Innate-like T lymphocytes in chronic liver disease.

In addition to its metabolic activities, it is now clear that the liver hosts a number of diverse immune cell types that control tissue homeostasis. Foremost among these are innate-like T lymphocytes, including natural killer T (NKT) and mucosal-associated innate T (MAIT) cells, which are a population of specialized T cells with innate characteristics that express semi-invariant T cell receptors with non-peptide antigen specificity. As primary liver residents, innate-like T cells have been associated with immune tolerance in the liver, but also with a number of hepatic diseases. Here, we focus on the biology of NKT and MAIT cells and how they operate during the course of chronic inflammatory diseases that eventually lead to hepatocellular carcinoma.

APOBEC3B Is Co-Expressed with PKCα/NF-κB in Oral and Oropharyngeal Squamous Cell Carcinomas.

The enzymatic activity of APOBEC3B (A3B) has been implicated as a prime source of mutagenesis in head and neck squamous cell carcinoma (HNSCC). The expression of Protein Kinase C α (PKCα) and Nuclear Factor-κΒ p65 (NF-κΒ p65) has been linked to the activation of the classical and the non-canonical NF-κB signaling pathways, respectively, both of which have been shown to lead to the upregulation of A3B. Accordingly, the aim of the present study was to evaluate the expression of PKCα, NF-κΒ p65 and A3B in non-HPV related oral and oropharyngeal squamous cell carcinomas (SCC), by means of immunohistochemistry and in silico methods. PKCα was expressed in 29/36 (80%) cases of oral and oropharyngeal SCCs, with 25 (69%) cases showing a PKCα+/A3B+ phenotype and only 6/36 (17%) cases showing a PKCα-/A3B+ phenotype. Εxpression of NF-κB p65 was seen in 33/35 (94%) cases of oral and oropharyngeal SCCs, with 30/35 (86%) cases showing an NF-κB p65+/A3B+ phenotype and only 2/35 (6%) cases showing an NF-κB p65-/A3B+ phenotype. In addition, mRNA expression analysis, using the UALCAN database, revealed strong expression of all three genes. These findings indicate that the expression of A3B is associated with PKCα/NF-κB p65 expression and suggest a potential role for the PKC/NF-κB signaling pathway in the development of oral and oropharyngeal cancer.

Oncogenic and Tumor Suppressor Functions for Lymphoid Enhancer Factor 1 in E2a-/- T Acute Lymphoblastic Leukemia.

T lymphocyte acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease affecting T cells at multiple stages of their development and is characterized by frequent genomic alterations. The transcription factor LEF1 is inactivated through mutation in a subset of T-ALL cases but elevated LEF1 expression and activating mutations have also been identified in this disease. Here we show, in a murine model of T-ALL arising due to E2a inactivation, that the developmental timing of Lef1 mutation impacts its ability to function as a cooperative tumor suppressor or oncogene. T cell transformation in the presence of LEF1 allows leukemic cells to become addicted to its presence. In contrast, deletion prior to transformation both accelerates leukemogenesis and results in leukemic cells with altered expression of genes controlling receptor-signaling pathways. Our data demonstrate that the developmental timing of Lef1 mutations impact its apparent oncogenic or tumor suppressive characteristics and demonstrate the utility of mouse models for understanding the cooperation and consequence of mutational order in leukemogenesis.

The transcription factor BCL-6 controls early development of innate-like T cells.

Innate T cells, including invariant natural killer T (iNKT) and mucosal-associated innate T (MAIT) cells, are a heterogeneous T lymphocyte population with effector properties preprogrammed during their thymic differentiation. How this program is initiated is currently unclear. Here, we show that the transcription factor BCL-6 was transiently expressed in iNKT cells upon exit from positive selection and was required for their proper development beyond stage 0. Notably, development of MAIT cells was also impaired in the absence of Bcl6. BCL-6-deficient iNKT cells had reduced expression of genes that were associated with the innate T cell lineage, including Zbtb16, which encodes PLZF, and PLZF-targeted genes. BCL-6 contributed to a chromatin accessibility landscape that was permissive for the expression of development-related genes and inhibitory for genes associated with naive T cell programs. Our results revealed new functions for BCL-6 and illuminated how this transcription factor controls early iNKT cell development.

