NF-κB Signaling is Required for X-Chromosome Inactivation Maintenance Following T cell Activation.

X Chromosome Inactivation (XCI) is a female-specific process which balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of Xist RNA and heterochromatic modifications on the inactive X chromosome (Xi), and these modifications become enriched at the Xi after cell stimulation. Here, we examined allele-specific gene expression and the epigenomic profiles of the Xi following T cell stimulation. We found that the Xi in unstimulated T cells is largely dosage compensated and is enriched with the repressive H3K27me3 modification, but not the H2AK119-ubiquitin (Ub) mark, even at promoters of XCI escape genes. Upon CD3/CD28-mediated T cell stimulation, the Xi accumulates H2AK119-Ub and H3K27me3 across the Xi. Next, we examined the T cell signaling pathways responsible for Xist RNA localization to the Xi and found that T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of TCR, is required. Disruption of NF-κB signaling, using inhibitors or genetic deletions, in mice and patients with immunodeficiencies prevents Xist/XIST RNA accumulation at the Xi and alters expression of some X-linked genes. Our findings reveal a novel connection between NF-κB signaling pathways which impact XCI maintenance in female T cells.

Lymph node formation and B cell homeostasis require IKK-α in distinct endothelial cell-derived compartments.

Global inactivation of IκB kinase (IKK)-α results in defective lymph node (LN) formation and B cell maturation, and loss of IKK-α-dependent noncanonical NF-κB signaling in stromal organizer and hematopoietic cells is thought to underlie these distinct defects. We previously demonstrated that this pathway is also activated in vascular endothelial cells (ECs). To determine the physiologic function of EC-intrinsic IKK-α, we crossed IkkαF/F mice with Tie2-cre or Cdh5-cre mice to ablate IKK-α in ECs. Notably, the compound defects of global IKK-α inactivation were recapitulated in IkkαTie2 and IkkαCdh5 mice, as both lacked all LNs and mature follicular and marginal zone B cell numbers were markedly reduced. However, as Tie2-cre and Cdh5-cre are expressed in all ECs, including blood forming hemogenic ECs, IKK-α was also absent in hematopoietic cells (HC). To determine if loss of HC-intrinsic IKK-α affected LN development, we generated IkkαVav mice lacking IKK-α in only the hematopoietic compartment. While mature B cell numbers were significantly reduced in IkkαVav mice, LN formation was intact. As lymphatic vessels also arise during development from blood ECs, we generated IkkαLyve1 mice lacking IKK-α in lymphatic ECs (LECs) to determine if IKK-α in lymphatic vessels impacts LN development. Strikingly, while mature B cell numbers were normal, LNs were completely absent in IkkαLyve1 mice. Thus, our findings reveal that IKK-α in distinct EC-derived compartments is uniquely required to promote B cell homeostasis and LN development, and we establish that LEC-intrinsic IKK-α is absolutely essential for LN formation.

Analysis of Calcium Control of Canonical NF-κB Signaling in B Lymphocytes.

The central role of calcium (Ca2+) signaling in lymphocyte development and acquisition of functional immunity and tolerance is well established. Ca2+ signals are initiated upon antigen binding to cognate receptors on lymphocytes that trigger store operated Ca2+ entry (SOCE). The underlying mechanism of SOCE in lymphocytes involves TCR and BCR mediated activation of Stromal Interaction Molecule 1 and 2 (STIM1/2) embedded in the ER membrane. Once activated, STIM proteins oligomerize and re-localize to ER domains juxtaposed to the plasma membrane where they activate Orai channels to allow Ca2+ to enter the cell across the plasma membrane. Importantly, STIM/Orai-dependent Ca2+ signals guide antigen induced lymphocyte development and function principally by regulating the activity of transcription factors.The most widely studied of these transcription factors is the Nuclear Factor of Activated T cells (NFAT). NFAT is expressed ubiquitously and the mechanism by which Ca2+ regulates NFAT activation and signaling is well known. By contrast, a mechanistic understanding of how Ca2+ signals also shape the activation and specificity of NF-κB to control the expression of pro-inflammatory genes has lagged. Here we discuss the methodology used to investigate Ca2+ dependent mechanisms of NF-κB activation in lymphocytes. Our approach focuses on three main areas of signal transduction and signaling: (1) antigen receptor engagement and Ca2+ dependent initiation of NF-kB signaling, (2) Ca2+ dependent induction of NF-κB heterodimer activation and nuclear localization, and (3) and how Ca2+ regulates NF-κB dependent expression of target genes and proteins.

