Summary An open chromatin largely devoid of heterochromatin is a hallmark of stem cells, from Planarians to Mammals. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and what are the molecules that maintain open chromatin in stem cells. Here we show that the chromatin remodeling factor Chd1 is required to maintain the open chromatin state of pluripotent mouse Embryonic Stem (ES) cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and down-regulation of Chd1 leads to accumulation of heterochromatin in ES cells. Chd1-deficient ES cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of ES cells, and for somatic cell reprogramming to the pluripotent state. The data suggest that pluripotent stem cells exist in a dynamic state of opposing epigenetic influences of euchromatin and heterochromatin.

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NF-κB-inducing kinase (NIK) is an essential upstream kinase in noncanonical NF-κB signaling. NIK-dependent NF-κB activation downstream of several TNF receptor family members mediates lymphoid organ development and B cell homeostasis. Peripheral T cell populations are normal in the absence of NIK, but the role of NIK during in vivo T cell responses to antigen has been obscured by other developmental defects in NIKdeficient mice. Here, we have identified a T cell-intrinsic requirement for NIK in graft-versus-host disease (GVHD), wherein NIK-deficient mouse T cells transferred into MHC class II mismatched recipients failed to cause GVHD. Although NIK was not necessary for antigen receptor signaling, it was absolutely required for costimulation through the TNF receptor family member OX40 (also known as CD134). When we conditionally overexpressed NIK in T cells, mice suffered rapid and fatal autoimmunity characterized by hyperactive effector T cells and poorly suppressive Foxp3 + Tregs. Together, these data illuminate a critical T cell-intrinsic role for NIK during immune responses and suggest that its tight regulation is critical for avoiding autoimmunity.

The protective capability of tumor antigen–specific T cells is regulated by costimulatory and inhibitory signals. Current approaches in cancer immunotherapy seek to restore the function of unresponsive T cells by blocking inhibitory pathways. In contrast, providing exogenous costimulatory signals to T cells also enhances antitumor functionality. By combining these two clinical approaches, we demonstrate the synergy of targeting PD-L1 together with the costimulatory molecule OX40, to enhance antitumor immunity. Concurrently blocking PD-L1 and providing a costimulatory agonist to OX40 increased the presence and functionality of tumor antigen–specific CD8+ T cells with simultaneous enhancement of T-helper type 1 (Th1)-skewed CD4+ T cells. This shift was functionally supported by increased glucose metabolism of antigen-specific CD8+ T cells and the acquisition of granzyme B by regulatory T cells. Together, this mechanism promoted tumor regression of late-stage tumors beyond that achieved by either blockade as monotherapy. These findings indicate that targeting both T-cell intrinsic (OX40) and extrinsic (PD-L1) regulatory molecules increases the bioenergetic potential of T cells, thereby expanding functional and tumor antigen–specific T cells.

Cancer cells harbor high affinity tumor-associated antigens capable of eliciting potent anti-tumor T cell responses yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and PD-1 have been used. We report here that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor-antigen specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Co-expression of Nur77GFP and OX40 identifies a polyclonal population of high affinity tumor-associated antigen-specific CD8+ T cells, which produce more IFNγ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or PD-L1. Moreover, expansion of these high affinity CD8 T cells prolongs survival of tumor bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor resident CD8 T cells thereby increasing the frequency of cytolytic, high affinity, tumor-associated antigen-specific cells.

OX40 is a costimulatory molecule from the TNFR family. In mice, it is expressed on Foxp3+ regulatory T cells (Tregs) constitutively and on conventional CD4 (Tconv) and CD8 T cells after Ag encounter. OX40 agonists are in clinical development to enhance antitumor immune responses, and one proposed mechanism of action is loss of Treg suppressive function. Studies have postulated that agonist OX40 therapy can impair Treg suppressive function. Using tools developed since the initial studies were published, we evaluated a direct effect of OX40 agonism on Treg function. We conclude that OX40 agonist Abs do not intrinsically impair Treg function but rather enhance Tconv cell IL-2 production, increasing Treg and Tconv cell proliferation. OX40-stimulated Tregs retain suppressive function, but also gain IFN-γ, TNF-α, and granzyme B expression. These data help resolve mechanistic questions regarding OX40 agonist immunotherapy and thus are relevant to developing combination therapies that target distinct T cell functions.

CD4+Foxp3+ regulatory T cells (Tregs) are indispensable negative regulators of immune responses. To understand Treg biology in health and disease, it is critical to elucidate factors that affect Treg homeostasis and suppressive function. Tregs express several costimulatory TNF receptor family members that activate non-canonical NF-κB via accumulation of NF-κB inducing kinase (NIK). We previously showed that constitutive NIK expression in all T cells causes fatal multi-organ autoimmunity associated with hyperactive conventional T cell responses and poor Treg-mediated suppression. Here, we show that constitutive NIK expression that is restricted to Tregs via a Cre-inducible transgene causes an autoimmune syndrome. We found that constitutive NIK expression decreased expression of numerous Treg signature genes and microRNAs involved in Treg homeostasis and suppressive phenotype. NIK transgenic Tregs competed poorly with WT Tregs in vivo and produced pro-inflammatory cytokines upon stimulation. Lineage tracing experiments revealed accumulation of ex-Foxp3+ T cells in mice expressing NIK constitutively in Tregs, and these former Tregs produced copious IFNγ and IL-2. Our data indicate that under inflammatory conditions in which NIK is activated, Tregs may lose suppressive function and may actively contribute to inflammation.

Midkine (MDK) is a heparin-binding growth factor involved in growth, survival, migration, and differentiation of various target cells and dysregulation of MDK signaling is implicated in a variety of inflammatory diseases and cancers. Although MDK has been reported to act on endothelial cells and to have proangiogenic effects, the exact role of MDK in angiogenesis is poorly defined. Here, we report that MDK is actually a modulator of angiogenesis and that it can abrogate the vascular endothelial growth factor A (VEGF-A)-induced proliferation of human microvascular endothelial cells in vitro through the downregulation of proangiogenic cytokines and through the upregulation of the antiangiogenic factor, tissue inhibitor of metalloproteinase 2. Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and of downstream signaling molecules, such as phosphatidylinositol-3-kinase and mitogen-activated protein kinases, is also impaired. Moreover, MDK downregulates VEGF-A-induced neovascularization and vascular permeability in vivo. We propose a model in which MDK is a new modulator of the VEGF-A-VEGFR-2 axis.