NF-κB subunits RelA and c-Rel selectively control CD4+ T cell function in multiple sclerosis and cancer.

The outcome of cancer and autoimmunity is often dictated by the effector functions of CD4+ conventional T cells (Tconv). Although activation of the NF-κB signaling pathway has long been implicated in Tconv biology, the cell-autonomous roles of the separate NF-κB transcription-factor subunits are unknown. Here, we dissected the contributions of the canonical NF-κB subunits RelA and c-Rel to Tconv function. RelA, rather than c-Rel, regulated Tconv activation and cytokine production at steady-state and was required for polarization toward the TH17 lineage in vitro. Accordingly, RelA-deficient mice were fully protected against neuroinflammation in a model of multiple sclerosis due to defective transition to a pathogenic TH17 gene-expression program. Conversely, Tconv-restricted ablation of c-Rel impaired their function in the microenvironment of transplanted tumors, resulting in enhanced cancer burden. Moreover, Tconv required c-Rel for the response to PD-1-blockade therapy. Our data reveal distinct roles for canonical NF-κB subunits in different disease contexts, paving the way for subunit-targeted immunotherapies.

The NF-κB RelA transcription factor is not required for CD8+ T-cell function in acute viral infection and cancer.

CD8+ T cells are critical mediators of pathogen clearance and anti-tumor immunity. Although signaling pathways leading to the activation of NF-κB transcription factors have crucial functions in the regulation of immune responses, the CD8+ T cell-autonomous roles of the different NF-κB subunits, are still unresolved. Here, we investigated the function of the ubiquitously expressed transcription factor RelA in CD8+ T-cell biology using a novel mouse model and gene-edited human cells. We found that CD8+ T cell-specific ablation of RelA markedly altered the transcriptome of ex vivo stimulated cells, but maintained the proliferative capacity of both mouse and human cells. In contrast, in vivo experiments showed that RelA deficiency did not affect the CD8+ T-cell response to acute viral infection or transplanted tumors. Our data suggest that in CD8+ T cells, RelA is dispensable for their protective activity in pathological contexts.

Xevinapant Combined with Pembrolizumab in Patients with Advanced, Pretreated, Colorectal and Pancreatic Cancer: Results of the Phase Ib/II CATRIPCA Trial.

PURPOSE: Xevinapant is an orally available inhibitor of apoptosis proteins (IAP) inhibitor. Preclinical data suggest that IAP antagonism may synergize with immune checkpoint blockers by modulating the NFκB pathway in immune cells. PATIENTS AND METHODS: Adult patients with non-high microsatellite instability advanced/metastatic pancreatic ductal adenocarcinoma (PDAC) or colorectal cancer were enrolled in this phase Ib/II study and received pembrolizumab 200 mg every 3 weeks intravenously, and ascending doses of oral xevinapant (100, 150, and 200 mg daily for 14 days on/7 days off). Dose escalation followed a 3+3 design with a 21-day dose-limiting toxicity (DLT) evaluation period. Following the determination of the recommended phase II dose (RP2D), 14 patients with PDAC and 14 patients with colorectal cancer were enrolled in expansion cohorts to assess preliminary efficacy. RESULTS: Forty-one patients (26 males) with a median age of 64 years were enrolled: 13 in the dose escalation and 28 in the two expansion cohorts. No DLT was observed during dose escalation. The RP2D was identified as xevinapant 200 mg/day + pembrolizumab 200 mg every 3 weeks. The most common adverse events (AE) were fatigue (37%), gastrointestinal AE (decreased appetite in 37%, nausea in 24%, stomatitis in 12%, and diarrhea and vomiting in 10% each), and cutaneous AE (pruritus, dry skin, and rash seen in 20%, 15%, and 15% of patients, respectively). The best overall response according to RECIST1.1 was partial response (confirmed) in 1 (3%), stable disease in 4 (10%), and progressive disease in 35 (88%). CONCLUSIONS: Xevinapant combined with pembrolizumab was well tolerated with no unexpected AEs. However, antitumor activity was low.

