Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control

T-lymphocyte development branches off from other lymphoid developmental programs through its requirement for sustained environmental signals through the Notch pathway. In the thymus, Notch signaling induces a succession of T-lineage regulatory factors that collectively create the T-cell identity through distinct steps. This process involves both the staged activation of T-cell identity genes and the staged repression of progenitor-cell-inherited regulatory genes once their roles in self-renewal and population expansion are no longer needed. With the recent characterization of innate lymphoid cells (ILCs) that share transcriptional regulation programs extensively with T-cell subsets, T-cell identity can increasingly be seen as defined in modular terms, as the processes selecting and actuating effector function are potentially detachable from the processes generating and selecting clonally unique T-cell receptor structures. The developmental pathways of different classes of T cells and ILCs are distinguished by the numbers of prerequisites of gene rearrangement, selection, and antigen contact before the cells gain access to nearly common regulatory mechanisms for choosing effector function. Here, the major classes of transcription factors that interact with Notch signals during T-lineage specification are discussed in terms of their roles in these programs, the evidence for their spectra of target genes at different stages, and their cross-regulatory and cooperative actions with each other. Specific topics include Notch modulation of PU.1 and GATA-3, PU.1-Notch competition, the relationship between PU.1 and GATA-3, and the roles of E proteins, Bcl11b, and GATA-3 in guiding acquisition of T-cell identity while avoiding redirection to an ILC fate

Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU.1

The ETS family transcription factor PU.1 is essential for the development of several blood lineages, including T cells, but its function in intrathymic T-cell precursors has been poorly defined. In the thymus, high PU.1 expression persists through multiple cell divisions in early stages but then falls sharply during T-cell lineage commitment. PU.1 silencing is critical for T-cell commitment, but it has remained unknown how PU.1 activities could contribute positively to T-cell development. Here we employed conditional knockout and modified antagonist PU.1 constructs to perturb PU.1 function stage-specifically in early T cells. We show that PU.1 is needed for full proliferation, restricting access to some non-T fates, and controlling the timing of T-cell developmental progression such that removal or antagonism of endogenous PU.1 allows precocious access to T-cell differentiation. Dominant-negative effects reveal that this repression by PU.1 is mediated indirectly. Genome-wide transcriptome analysis identifies novel targets of PU.1 positive and negative regulation affecting progenitor cell signaling and cell biology and indicating distinct regulatory effects on different subsets of progenitor cell transcription factors. Thus, in addition to supporting early T-cell proliferation, PU.1 regulates the timing of activation of the core T-lineage developmental program

Transcriptional Establishment of Cell-Type Identity: Dynamics and Causal Mechanisms of T-Cell Lineage Commitment

Precursor cell entry into the T-cell developmental pathway can be divided into two phases by the closure of T-lineage commitment. As cells decide against the last alternative options to the T-cell fate, they turn on the transcription factor Bcl11b and silence expression of a group of multipotent progenitor regulatory factors that include hematopoietic transcription factor PU.1. Functional perturbation tests show that Bcl11b is needed for commitment while PU.1 actively participates in keeping open access to alternative fates, until it is silenced; however, PU.1 and Bcl11b both contribute positively to T-cell development. Our recent work reviewed here sheds light on the transcriptional regulatory network that determines the timing and irreversibility of Bcl11b activation, the ways that Notch signaling from the thymic microenvironment restricts the action of PU.1 to prevent it from diverting cells to non-T fates, and the target genes that PU.1 still regulates under the influence of Notch signaling to contribute to T-cell generation. We argue that T-cell development depends on the sequential operation of two interlaced, but mutually antagonistic, gene regulatory networks, one initially supporting expansion before commitment and the other imposing a "terminal" differentiation process on committed cells

GATA-3 Dose-Dependent Checkpoints in Early T Cell Commitment

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4CD8 (double-negative [DN]) stages 1–3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3–deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3–deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro–T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System

PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471

Regulation of Notch signaling by the E3 ubiquitin ligase Cbl-b and its role in CD4+ T cell anergy

Notch signaling is an evolutionarily conserved pathway that mediates a variety of cell fate decisions. In the immune system, the Notch pathway plays an important role throughout the life of T cells. Currently, the role of Notch signaling in peripheral T cells is being actively investigated. The Notch pathway has already been shown to be important for proliferation, IL-2 production and helper T cell differentiation following peripheral T cell activation. In this study, we show that the E3 ubiquitin ligase Cbl-b regulates Notch protein levels by targeting it for proteosomal degradation. Notch and Cbl-b associate with each other in bulk splenocytes and when co-transfected in 293T cells. Cbl-b co-expression led to reduction in the activity of a Notch reporter construct and enhanced the ubiquitination of the Notch protein. We have also used various deletion mutants of Notch and Cbl-b to localize the interacting regions on both proteins. ^ T cell anergy is a state of T-cell hyporesponsiveness following incomplete activation. Cbl-b has been shown to be upregulated in anergic T cells and is crucial for anergy induction. As Notch is important for effector functions of peripheral T cells, our discovery that Cbl-b negatively regulates Notch signaling has led us to investigate the role of Notch in anergy induction. Using an ex-vivo system to induce anergy in CD4+ T cells we show that Notch expression is down regulated upon induction of anergy. Also, Notch and CSL bind to the IL-2 promoter and positively regulate transcription and Notch also augments NF-κB binding to this promoter. Finally, we show that Notch expression during anergy induction rescues cytokine production and proliferation of these cells upon restimulation. Our findings suggest a novel role for Notch signaling in opposing the establishment of anergy.

Differential control of peripheral circadian rhythms by suprachiasmatic-dependent neural signals

Although dependent on the integrity of a central pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN), endogenous daily (circadian) rhythms are expressed in a wide variety of peripheral organs. The pathways by which the pacemaker controls the periphery are unclear. Here, we used parabiosis between intact and SCN-lesioned mice to show that nonneural (behavioral or bloodborne) signals are adequate to maintain circadian rhythms of clock gene expression in liver and kidney, but not in heart, spleen, or skeletal muscle. These results indicate that the SCN regulates expression of circadian oscillations in different peripheral organs by diverse pathways

ERK mediates interferon gamma-induced melanoma cell death.

BackgroundInterferon-gamma (IFNγ) exerts potent growth inhibitory effects on a wide range of cancer cells through unknown signaling pathways. We pursued complementary screening approaches to characterize the growth inhibition pathway.MethodsWe performed chemical genomics and whole genome targeting CRISPR/Cas9 screens using patient-derived melanoma lines to uncover essential nodes in the IFNγ-mediated growth inhibition pathway. We used transcriptomic profiling to identify cell death pathways activated upon IFNγ exposure. Live imaging experiments coupled with apoptosis assays confirmed the involvement of these pathways in IFNγ-mediated cell death.ResultsWe show that IFNγ signaling activated ERK. Blocking ERK activation rescued IFNγ-mediated apoptosis in 17 of 23 (~ 74%) cell lines representing BRAF, NRAS, NF1 mutant, and triple wild type subtypes of cutaneous melanoma. ERK signaling induced a stress response, ultimately leading to apoptosis through the activity of DR5 and NOXA proteins.ConclusionsOur results provide a new understanding of the IFNγ growth inhibition pathway, which will be crucial in defining mechanisms of immunotherapy response and resistance