Inhibition of TYK2 and JAK1 ameliorates imiquimod-induced psoriasis-like dermatitis by inhibiting IL-22 and the IL-23/IL-17 axis.

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making JAK inhibition an appealing strategy for the treatment of psoriasis. In this study, we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and tyrosine kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose dependently (1 nM-10 µM) inhibited JAK1- and/or TYK2-dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild-type mice with SAR-20347 significantly reduced IL-12-induced IFN-γ production and IL-22-dependent serum amyloid A to similar extents, indicating that, in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes and proinflammatory cytokine levels compared with both TYK2 mutant mice and wild-type controls. Taken together, these data indicate that targeting both JAK1- and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis.

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.

Innate PLZF+CD4+ αß 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 αß 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 αß TCRs, neither ß2-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.

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 αß 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.

Intrathymic programming of effector fates in three molecularly distinct γδ T cell subtypes.

Innate γδ T cells function in the early phase of immune responses. Although innate γδ T cells have often been studied as one homogenous population, they can be functionally classified into effector subsets on the basis of the production of signature cytokines, analogous to adaptive helper T cell subsets. However, unlike the function of adaptive T cells, γδ effector T cell function correlates with genomically encoded T cell antigen receptor (TCR) chains, which suggests that clonal TCR selection is not the main determinant of the differentiation of γδ effector cells. A high-resolution transcriptome analysis of all emergent γδ thymocyte subsets segregated on the basis of use of the TCR γ-chain or δ-chain indicated the existence of three separate subtypes of γδ effector cells in the thymus. The immature γδ subsets were distinguished by unique transcription-factor modules that program effector function.

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 gammadeltaT cells is pivotal for controlling IgE production in vivo.

In conventional alphabeta T cells, the Tec family tyrosine kinase Itk is required for signaling downstream of the T cell receptor (TCR). Itk also regulates alphabeta 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 gammadelta T cells, as normal IgE levels are observed in Itk(-/-)Tcrd(-/-) mice. When stimulated through the gammadelta TCR, Itk(-/-) gammadelta T cells produce high levels of Th2 cytokines, but diminished IFNgamma. In addition, activated Itk(-/-) gammadelta 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 gammadelta T cells numbers are increased in Itk(-/-) mice, most notably the Vgamma1.1(+)Vdelta6.3(+) subset that represents the dominant population of gammadelta NKT cells. Itk(-/-) gammadelta NKT cells also have increased expression of PLZF, a transcription factor required for alphabeta NKT cells, indicating a common molecular program between alphabeta and gammadelta NKT cell lineages. Together, these data indicate that Itk signaling regulates gammadelta 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.

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.

The Tec family tyrosine kinases Itk and Rlk regulate the development of conventional CD8+ T cells.

The Tec family tyrosine kinases, Itk and Rlk, are expressed in thymocytes and peripheral T cells and regulate thresholds of T cell receptor signaling. Yet little is known about the specific role of Itk- and Rlk-dependent signals in CD8(+) T cell maturation. We show here that Itk(-/-) and Rlk(-/-)Itk(-/-) mice were nearly devoid of conventional CD8(+) T cells and, instead, contained a large population of CD8(+) T cells that bear striking similarity to lineages of innate lymphocytes. Itk(-/-) and Rlk(-/-)Itk(-/-) CD8(+) thymocytes and T cells were CD44(hi), CD122(+), and NK1.1(+); were able to produce interferon-gamma directly ex vivo; and were dependent on interleukin-15. Itk(-/-) and Rlk(-/-)Itk(-/-) CD8(+) thymocytes expressed abundant transcripts for the T box transcription factor, eomesodermin, correlating with their phenotype and function. These data indicate a critical role for Itk and Rlk in conventional CD8(+) T cell development in the thymus.

Frameshift mutations induced by four isomeric nitroacridines and their des-nitro counterpart in the lacZ reversion assay in Escherichia coli.

Acridines are well-known as compounds that intercalate noncovalently between DNA base pairs and induce +/-1 frameshift mutations at sites of monotonous repeats of a single base. Reactive derivatives of acridines, including acridine mustards and nitroacridines, form covalent adducts in DNA and exhibit mutagenic properties different from the simple intercalators. We compared the frameshift mutagenicity of the cancer chemotherapy drug nitracrine (1-nitro-9-(3'-dimethylaminopropylamino)-acridine), its des-nitro counterpart 9-(3'-dimethylaminopropylamino)-acridine (DAPA), and its 2-, 3-, and 4-nitro isomers (2-, 3-, and 4-nitro-DAPA) in the lacZ reversion assay in Escherichia coli. DAPA is a simple intercalator, much like the widely studied 9-aminoacridine. It most strongly induced +/-1 frameshift mutations in runs of guanine residues and more weakly induced -1 frameshifts in a run of adenine residues. A nitro group in the 1, 3, or 4 position of DAPA reduced the yield of +/-1 frameshift mutations. DAPA weakly induced -2 frameshifts in an alternating CG sequence. In contrast, nitracrine and its 3-nitro isomer resembled the 3-nitroacridine Entozon in effectively inducing -2 frameshift mutations. The 2- and 4-nitro isomers were less effective than the 1- and 3-nitro compounds in -2 frameshift mutagenesis. The results are interpreted with respect to intercalation, steric interactions, effects of base strength on DNA binding, enzymatic processing, and a slipped mispairing model of frameshift mutagenesis.