BCR-Induced Ca2+ Signals Dynamically Tune Survival, Metabolic Reprogramming, and Proliferation of Naive B Cells.

B cell receptor (BCR) engagement induces naive B cells to differentiate and perform critical immune-regulatory functions. Acquisition of functional specificity requires that a cell survive, enter the cell cycle, and proliferate. We establish that quantitatively distinct Ca2+ signals triggered by variations in the extent of BCR engagement dynamically regulate these transitions by controlling nuclear factor κB (NF-κB), NFAT, and mTORC1 activity. Weak BCR engagement induces apoptosis by failing to activate NF-κB-driven anti-apoptotic gene expression. Stronger signals that trigger more robust Ca2+ signals promote NF-κB-dependent survival and NFAT-, mTORC1-, and c-Myc-dependent cell-cycle entry and proliferation. Finally, we establish that CD40 or TLR9 costimulation circumvents these Ca2+-regulated checkpoints of B cell activation and proliferation. As altered BCR signaling is linked to autoimmunity and B cell malignancies, these results have important implications for understanding the pathogenesis of aberrant B cell activation and differentiation and therapeutic approaches to target these responses.

4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling.

Clinical response to chimeric antigen receptor (CAR) T cell therapy is correlated with CAR T cell persistence, especially for CAR T cells that target CD19+ hematologic malignancies. 4-1BB-costimulated CAR (BBζ) T cells exhibit longer persistence after adoptive transfer than do CD28-costimulated CAR (28ζ) T cells. 4-1BB signaling improves T cell persistence even in the context of 28ζ CAR activation, which indicates distinct prosurvival signals mediated by the 4-1BB cytoplasmic domain. To specifically study signal transduction by CARs, we developed a cell-free, ligand-based activation and ex vivo culture system for CD19-specific CAR T cells. We observed greater ex vivo survival and subsequent expansion of BBζ CAR T cells when compared to 28ζ CAR T cells. We showed that only BBζ CARs activated noncanonical nuclear factor κB (ncNF-κB) signaling in T cells basally and that the anti-CD19 BBζ CAR further enhanced ncNF-κB signaling after ligand engagement. Reducing ncNF-κB signaling reduced the expansion and survival of anti-CD19 BBζ T cells and was associated with a substantial increase in the abundance of the most pro-apoptotic isoforms of Bim. Although our findings do not exclude the importance of other signaling differences between BBζ and 28ζ CARs, they demonstrate the necessary and nonredundant role of ncNF-κB signaling in promoting the survival of BBζ CAR T cells, which likely underlies the engraftment persistence observed with this CAR design.

STIM- and Orai-mediated calcium entry controls NF-κB activity and function in lymphocytes.

The central role of Ca2+ signaling in the development of functional immunity and tolerance is well established. These signals are initiated by antigen binding to cognate receptors on lymphocytes that trigger store operated Ca2+ entry (SOCE). The underlying mechanism of SOCE in lymphocytes involves TCR and BCR mediated activation of Stromal Interaction Molecule 1 and 2 (STIM1/2) molecules embedded in the ER membrane leading to their activation of Orai channels in the plasma membrane. STIM/Orai dependent Ca2+ signals guide key antigen induced lymphocyte development and function principally through direct regulation of Ca2+ dependent transcription factors. The role of Ca2+ signaling in NFAT activation and signaling is well known and has been studied extensively, but a wide appreciation and mechanistic understanding of how Ca2+ signals also shape the activation and specificity of NF-κB dependent gene expression has lagged. Here we discuss and interpret what is known about Ca2+ dependent mechanisms of NF-kB activation, including what is known and the gaps in our understanding of how these signals control lymphocyte development and function.