Transcription factor ID2 prevents E proteins from enforcing a naïve T lymphocyte gene program during NK cell development.

All innate lymphoid cells (ILCs) require the small helix-loop-helix transcription factor ID2, but the functions of ID2 are not well understood in these cells. We show that mature natural killer (NK) cells, the prototypic ILCs, developed in mice lacking ID2 but remained as precursor CD27+CD11b- cells that failed to differentiate into CD27-CD11b+ cytotoxic effectors. We show that ID2 limited chromatin accessibility at E protein binding sites near naïve T lymphocyte-associated genes including multiple chemokine receptors, cytokine receptors, and signaling molecules and altered the NK cell response to inflammatory cytokines. In the absence of ID2, CD27+CD11b- NK cells expressed ID3, a helix-loop-helix protein associated with naïve T cells, and they transitioned from a CD8 memory precursor-like to a naïve-like chromatin accessibility state. We demonstrate that ID3 was required for the development of ID2-deficient NK cells, indicating that completely unfettered E protein function is incompatible with NK cell development. These data solidify the roles of ID2 and ID3 as mediators of effector and naïve gene programs, respectively, and revealed a critical role for ID2 in promoting a chromatin state and transcriptional program in CD27+CD11b- NK cells that supports cytotoxic effector differentiation and cytokine responses.

Transcriptional and epigenetic regulation of innate-like T lymphocyte development.

Invariant Natural Killer T (iNKT) cells are a heterogeneous innate T cell population that recognizes lipid antigens. Despite the monospecific nature of their T cell receptor, iNKT cells differentiate into stable sublineages during thymic development, before foreign antigen encounter. How iNKT cell subsets acquire and maintain their functional programs is a central question in innate lymphocyte biology. Global transcriptional and epigenetic profiling of iNKT subsets has provided insights into the internal wiring of these subsets that defines their identity. Comparison of the iNKT transcriptional programs with those of other adaptive and innate lymphocyte lineages revealed common core regulatory circuits that may dictate effector functions. In this review, we summarize recent advances on the molecular mechanisms involved in iNKT cell development.

Applying the TOR(C)QUE in iNKT cells: A new twist in an old tale.

The mammalian Target of Rapamycin (mTOR) protein controls the machinery necessary for T-cell activation, differentiation, and memory formation, as a component of mTOR complex 1 (mTORC1) and mTORC2, which function both downstream and upstream of AKT. Invariant natural killer T (iNKT) cells are a unique T-cell subset that exist in a primed state, capable of rapid activation, and produce large quantities of cytokines. iNKT-cell effector differentiation is dependent on the mTORC1 complex; however, the requirements for mTORC2 in iNKT cells have been controversial. In this issue, Sklarz et al. [Eur. J. Immunol. 2017. 47: 516-526] provide a careful analysis of the requirements for the mTORC2 component Rictor in iNKT cells, providing a new twist in this unfolding tale. The authors demonstrate that Rictor is required for iNKT-cell proliferation and survival during the key stage of intrathymic expansion and that Rictor supports the development of NKT17 cells, an effector subset which depends on the transcription factor RORγt and produces interleukin (IL)-17, in both the thymus and the lung. IL-4-producing NKT2 cells develop in the absence of Rictor but the cytotoxic potential of iNKT cells is Rictor-dependent.

The transcription factor lymphoid enhancer factor 1 controls invariant natural killer T cell expansion and Th2-type effector differentiation.

Invariant natural killer T cells (iNKT cells) are innate-like T cells that rapidly produce cytokines that impact antimicrobial immune responses, asthma, and autoimmunity. These cells acquire multiple effector fates during their thymic development that parallel those of CD4(+) T helper cells. The number of Th2-type effector iNKT cells is variable in different strains of mice, and their number impacts CD8 T, dendritic, and B cell function. Here we demonstrate a unique function for the transcription factor lymphoid enhancer factor 1 (LEF1) in the postselection expansion of iNKT cells through a direct induction of the CD127 component of the receptor for interleukin-7 (IL-7) and the transcription factor c-myc. LEF1 also directly augments expression of the effector fate-specifying transcription factor GATA3, thus promoting the development of Th2-like effector iNKT cells that produce IL-4, including those that also produce interferon-γ. Our data reveal LEF1 as a central regulator of iNKT cell number and Th2-type effector differentiation.