MDR1-EXPRESSING CD4+ T CELLS WITH TH1.17 FEATURES RESIST TO NEOADJUVANT CHEMOTHERAPY AND ARE ASSOCIATED WITH BREAST CANCER CLINICAL RESPONSE.

BACKGROUND: Multidrug resistance-1 (MDR1) transporter limits the intracellular accumulation of chemotherapies (paclitaxel, anthracyclines) used in breast cancer (BC) treatment. In addition to tumor cells, MDR1 is expressed on immune cell subsets in which it confers chemoresistance. Among human T cells, MDR1 is expressed by most CD8+ T cells, and a subset of CD4+ T helper (Th) cells. Here we explored the expression, function and regulation of MDR1 on CD4+ T cells and investigated the role of this population in response to neoadjuvant chemotherapy (NAC) in BC. METHODS: Phenotypic and functional characteristics of MDR1+ CD4 Th cells were assessed on blood from healthy donors and patients with BC by flow cytometry. These features were extended to CD4+ Th cells from untreated breast tumor by flow cytometry and RNA-sequencing (RNA-seq). We performed in vitro polarization assays to decipher MDR1 regulation on CD4 Th cells. We evaluated in vitro the impact of chemotherapy agents on MDR1+ CD4+ Th cells. We analyzed the impact of NAC treatment on MDR1+ CD4+ Th cells from blood and tumors and their association with treatment efficacy in two independent BC cohorts and in a public RNA-seq data set of BC tumor biopsies before and after NAC. Finally, we performed single cell (sc) RNAseq of blood CD4+ memory T cells from NAC-treated patients and combined them with an scRNAseq public data set. RESULTS: MDR1+ CD4 Th cells were strongly enriched in Th1.17 polyfunctional cells but also in Th17 cells, both in blood and untreated breast tumor tissues. Mechanistically, Tumor growth factor (TGF)-ß1 was required for MDR1 induction during in vitro Th17 or Th1.17 polarization. MDR1 expression conferred a selective advantage to Th1.17 and Th17 cells following paclitaxel treatment in vitro and in vivo in NAC-treated patients. scRNAseq demonstrated MDR1 association with tumor Th1.17 and Th with features of cytotoxic cells. Enrichment in MDR1+ CD4+ Th1.17 and Th17 cells, in blood and tumors positively correlated with pathological response. Absence of early modulation of Th1.17 and Th17 in NAC-resistant patients, argue for its use as a biomarker for chemotherapy regimen adjustment. CONCLUSION: MDR1 favored the enrichment of Th1.17 and Th17 in blood and tumor after NAC that correlated to clinical response.

A pan-cancer analysis implicates human NKIRAS1 as a tumor-suppressor gene.

The NF-κB family of transcription factors and the Ras family of small GTPases are important mediators of proproliferative signaling that drives tumorigenesis and carcinogenesis. The κB-Ras proteins were previously shown to inhibit both NF-κB and Ras activation through independent mechanisms, implicating them as tumor suppressors with potentially broad relevance to human cancers. In this study, we have used two mouse models to establish the relevance of the κB-Ras proteins for tumorigenesis. Additionally, we have utilized a pan-cancer bioinformatics analysis to explore the role of the κB-Ras proteins in human cancers. Surprisingly, we find that the genes encoding κB-Ras 1 (NKIRAS1) and κB-Ras 2 (NKIRAS2) are rarely down-regulated in tumor samples with oncogenic Ras mutations. Reduced expression of human NKIRAS1 alone is associated with worse prognosis in at least four cancer types and linked to a network of genes implicated in tumorigenesis. Our findings provide direct evidence that loss of NKIRAS1 in human tumors that do not carry oncogenic RAS mutations is associated with worse clinical outcomes.

TCR-independent CD137 (4-1BB) signaling promotes CD8+-exhausted T cell proliferation and terminal differentiation.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

ZEB1 transcription factor promotes immune escape in melanoma.