T Cell Receptor-induced Nuclear Factor κB (NF-κB) Signaling and Transcriptional Activation Are Regulated by STIM1- and Orai1-mediated Calcium Entry.

T cell activation following antigen binding to the T cell receptor (TCR) involves the mobilization of intracellular Ca(2+) to activate the key transcription factors nuclear factor of activated T lymphocytes (NFAT) and NF-κB. The mechanism of NFAT activation by Ca(2+) has been determined. However, the role of Ca(2+) in controlling NF-κB signaling is poorly understood, and the source of Ca(2+) required for NF-κB activation is unknown. We demonstrate that TCR- but not TNF-induced NF-κB signaling upstream of IκB kinase activation absolutely requires the influx of extracellular Ca(2+) via STIM1-dependent Ca(2+) release-activated Ca(2+)/Orai channels. We further show that Ca(2+) influx controls phosphorylation of the NF-κB protein p65 on Ser-536 and that this posttranslational modification controls its nuclear localization and transcriptional activation. Notably, our data reveal that this role for Ca(2+) is entirely separate from its upstream control of IκBα degradation, thereby identifying a novel Ca(2+)-dependent distal step in TCR-induced NF-κB activation. Finally, we demonstrate that this control of distal signaling occurs via Ca(2+)-dependent PKCα-mediated phosphorylation of p65. Thus, we establish the source of Ca(2+) required for TCR-induced NF-κB activation and define a new distal Ca(2+)-dependent checkpoint in TCR-induced NF-κB signaling that has broad implications for the control of immune cell development and T cell functional specificity.

Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity.

Innate lymphoid cells (ILCs) are critical for maintaining epithelial barrier integrity at mucosal surfaces; however, the tissue-specific factors that regulate ILC responses remain poorly characterized. Using mice with intestinal epithelial cell (IEC)-specific deletions in either inhibitor of κB kinase (IKK)α or IKKß, two critical regulators of NFκB activation, we demonstrate that IEC-intrinsic IKKα expression selectively regulates group 3 ILC (ILC3)-dependent antibacterial immunity in the intestine. Although IKKß(ΔIEC) mice efficiently controlled Citrobacter rodentium infection, IKKα(ΔIEC) mice exhibited severe intestinal inflammation, increased bacterial dissemination to peripheral organs, and increased host mortality. Consistent with weakened innate immunity to C. rodentium, IKKα(ΔIEC) mice displayed impaired IL-22 production by RORγt(+) ILC3s, and therapeutic delivery of rIL-22 or transfer of sort-purified IL-22-competent ILCs from control mice could protect IKKα(ΔIEC) mice from C. rodentium-induced morbidity. Defective ILC3 responses in IKKα(ΔIEC) mice were associated with overproduction of thymic stromal lymphopoietin (TSLP) by IECs, which negatively regulated IL-22 production by ILC3s and impaired innate immunity to C. rodentium. IEC-intrinsic IKKα expression was similarly critical for regulation of intestinal inflammation after chemically induced intestinal damage and colitis. Collectively, these data identify a previously unrecognized role for epithelial cell-intrinsic IKKα expression and TSLP in regulating ILC3 responses required to maintain intestinal barrier immunity.

Understanding high endothelial venules: Lessons for cancer immunology.

