Themis (Thymocyte expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis is expressed in high amounts in thymocytes between the pre-T cell receptor (TCR) and positive selection checkpoints, and in low amounts in mature T cells. Themis-deficient thymocytes exhibit defective positive selection, which results in reduced numbers of mature thymocytes. Negative selection is also impaired in Themis-deficient mice. A higher percentage of Themis-deficient T cells exhibit CD4+CD25+Foxp3+ regulatory and CD62LloCD44hi memory phenotypes than in wild-type mice. Supporting a role for Themis in TCR signaling, this protein is phosphorylated quickly after TCR stimulation, and is needed for optimal TCR-driven Ca2+ mobilization and Erk activation.

The Tec family kinase Itk regulates the development of conventional and innate CD8+ T cells. However, little is known about the role of Itk in the development of CD4+ T cell lineages, although the role of Itk in the T cell activation and function is well defined. We show in this study that Itk null mice have increased percentage of CD62LlowCD44high memory phenotype CD4+ T cells compared with wild-type mice. These cells arise directly in the thymus, express high levels of transcripts for the T-bet and IFN-γ and are able to produce IFN-γ directly ex vivo in response to stimulation. Itk deficiency greatly decreases the number of CD4+ T cells with CD62LhighCD44low naive phenotype, but has no effect on the number of memory phenotype CD4+ T cells, indicating that the development of memory phenotype CD4+ T cells is Itk-independent. We further show that the development of the naive phenotype CD4+ T cells is dependent on active Itk signals and can be rescued by expression of Itk specifically in T cells. Our data also show that Itk is required for functional TCR signaling in these cells, but not for the innate function in response to IL-12/IL-18 or Listeria monocytogenes stimulation. These results indicate that CD62LhighCD44low “naive” CD4+ and CD62LlowCD44high “innate memory phenotype” CD4+ T cells may be independent populations that differ in their requirement for Itk signals for development. Our data also suggest that CD4+CD62LlowCD44high memory phenotype T cells have innate immune function.

T cells with a memory-like phenotype and possessing innate immune function have been previously identified as CD8(+)CD44(hi) cells. These cells rapidly secrete IFN-gamma upon stimulation with IL-12/IL-18 and are involved in innate responses to infection with Listeria monocytogenes. The signals regulating these cells are unclear. The Tec kinase Itk regulates T cell activation and we report here that a majority of the CD8(+) T cells in Itk null mice have a phenotype of CD44(hi) similar to memory-like innate T cells. These cells are observed in mice carrying an Itk mutant lacking the kinase domain, indicating that active Tec kinase signaling suppresses their presence. These cells carry preformed message for and are able to rapidly produce IFN-gamma upon stimulation in vitro with IL-12/IL-18, and endow Itk null mice the ability to effectively respond to infection with L. monocytogenes or exposure to lipopolysaccharides by secretion of IFN-gamma. Transfer of these cells rescues the ability of IFN-gamma null mice to reduce bacterial burden following L. monocytogenes infection, indicating that these cells are functional CD8(+)CD44(hi) T cells previously detected in vivo. These results indicate that active signals from Tec kinases regulate the development of memory-like CD8(+) T cells with innate function.

IntroductionThe tyrosine kinase Itk, a member of the Tec family of nonreceptor tyrosine kinases, is expressed in T cells and regulates signaling via the T-cell receptor. 1 On T-cell receptor activation, Itk phosphorylates and activates phospholipase-␥1, leading to calcium influx, as well as activation of nuclear factor-B, nuclear factor of activated T cell, and Ras-dependent signaling pathways. Signals regulated by Itk control the development of ␣␤ T cells such that positive and negative selection is affected in the absence of Itk. In addition, Itk also controls the development of populations of T cells that have a naive phenotype (CD62L hi /CD44 lo ), such that, in its absence, nonconventional CD4 ϩ and CD8 ϩ T cells carrying a memory phenotype (CD62L lo /CD44 hi ) and exhibiting innate function predominate. [2][3][4][5] Itk signals also regulate the development of Th2 cells such that, in its absence, T cells from Itk null mice have defects in the production of Th2 cytokines, and these mice have defects in generating Th2 response in several infection and allergic asthma models. [6][7][8][9] Despite this defect in the generation of effective Th2 responses and secretion of Th2 cytokines, Itk null mice paradoxically exhibit increased class switch in B cells to IgE and elevated levels of serum IgE. 10,11 What is not clear is the source of cytokines that could drive the increase in class switch to IgE.T cells are divided into ␣␤ T cells and ␥␦ T cells according to their TCR expression. Both ␣␤ and ␥␦ T cells arise from the most immature CD4 Ϫ CD8 Ϫ double-negative (DN) thymocytes in the thymus. DN thymocytes are divided into 4 developmental stages according to the surface expression of CD25 and CD44, from most immature DN1 to more mature DN4 cells. ␥␦ T cells separate from ␣␤ T cells at DN stages, although the exact time point and the mechanisms involved in this process are still elusive. 12,13 Several studies have shown that the strength of the TCR signal is important for T-cell lineage commitment. Stronger TCR signals favor the development of ␥␦ T cells, whereas the weaker signals favor the development of ␣␤ T cells. 14-16 Studies on ␥␦ TCR transgenic mice have demonstrated that negative selection occurs during the development of ␥␦ T cells in adult thymus, but whether positive selection is necessary is still controversial.␥␦ T cells can produce Th1, Th2, and Th17 cytokines thus having multiple functions in the modulating immune responses, such as host defense and tumor immunity. [17][18][19][20][21][22][23] Both murine and human ␥␦ T cells have been suggested to provide help to B cells, which is correlated with their production of the Th2 cytokine interleukin-4 (IL-4). [24][25][26] More interestingly, several studies showed that only the CD4 ϩ ␥␦ T cells are able to produce 21 We show here that mice lacking Itk have altered ␥␦ T-cell development such that they have more of these cells. We also show that the CD4 ϩ population of ␥␦ T cells is expanded in the absence of Itk and that this population can induce B cell...