Catherine C. Yin

NOD2 pathway activation by MDP or Mycobacterium tuberculosis infection involves the stable polyubiquitination of Rip2

The Rip2 kinase contains a caspase recruitment domain and has been implicated in the activation of the transcriptional factor NF-κB downstream of Toll-like receptors, Nod-like receptors, and the T cell receptor. Although Rip2 has been linked to Nod signaling, how Nod-Rip2 proteins mediate NF-κB activation has remained unclear. We find Rip2 required for Nod2-mediated NF-κB activation and to a lesser extent mitogen-activated protein kinase activation. We demonstrate that Rip2 and IκB kinase-γ become stably polyubiquitinated upon treatment of cells with the NOD2 ligand, muramyl dipeptide. We also demonstrate a requirement for the E2-conjugating enzyme Ubc13, the E3 ubiquitin ligase Traf6, and the ubiquitin-activated kinase Tak1 in Nod2-mediated NF-κB activation. Rip2 polyubiquitination is also stimulated when macrophages are infected with live Mycobacterium tuberculosis but not when infected with heat-killed bacteria. Consistent with our data linking Rip2 to NOD and not Toll-like receptor signaling, M. tuberculosis-induced Rip2 polyubiquitination appears MyD88-independent. Collectively, these data reveal that the NOD2 pathway is ubiquitin-regulated and that Rip2 employs a ubiquitin-dependent mechanism to achieve NF-κB activation.

Asymmetric Proteasome Segregation as a Mechanism for Unequal Partitioning of the Transcription Factor T-bet during T Lymphocyte Division

Polarized segregation of proteins in T cells is thought to play a role in diverse cellular functions including signal transduction, migration, and directed secretion of cytokines. Persistence of this polarization can result in asymmetric segregation of fate-determining proteins during cell division, which may enable a T cell to generate diverse progeny. Here, we provide evidence that a lineage-determining transcription factor, T-bet, underwent asymmetric organization in activated T cells preparing to divide and that it was unequally partitioned into the two daughter cells. This unequal acquisition of T-bet appeared to result from its asymmetric destruction during mitosis by virtue of concomitant asymmetric segregation of the proteasome. These results suggest a mechanism by which a cell may unequally localize cellular activities during division, thereby imparting disparity in the abundance of cell fate regulators in the daughter cells.

Tec kinase Itk in γδT cells is pivotal for controlling IgE production in vivo

In conventional αβ T cells, the Tec family tyrosine kinase Itk is required for signaling downstream of the T cell receptor (TCR). Itk also regulates αβ T cell development, lineage commitment, and effector function. A well established feature of Itk−/− mice is their inability to generate T helper type 2 (Th2) responses that produce IL-4, IL-5, and IL-13; yet these mice have spontaneously elevated levels of serum IgE and increased numbers of germinal center B cells. Here we show that the source of this phenotype is γδ T cells, as normal IgE levels are observed in Itk−/−Tcrd−/− mice. When stimulated through the γδ TCR, Itk−/− γδ T cells produce high levels of Th2 cytokines, but diminished IFNγ. In addition, activated Itk−/− γδ T cells up-regulate costimulatory molecules important for B cell help, suggesting that they may directly promote B cell activation and Ig class switching. Furthermore, we find that γδ T cells numbers are increased in Itk−/− mice, most notably the Vγ1.1+Vδ6.3+ subset that represents the dominant population of γδ NKT cells. Itk−/− γδ NKT cells also have increased expression of PLZF, a transcription factor required for αβ NKT cells, indicating a common molecular program between αβ and γδ NKT cell lineages. Together, these data indicate that Itk signaling regulates γδ T cell lineage development and effector function and is required to control IgE production in vivo.

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

Summary:  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, Ca2+ 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 γδ 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.

