decision is also influenced by signaling via signaling lymphocytic activation molecule (SLAM) family receptors. Here we discuss these two signaling pathways that each contribute to conventional versus innate T-cell lineage commitment.

Fig. 1. Role of Tec kinases in TCR signaling

Upon stimulation of the TCR, Tec kinases (only Itk is shown for simplicity) are activated by the Src kinase, Lck, and bind to a complex involving LAT and SLP-76. Itk then phosphorylates PLCγ to induce Ca²⁺ mobilization and MAPK activation, resulting in the activation of transcription factors, such as NFAT and AP-1.

Fig. 2. Structures of the Tec family of non-receptor protein tyrosine kinases

The five members of the Tec family of kinases are depicted, indicating their domain structure. Each member contains C-terminal kinase domain, an SH2 domain for interaction with phosphorylated tyrosines, and an SH3 domain for interaction with proline-rich regions (PRRs). Other domains include a Tec homology (TH) domain that may contain a Btk homology domain plus one or two PRRs. With the exception of Rlk, each contains an N-terminal pleckstrin homology domain for localization to the cell membrane upon activation. Rlk instead contains an N-terminal cysteine-string motif that becomes palmitoylated to localize Rlk to the cell membrane.

Fig. 3

Rlk/Itk^−/−mice have an increased proportion of γδ T cells in the thymus. Cells were prepared from thymus and spleen of wildtype (WT) and Rlk/Itk^−/−mice. (A) Cells were stained with anti-TCRγ and anti-TCRβ antibodies and analyzed by flow cytometry. Numbers in quadrants indicate the percentage of cells in each subset. Data are representative of 2 independent experiments with 2–3 mice per group. (B) Percentages and absolute numbers of TCRγ⁺ cells, based on TCRγ⁺TCRβ staining, are indicated for the thymus and spleen. Each data point represents a different animal, and the bars represent the mean. p values were determined using the Mann-Whitney test.

Fig. 4. Altered γδ T-cell subsets in the spleen and thymus of Rlk/Itk^−/−mice

Cells were prepared from thymus and spleen of wildtype (WT) and Rlk/Itk^{−/ −}mice. (A) CD4 versus CD8 expression (left panels) and CD4 versus CD44 expression (right panels) on gated TCRγ⁺TCRβ cells. (B) The percentages of CD4⁺TCRγ⁺ cells in the thymus (n=5–6 mice) and spleen (n=4–5 mice) are shown. Each data point represents a different animal, and the bars represent the mean. Data are representative of 2 independent experiments with 2–3 mice per group. Statistically significant differences are shown; p values were determined using the Mann-Whitney test.

Fig. 5

Rlk/Itk^{−/ −}γδ T cells show impaired production of cytokines following ex vivo activation. Lymph node and spleen cells from WT and Rlk/Itk^{−/ −}mice were pooled and TCRγ⁺ cells were isolated by cell sorting. 5× 10⁴ cells were stimulated with 10 μg/ml of anti-TCRγ antibody for 72 h, and supernatants were analyzed for the presence of IL-4, IL-10, IL-13, and IFNγ by cytometric bead array (CBA).

Fig. 6. Model depicting the role of TCR signaling in thymocyte lineage differentiation

(A). In the presence of Itk and Rlk, strong signaling thresholds promote the development of Tregs, NKT, and CD4⁺ T cells, while weaker signaling promotes the development of conventional CD8⁺ and γδ T cells. Few innate CD8⁺ T cells develop under these conditions.(B) In the absence of Tec kinases, the signaling threshold for development in altered. Although a reduced population of conventional αβ CD4⁺ T cells and Tregs remain, NKT and conventional αβ CD8⁺ T cells are severely diminished. Populations of innate CD8⁺ and CD4⁺ T cells develop instead, and there is an increase in the γδ T-cell population. Thus, by lowering the signaling threshold during development, innate-like lymphocytes are able to develop at the expense of conventional T cells.

Fig. 6. Model depicting the role of TCR signaling in thymocyte lineage differentiation

(A). In the presence of Itk and Rlk, strong signaling thresholds promote the development of Tregs, NKT, and CD4⁺ T cells, while weaker signaling promotes the development of conventional CD8⁺ and γδ T cells. Few innate CD8⁺ T cells develop under these conditions.(B) In the absence of Tec kinases, the signaling threshold for development in altered. Although a reduced population of conventional αβ CD4⁺ T cells and Tregs remain, NKT and conventional αβ CD8⁺ T cells are severely diminished. Populations of innate CD8⁺ and CD4⁺ T cells develop instead, and there is an increase in the γδ T-cell population. Thus, by lowering the signaling threshold during development, innate-like lymphocytes are able to develop at the expense of conventional T cells.

Fig. 7. TCR and SLAM family receptor signaling in conventional and innate T cell development

During the DN2 to DN3 stages of thymocytes development, γδ T cells are thought to differentiate from αβ T cells. In the absence of the Tec family kinases Itk and Rlk, the γδ T-cell population increases. In addition to the decision between γδ or αβ T-cell development, Itk and Rlk are also involved at the DP stage of development during the decision of an αβ T cell to become a conventional or innate T cell. Strong TCR signaling inhibits SLAM family signaling that potentially leads to the induction of the transcription factor Eomes. This inhibition leads to the development of conventional αβ CD4⁺ and CD8⁺ T cells. Interestingly, NKT cells appear to require both strong TCR signaling and SLAM family signals for their development. However, in conditions where TCR signaling is weak but SLAM family signaling is intact, innate-like CD4⁺ and CD8⁺ T cells develop. Thus, TCR signaling plays an important role in this developmental decision.