Takuya Ohtani

Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A

Precise control of the timing and magnitude of Notch signaling is essential for the normal development of many tissues, but the feedback loops that regulate Notch are poorly understood. Developing T cells provide an excellent context to address this issue. Notch1 signals initiate T-cell development and increase in intensity during maturation of early T-cell progenitors (ETP) to the DN3 stage. As DN3 cells undergo beta-selection, during which cells expressing functionally rearranged TCRbeta proliferate and differentiate into CD4(+)CD8(+) progeny, Notch1 signaling is abruptly down-regulated. In this report, we investigate the mechanisms that control Notch1 expression during thymopoiesis. We show that Notch1 and E2A directly regulate Notch1 transcription in pre-beta-selected thymocytes. Following successful beta-selection, pre-TCR signaling rapidly inhibits Notch1 transcription via signals that up-regulate Id3, an E2A inhibitor. Consistent with a regulatory role for Id3 in Notch1 down-regulation, post-beta-selected Id3-deficient thymocytes maintain Notch1 transcription, whereas enforced Id3 expression decreases Notch1 expression and abrogates Notch1-dependent T-cell survival. These data provide new insights into Notch1 regulation in T-cell progenitors and reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development. Our findings also suggest new strategies for inhibiting Notch1 signaling in pathologic conditions.

Notch regulation of early thymocyte development

Notch signaling plays multiple roles in T cell development. Following thymic entry, Notch signals are required to specify the T cell fate from a multipotent hematopoietic progenitor. At subsequent steps in early T cell development, Notch provides important differentiation, survival, proliferation and metabolic signals. This review focuses on the multiple functions of Notch in early T cell development, from T cell specification in the thymus through beta selection.

Dynamic Interactions between TIP60 and p300 Regulate FOXP3 Function through a Structural Switch Defined by a Single Lysine on TIP60

The human FOXP3 molecule is an oligomeric transcriptional factor able to mediate activities that characterize T regulatory cells, a class of lymphocytes central to the regulation of immune responses. The activity of FOXP3 is regulated at the posttranslational level, in part by two histone acetyltransferases (HATs): TIP60 and p300. TIP60 and p300 work cooperatively to regulate FOXP3 activity. Initially, p300 and TIP60 interactions lead to the activation of TIP60 and facilitate acetylation of K327 of TIP60, which functions as a molecular switch to allow TIP60 to change binding partners. Subsequently, p300 is released from this complex, and TIP60 interacts with and acetylates FOXP3. Maximal induction of FOXP3 activities is observed when both p300 and TIP60 are able to undergo cooperative interactions. Conditional knockout of TIP60 in Treg cells significantly decreases the Treg population in the peripheral immune organs, leading to a scurfy-like fatal autoimmune disease.

Pim-2 Kinase Influences Regulatory T Cell Function and Stability by Mediating Foxp3 Protein N-terminal Phosphorylation

Background: Foxp3 activity is regulated by various posttranslational modifications. Results: Pim-2 kinase phosphorylates the Foxp3 N-terminal domain and influences the Foxp3 level in vivo. Conclusion: Pim-2 is a negative regulator of Foxp3 activity. Significance: Phosphorylation of Foxp3 by Pim-2 kinase negatively regulates Treg cell suppressive function and stability. Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4+CD25+Foxp3+ regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses.

Trib2 suppresses tumor initiation in Notch-driven T-ALL

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes.

Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns

The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56hi versus CD56dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings.

Epigenomic-Guided Mass Cytometry Profiling Reveals Disease-Specific Features of Exhausted CD8 T Cells

SUMMARY Exhausted CD8 T (Tex) cells are immunotherapy targets in chronic infection and cancer, but a comprehensive assessment of Tex cell diversity in human disease is lacking. Here, we developed a transcriptomic‐ and epigenetic‐guided mass cytometry approach to define core exhaustion‐specific genes and disease‐induced changes in Tex cells in HIV and human cancer. Single‐cell proteomic profiling identified 9 distinct Tex cell clusters using phenotypic, functional, transcription factor, and inhibitory receptor co‐expression patterns. An exhaustion severity metric was developed and integrated with high‐dimensional phenotypes to define Tex cell clusters that were present in healthy subjects, common across chronic infection and cancer or enriched in either disease, linked to disease severity, and changed with HIV therapy. Combinatorial patterns of immunotherapy targets on different Tex cell clusters were also defined. This approach and associated datasets present a resource for investigating human Tex cell biology, with implications for immune monitoring and immunomodulation in chronic infections, autoimmunity, and cancer. Graphical Abstract Figure. No Caption available. HighlightsUnbiased identification of unique Tex genes using transcriptomics and epigenomicsHigh‐dimensional CyTOF profiling of human Tex gene products reveals heterogeneityIdentification of key disease‐relevant Tex cell populations in HIV and lung cancerDevelopment of exhaustion metrics applicable to human immune monitoring &NA; Exhausted T (Tex) cells have poor function in chronic infections and cancer but can be therapeutically re‐invigorated. Bengsch et al. use genes modified epigenetically during exhaustion and high‐dimensional CyTOF profiling to define Tex cell heterogeneity in humans with HIV or lung cancer and link Tex cell features to disease progression and response to immunotherapy.

Monoclonal Antibodies for Cancer Therapy and Prevention: Paradigm Studies in Targeting the neu/ERBB2/HER2 Oncoprotein

Abstract The foundation for targeted therapy of cancers driven by members of the ErbB oncoprotein family was established initially by the demonstration that ectodomain binding monoclonal antibodies (mAb) could disable the protein kinase encoded by the HER2/neu oncogene. Homomeric and heteromeric erbB kinases play critical roles in the development of cancer and in the spread of early lesions. In particular, antibodies targeting the p185erbB2/neu receptor provide major clinical benefits in the treatment of breast cancer and also stomach cancer. As suggested by our study with oncogenic neu transgenic mice, anti-p185erbB2/neu antibodies are also effective in preventing the tissue hyperplasia that precedes tumorigenesis, tumor growth and the dissemination of ErbB2/neu kinase-positive cells into other tissues. As a therapeutic principle, “reversion of phenotype” for established tumors and “prevention” of tumorigenesis and spread can explain the basis for the benefits invoked by therapeutic and adjuvant therapies for breast cancer patients after cancers are surgically removed. These emerging principles being enlightened by ongoing studies of monoclonal antibody therapy will continue to provide guidance for the development of new targeted therapies for resistant tumors that arise after treatment.