High endothelial venules (HEVs) are blood vessels especially adapted for lymphocyte trafficking which are normally found in secondary lymphoid organs such as lymph nodes (LN) and Peyer's patches. It has long been known that HEVs develop in non-lymphoid organs during chronic inflammation driven by autoimmunity, infection or allografts. More recently, HEVs have been observed in solid, vascularized tumors and their presence correlated with reduced tumor size and improved patient outcome. It is proposed that newly formed HEV promote antitumor immunity by recruiting naive lymphocytes into the tumor, thus allowing the local generation of cancerous tissue-destroying lymphocytes. Understanding how HEVs develop and function are therefore important to unravel their role in human cancers. In LN, HEVs develop during embryonic and early post-natal life and are actively maintained by the LN microenvironment. Systemic blockade of lymphotoxin-ß receptor leads to HEV de-differentiation, but the LN components that induce HEV differentiation have remained elusive. Recent elegant studies using gene-targeted mice have demonstrated clearly that triggering the lymphotoxin-ß receptor in endothelial cells (EC) induces the differentiation of HEV and that CD11c+ dendritic cells play a crucial role in this process. It will be important to determine whether lymphotoxin-ß receptor-dependent signaling in EC drives the development of HEV during tumorigenesis and which cells have HEV-inducer properties. This may reveal therapeutic approaches to promote HEV neogenesis and determine the impact of newly formed HEV on tumor immunity.

Noncanonical NF-κB activation and SDF-1 expression in human endothelial cells.

NF-κB is a family of transcription factors regulated through two distinct signaling cascades, the classical and the Noncanonical NF-κB pathways. Noncanonical NF-κB plays important roles in the immune system, as it is necessary for lymphoid organogenesis and B-cell survival and differentiation, as well as osteoclastogenesis. In the last few years, there has been an increased number of studies focusing on both identifying the upstream events that regulate the noncanonical NF-κB pathway as well as determining the physiological roles of noncanonical NF-κB in normal and disease pathologies, such as cancer and autoimmune diseases. Dysregulation of noncanonical NF-κB has now been associated with the pathogenesis of several types of lymphomas and autoimmune diseases and is believed to contribute to chronic inflammatory diseases, including ulcerative colitis. These studies suggest that targeting the Noncanonical pathway, similar to classical NF-κB, may have some therapeutic potential in the future; however, there is still quite a bit about the regulation of the noncanonical signaling that remains to be defined. In this chapter we describe the use of HUVEC, as an in vitro model for examining noncanonical NF-κB signaling in response to different stimuli. We demonstrate two different methods to measure noncanonical NF-κB activation: the processing of p100 to p52, and noncanonical NF-κB-dependent gene expression of CXCL12. The first method examines a key regulatory requirement for noncanonical NF-κB activation, by which p100 undergoes proteolytic cleavage to relieve the inhibition of NF-κB dimers for nuclear translocation and activation of gene transcription. The latter demonstrates the downstream effects of activated noncanonical NF-κB in response to stimuli.

Stable reconstitution of IKK-deficient mouse embryonic fibroblasts.

Retroviral transduction is an invaluable technique in molecular biology used to express proteins encoded by nonviral genes in mammalian cells. A key feature of this technique is the ability to create cell lines that stably express the protein of interest and can be cultured long term. Here we describe a retroviral transduction procedure for mouse embryonic fibroblasts (MEFs) that uses Platinum-E cells to rapidly package high-titer, helper-free retrovirus. This technique is useful to study the role of key signaling kinases in the NF-κB signal transduction pathway.

NEMO-binding domain peptide inhibition of inflammatory signal-induced NF-κB activation in vivo.