ID'ing innate and innate-like lymphoid cells.

The immune system can be divided into innate and adaptive components that differ in their rate and mode of cellular activation, with innate immune cells being the first responders to invading pathogens. Recent advances in the identification and characterization of innate lymphoid cells have revealed reiterative developmental programs that result in cells with effector fates that parallel those of adaptive lymphoid cells and are tailored to effectively eliminate a broad spectrum of pathogenic challenges. However, activation of these cells can also be associated with pathologies such as autoimmune disease. One major distinction between innate and adaptive immune system cells is the constitutive expression of ID proteins in the former and inducible expression in the latter. ID proteins function as antagonists of the E protein transcription factors that play critical roles in lymphoid specification as well as B- and T-lymphocyte development. In this review, we examine the transcriptional mechanisms controlling the development of innate lymphocytes, including natural killer cells and the recently identified innate lymphoid cells (ILC1, ILC2, and ILC3), and innate-like lymphocytes, including natural killer T cells, with an emphasis on the known requirements for the ID proteins.

Essential functions for ID proteins at multiple checkpoints in invariant NKT cell development.

Invariant NKT (iNKT) cells display characteristics of both adaptive and innate lymphoid cells (ILCs). Like other ILCs, iNKT cells constitutively express ID proteins, which antagonize the E protein transcription factors that are essential for adaptive lymphocyte development. However, unlike ILCs, ID2 is not essential for thymic iNKT cell development. In this study, we demonstrated that ID2 and ID3 redundantly promoted iNKT cell lineage specification involving the induction of the signature transcription factor PLZF and that ID3 was critical for development of TBET-dependent NKT1 cells. In contrast, both ID2 and ID3 limited iNKT cell numbers by enforcing the postselection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development.

SAP protein-dependent natural killer T-like cells regulate the development of CD8(+) T cells with innate lymphocyte characteristics.

CD8(+) T cells are selected via low-affinity interaction with MHC class I molecules on thymic epithelial cells (TECs). However, compromised T cell receptor signaling was proposed to force CD8(+) T cell selection on hematopoietic cells through a SLAM-associated protein (SAP)-dependent mechanism similar to NKT cells. The outcome is an unconventional CD8(+) T cell with phenotypic and functional characteristics of innate lymphocytes. Here we showed that Id3(-/-) CD8(+) T cells had an innate-like phenotype and required SAP for their development. However, like conventional CD8(+) T cells, Id3(-/-) CD8(+) thymocytes were selected on TECs. The requirement for SAP and the innate-like phenotype was not intrinsic to Id3(-/-) CD8(+) thymocytes. Rather, an expanded population of NKT-like cells induced the innate phenotype on CD8(+) T cells through production of interleukin-4. Our findings reveal that accumulation of NKT-like cells promotes conventional CD8(+) thymocytes to acquire innate lymphocyte characteristics.

Inhibitor of DNA binding 3 limits development of murine slam-associated adaptor protein-dependent "innate" gammadelta T cells.

BACKGROUND: Id3 is a dominant antagonist of E protein transcription factor activity that is induced by signals emanating from the alphabeta and gammadelta T cell receptor (TCR). Mice lacking Id3 were previously shown to have subtle defects in positive and negative selection of TCRalphabeta+ T lymphocytes. More recently, Id3(-/-) mice on a C57BL/6 background were shown to have a dramatic expansion of gammadelta T cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that mice lacking Id3 have reduced thymocyte numbers but increased production of gammadelta T cells that express a Vgamma1.1+Vdelta6.3+ receptor with restricted junctional diversity. These Vgamma1.1+Vdelta6.3+ T cells have multiple characteristics associated with "innate" lymphocytes such as natural killer T (NKT) cells including an activated phenotype, expression of the transcription factor PLZF, and rapid production of IFNg and interleukin-4. Moreover, like other "innate" lymphocyte populations, development of Id3(-/-) Vgamma1.1+Vdelta6.3+ T cells requires the signaling adapter protein SAP. CONCLUSIONS: Our data provide novel insight into the requirements for development of Vgamma1.1+Vdelta6.3+ T cells and indicate a role for Id3 in repressing the response of "innate" gammadelta T cells to SAP-mediated expansion or survival.