The Tec Kinase ITK Regulates Thymic Expansion, Emigration, and Maturation of γδ NKT Cells

The Tec family tyrosine kinase, Itk, regulates signaling downstream of the TCR. The absence of Itk in CD4+ T cells results in impaired Th2 responses along with defects in maturation, cytokine production, and survival of iNKT cells. Paradoxically, Itk−/− mice have spontaneously elevated serum IgE levels, resulting from an expansion of the Vγ1.1+Vδ6.3+ subset of γδ T cells, known as γδ NKT cells. Comparisons between γδ NKT cells and αβ iNKT cells showed convergence in the pattern of cell surface marker expression, cytokine profiles, and gene expression, suggesting that these two subsets of NKT cells undergo similar differentiation programs. Hepatic γδ NKT cells have an invariant TCR and are derived predominantly from fetal progenitors that expand in the thymus during the first weeks of life. The adult thymus contains these invariant γδ NKT cells plus a heterogeneous population of Vγ1.1+Vδ6.3+ T cells with diverse CDR3 sequences. This latter population, normally excluded from the liver, escapes the thymus and homes to the liver when Itk is absent. In addition, Itk−/− γδ NKT cells persistently express high levels of Zbtb16 (PLZF) and Il4, genes that are normally downregulated in the most mature subsets of NKT cells. These data indicate that Itk signaling is required to prevent the expansion of γδ NKT cells in the adult thymus, to block their emigration, and to promote terminal NKT cell maturation.

Innate PLZF+CD4+ αβ T cells develop and expand in the absence of Itk.

T cell development in the thymus produces multiple lineages of cells, including innate T cells. Studies in mice harboring alterations in TCR signaling proteins or transcriptional regulators have revealed an expanded population of CD4+ innate T cells in the thymus that produce IL-4 and express the transcription factor promyelocytic leukemia zinc finger (PLZF). In these mice, IL-4 produced by the CD4+PLZF+ T cell population leads to the conversion of conventional CD8+ thymocytes into innate CD8+ T cells resembling memory T cells expressing eomesodermin. The expression of PLZF, the signature invariant NKT cell transcription factor, in these innate CD4+ T cells suggests that they might be a subset of αβ or γδ TCR+ NKT cells or mucosal-associated invariant T (MAIT) cells. To address these possibilities, we characterized the CD4+PLZF+ innate T cells in itk−/− mice. We show that itk−/− innate PLZF+CD4+ T cells are not CD1d-dependent NKT cells, MR1-dependent MAIT cells, or γδ T cells. Furthermore, although the itk−/− innate PLZF+CD4+ T cells express αβ TCRs, neither β2-microglobulin–dependent MHC class I nor any MHC class II molecules are required for their development. In contrast to invariant NKT cells and MAIT cells, this population has a highly diverse TCRα-chain repertoire. Analysis of peripheral tissues indicates that itk−/− innate PLZF+CD4+ T cells preferentially home to spleen and mesenteric lymph nodes owing to increased expression of gut-homing receptors, and that their expansion is regulated by commensal gut flora. These data support the conclusion that itk−/− innate PLZF+CD4+ T cells are a novel subset of innate T cells.

Development of Innate CD4+ and CD8+ T Cells in Itk-Deficient Mice Is Regulated by Distinct Pathways

T cell development in the thymus produces multiple lineages of cells, including innate T cells such as γδ TCR+ cells, invariant NKT cells, mucosal-associated invariant T cells, and H2–M3-specific cells. Although innate cells are generally a minor subset of thymocytes, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional CD8+ T cells develop as innate cells with characteristics of memory T cells. Thus, in Itk-deficient mice, mature CD4−CD8+ (CD8 single-positive [SP]) thymocytes express high levels of the transcription factor eomesodermin (Eomes) and are dependent on IL-4 being produced in the thymic environment by a poorly characterized subset of CD4+ thymocytes expressing the transcriptional regulator promyelocytic leukemia zinc finger. In this study, we show that a sizeable proportion of mature CD4+CD8− (CD4SP) thymocytes in itk−/− mice also develop as innate Eomes-expressing T cells. These cells are dependent on MHC class II and IL-4 signaling for their development, indicating that they are conventional CD4+ T cells that have been converted to an innate phenotype. Surprisingly, neither CD4SP nor CD8SP innate Eomes+ thymocytes in itk−/− or SLP-76(Y145F) mice are dependent on γδ T cells for their development. Instead, we find that the predominant population of Eomes+ innate itk−/− CD4SP thymocytes is largely absent in mice lacking CD1d-specific invariant NKT cells, with no effect on innate itk−/− CD8SP thymocytes. In contrast, both subsets of innate Eomes+itk−/− T cells require the presence of a novel promyelocytic leukemia zinc finger–expressing, SLAM family receptor adapter protein–dependent thymocyte population that is essential for the conversion of conventional CD4+ and CD8+ T cells into innate T cells with a memory phenotype.