NF-κB comprises a family of transcription factors that regulate the expression of diverse gene families essential for inflammatory and immune responses as well as cell survival and cell death pathways. Aberrant NF-κB transcriptional activity plays pivotal roles in a large number of human pathologies, including a variety of cancers and chronic inflammatory diseases. Therefore, there has been a large increase in studies aimed at identifying and testing drugs or small molecule inhibitors that would specifically block NF-κB activation in inflammatory diseases and cancer. In this chapter, we describe an in vivo system to test the inhibitory effects of the NEMO-binding domain (NBD) peptide on NF-κB activation specifically in the vascular endothelium and lymphocytes in mice. We demonstrate that pretreatment of mice with the NBD peptide reduces the NF-κB induced gene expression of cell adhesion molecules and DNA-binding activity following systemic LPS stimulation. These methods can be further used to test alternate inhibitors for effects on NF-κB signaling in murine endothelium and immune cells.

Sneaking-ligand fusion proteins attenuate serum transfer arthritis by endothelium-targeted NF-κB inhibition.

The nuclear transcription factor κB (NF-κB) is a crucial mediator of the inflammatory and immune response. The contribution of dysregulated NF-κB is established in the pathogenesis of arthritis. Accordingly, NF-κB represents an attractive molecular target for the development of therapeutic interventions in inflammatory diseases. However, ubiquitous pharmacologic suppression of NF-κB activity is limited by the hazards of toxic side effects and profound immunosuppression. Cell type-specific NF-κB inhibition with the "sneaking-ligand" approach could identify disease-relevant cell types and improve risk-benefit ratios of therapeutic interventions. Vascular endothelial cells act as a gatekeeper and are crucial for leukocyte recruitment into sites of inflammation. The endothelium-specific NF-κB inhibitor SLC1 ameliorates serum transfer arthritis in mice and protects against inflammation and cartilage destruction. In this chapter, we describe the SLC1 treatment schedule in the K/BxN serum transfer arthritis and present the evaluation system to analyze arthritis severity and histopathological alterations.

Negative feedback regulation of NF-κB-inducing kinase is proteasome-dependent but does not require cellular inhibitors of apoptosis.

Non-canonical NF-κB signaling is controlled by the precise regulation of NF-κB inducing kinase (NIK) stability. NIK is constitutively ubiquitylated by cellular inhibitor of apoptosis (cIAP) proteins 1 and 2, leading to its complete proteasomal degradation in resting cells. Following stimulation, cIAP-mediated ubiquitylation of NIK ceases and NIK is stabilized, allowing for inhibitor of κB kinase (IKK)α activation and non-canonical NF-κB signaling. Non-canonical NF-κB signaling is terminated by feedback phosphorylation of NIK by IKKα that promotes NIK degradation; however, the mechanism of active NIK protein turnover remains unknown. To address this question, we established a strategy to precisely distinguish between basal degradation of newly synthesized endogenous NIK and induced active NIK in stimulated cells. Using this approach, we found that IKKα-mediated degradation of signal-induced activated NIK occurs through the proteasome. To determine whether cIAP1 or cIAP2 play a role in active NIK turnover, we utilized a Smac mimetic (GT13072), which promotes degradation of these E3 ubiquitin ligases. As expected, GT13072 stabilized NIK in resting cells. However, loss of the cIAPs did not inhibit proteasome-dependent turnover of signal-induced NIK showing that unlike the basal regulatory mechanism, active NIK turnover is independent of cIAP1 and cIAP2. Our results therefore establish that the negative feedback control of IKKα-mediated NIK turnover occurs via a novel proteasome-dependent and cIAP-independent mechanism.

A phase I clinical trial of systemically delivered NEMO binding domain peptide in dogs with spontaneous activated B-cell like diffuse large B-cell lymphoma.

Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL.

Triggering ubiquitination of IFNAR1 protects tissues from inflammatory injury.