The RAS-dependent ERF control of cell proliferation and differentiation is mediated by c-Myc repression.

The ERF transcriptional repressor is a downstream effector of the RAS/ERK pathway that interacts with and is directly phosphorylated by ERKs in vivo and in vitro. This phosphorylation results in its cytoplasmic export and inactivation, although lack of ERK activity allows its immediate nuclear accumulation and repressor function. Nuclear ERFs arrest cell cycle progression in G(1) and can suppress ras-dependent tumorigenicity. Here we provide evidence that ERF function is mediated by its ability to repress transcription of c-Myc. Promoter reporter assays indicate a DNA binding-dependent and repressor domain-dependent Myc transcriptional repression. Chromatin immunoprecipitations in primary cells suggest that ERF specifically binds on the c-Myc promoter in an E2F4/5-dependent manner and only under conditions that the physiological c-Myc transcription is stopped. Cellular systems overexpressing nuclear ERF exhibit reduced c-Myc mRNA and tumorigenic potential. Elimination of Erf in animal models results in increased c-Myc expression, whereas Erf(-)(/)(-) primary fibroblasts fail to down-regulate Myc in response to growth factor withdrawal. Finally, elimination of c-Myc in primary mouse embryo fibroblasts negates the ability of nuclear ERF to suppress proliferation. Thus Erf provides a direct link between the RAS/ERK signaling and the transcriptional regulation of c-Myc and suggests that RAS/ERK attenuation actively regulates cell fate.

Transcriptional repressor erf determines extraembryonic ectoderm differentiation.

Extraembryonic ectoderm differentiation and chorioallantoic attachment are fibroblast growth factor (FGF)- and transforming growth factor beta-regulated processes that are the first steps in the development of the placenta labyrinth and the establishment of the fetal-maternal circulation in the developing embryo. Only a small number of genes have been demonstrated to be important in trophoblast stem cell differentiation. Erf is a ubiquitously expressed Erk-regulated, ets domain transcriptional repressor expressed throughout embryonic development and adulthood. However, in the developing placenta, after 7.5 days postcoitum (dpc) its expression is restricted to the extraembryonic ectoderm, and its expression is restricted after 9.5 dpc in a subpopulation of labyrinth cells. Homozygous deletion of Erf in mice leads to a block of chorionic cell differentiation before chorioallantoic attachment, resulting in a persisting chorion layer, a persisting ectoplacental cone cavity, failure of chorioallantoic attachment, and absence of labyrinth. These defects result in embryo death by 10.5 dpc. Trophoblast stem cell lines derived from Erf(dl1/dl1) knockout blastocysts exhibit delayed differentiation and decreased expression of spongiotrophoblast markers, consistent with the persisting chorion layer, the expanded giant cell layer, and the diminished spongiotrophoblast layer observed in vivo. Our data suggest that attenuation of FGF/Erk signaling and consecutive Erf nuclear localization and function is required for extraembryonic ectoderm differentiation, ectoplacental cone cavity closure, and chorioallantoic attachment.

The transcriptional ETS2 repressor factor associates with active and inactive Erks through distinct FXF motifs.

The transcriptional ETS2 repressor factor (ERF) is phosphorylated by Erks both in vivo and in vitro. This phosphorylation determines the subcellular localization and biological function of ERF. Here, we show that active and inactive Erk2 proteins bind ERF with high affinity through a hydrophobic pocket formed by the alphaF and alphaG helices and the activation loop of Erk2. We have identified two FXF motifs on ERF that mediate the specific interaction with Erks. One of these motifs is utilized only by active Erks, whereas the other mediates the association with inactive Erks but also contributes to interaction with active Erks. Mutation of the phenylalanines of these motifs to alanines resulted in decreased association and phosphorylation of ERF by Erks both in cells and in vitro. ERF proteins carrying these mutations exhibited increased nuclear accumulation and increased inhibition of cellular proliferation. Expression of ERF regions harboring these motifs could inhibit Erk activity in cells. Our data suggest that, in the proper context, FXF motifs can mediate a strong and specific interaction not only with active but also inactive Erks and that these interactions determine protein function in vivo.