Type 1 interferons (IFN) protect the host against viruses by engaging a cognate receptor (consisting of IFNAR1/IFNAR2 chains) and inducing downstream signaling and gene expression. However, inflammatory stimuli can trigger IFNAR1 ubiquitination and downregulation thereby attenuating IFN effects in vitro. The significance of this paradoxical regulation is unknown. Presented here results demonstrate that inability to stimulate IFNAR1 ubiquitination in the Ifnar1(SA) knock-in mice renders them highly susceptible to numerous inflammatory syndromes including acute and chronic pancreatitis, and autoimmune and toxic hepatitis. Ifnar1(SA) mice (or their bone marrow-receiving wild type animals) display persistent immune infiltration of inflamed tissues, extensive damage and gravely inadequate tissue regeneration. Pharmacologic stimulation of IFNAR1 ubiquitination is protective against from toxic hepatitis and fulminant generalized inflammation in wild type but not Ifnar1(SA) mice. These results suggest that endogenous mechanisms that trigger IFNAR1 ubiquitination for limiting the inflammation-induced tissue damage can be purposely mimicked for therapeutic benefits.

Noncanonical NF-κB signaling is limited by classical NF-κB activity.

Precise regulation of nuclear factor κB (NF-κB) signaling is crucial for normal immune responses, and defective NF-κB activity underlies a range of immunodeficiencies. NF-κB is activated through two signaling cascades: the classical and noncanonical pathways. The classical pathway requires inhibitor of κB kinase ß (IKKß) and NF-κB essential modulator (NEMO), and hypomorphic mutations in the gene encoding NEMO (ikbkg) lead to inherited immunodeficiencies, collectively termed NEMO-ID. Noncanonical NF-κB activation requires NF-κB-inducing kinase (NIK) and IKKα, but not NEMO. We found that noncanonical NF-κB was basally active in peripheral blood mononuclear cells from NEMO-ID patients and that noncanonical NF-κB signaling was similarly enhanced in cell lines lacking functional NEMO. NIK, which normally undergoes constitutive degradation, was aberrantly present in resting NEMO-deficient cells, and regulation of its abundance was rescued by reconstitution with full-length NEMO, but not a mutant NEMO protein unable to physically associate with IKKα or IKKß. Binding of NEMO to IKKα was not required for ligand-dependent stabilization of NIK or noncanonical NF-κB signaling. Rather, an intact and functional IKK complex was essential to suppress basal NIK activity in unstimulated cells. Despite interacting with IKKα and IKKß to form an IKK complex, NEMO mutants associated with immunodeficiency failed to rescue classical NF-κB signaling or reverse the accumulation of NIK. Together, these findings identify a crucial role for classical NF-κB activity in the suppression of basal noncanonical NF-κB signaling.

NF-κB inhibitor targeted to activated endothelium demonstrates a critical role of endothelial NF-κB in immune-mediated diseases.

Activation of the nuclear transcription factor κB (NF-κB) regulates the expression of inflammatory genes crucially involved in the pathogenesis of inflammatory diseases. NF-κB governs the expression of adhesion molecules that play a pivotal role in leukocyte-endothelium interactions. We uncovered the crucial role of NF-κB activation within endothelial cells in models of immune-mediated diseases using a "sneaking ligand construct" (SLC) selectively inhibiting NF-κB in the activated endothelium. The recombinant SLC1 consists of three modules: (i) an E-selectin targeting domain, (ii) a Pseudomonas exotoxin A translocation domain, and (iii) a NF-κB Essential Modifier-binding effector domain interfering with NF-κB activation. The E-selectin-specific SLC1 inhibited NF-κB by interfering with endothelial IκB kinase 2 activity in vitro and in vivo. In murine experimental peritonitis, the application of SLC1 drastically reduced the extravasation of inflammatory cells. Furthermore, SLC1 treatment significantly ameliorated the disease course in murine models of rheumatoid arthritis. Our data establish that endothelial NF-κB activation is critically involved in the pathogenesis of arthritis and can be selectively inhibited in a cell type- and activation stage-dependent manner by the SLC approach. Moreover, our strategy is applicable to delineating other pathogenic signaling pathways in a cell type-specific manner and enables selective targeting of distinct cell populations to improve effectiveness and risk-benefit ratios of therapeutic interventions.