BACKGROUND: The efficacy of immunotherapies in metastatic melanoma depends on a robust T cell infiltration. Oncogenic alterations of tumor cells have been associated to T cell exclusion. Identifying novel cancer cell-intrinsic non-genetic mechanisms of immune escape, the targeting of which would reinstate T cell recruitment, would allow to restore the response to anti-programmed cell death protein 1 (PD-1) antibody therapy. The epithelial-to-mesenchymal transition (EMT)-inducing transcription factor ZEB1 is a major regulator of melanoma cell plasticity, driving resistance to mitogen-activated protein kinase (MAPK) targeted therapies. We thus wondered whether ZEB1 signaling in melanoma cells may promote immune evasion and resistance to immunotherapy. METHODS: We evaluated the putative correlation between ZEB1 expression in melanoma cells and the composition of the immune infiltrate in a cohort of 60 human melanoma samples by combining transcriptomic (RNA-sequencing) and seven-color spatial multi-immunofluorescence analyses. Algorithm-based spatial reconstitution of tumors allowed the quantification of CD8+, CD4+ T cells number and their activation state (PD-1, Ki67). ZEB1 gain-of-function or loss-of-function approaches were then implemented in syngeneic melanoma mouse models, followed by monitoring of tumor growth, quantification of immune cell populations frequency and function by flow cytometry, cytokines secretion by multiplex analyses. Chromatin-immunoprecipitation was used to demonstrate the direct binding of this transcription factor on the promoters of cytokine-encoding genes. Finally, the sensitivity to anti-PD-1 antibody therapy upon ZEB1 gain-of-function or loss-of-function was evaluated. RESULTS: Combined spatial and transcriptomic analyses of the immune infiltrates in human melanoma samples demonstrated that ZEB1 expression in melanoma cells is associated with decreased CD8+ T cell infiltration, independently of ß-catenin pathway activation. ZEB1 ectopic expression in melanoma cells impairs CD8+ T cell recruitment in syngeneic mouse models, resulting in tumor immune evasion and resistance to immune checkpoint blockade. Mechanistically, we demonstrate that ZEB1 directly represses the secretion of T cell-attracting chemokines, including CXCL10. Finally, Zeb1 knock-out, by promoting CD8+ T cell infiltration, synergizes with anti-PD-1 antibody therapy in promoting tumor regression. CONCLUSIONS: We identify the ZEB1 transcription factor as a key determinant of melanoma immune escape, highlighting a previously unknown therapeutic target to increase efficacy of immunotherapy in melanoma. TRIAL REGISTRATION NUMBER: NCT02828202.

A T cell-intrinsic function for NF-κB RelB in experimental autoimmune encephalomyelitis.

NF-kappaB (NF-κB) is a family of transcription factors with pleiotropic functions in immune responses. The alternative NF-κB pathway that leads to the activation of RelB and NF-κB2, was previously associated with the activation and function of T cells, though the exact contribution of these NF-κB subunits remains unclear. Here, using mice carrying conditional ablation of RelB in T cells, we evaluated its role in the development of conventional CD4+ T (Tconv) cells and their function in autoimmune diseases. RelB was largely dispensable for Tconv cell homeostasis, activation and proliferation, and for their polarization toward different flavors of Thelper cells in vitro. Moreover, ablation of RelB had no impact on the capacity of Tconv cells to induce autoimmune colitis. Conversely, clinical severity of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS) was significantly reduced in mice with RelB-deficient T cells. This was associated with impaired expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically in the central nervous system. Our data reveal a discrete role for RelB in the pathogenic function of Tconv cells during EAE, and highlight this transcription factor as a putative therapeutic target in MS.

The many-sided contributions of NF-κB to T-cell biology in health and disease.

T cells (or T lymphocytes) exhibit a myriad of functions in immune responses, ranging from pathogen clearance to autoimmunity, cancer and even non-lymphoid tissue homeostasis. Therefore, deciphering the molecular mechanisms orchestrating their specification, function and gene expression pattern is critical not only for our comprehension of fundamental biology, but also for the discovery of novel therapeutic targets. Among the master regulators of T-cell identity, the functions of the NF-κB family of transcription factors have been under scrutiny for several decades. However, a more precise understanding of their pleiotropic functions is only just emerging. In this review we will provide a global overview of the roles of NF-κB in the different flavors of mature T cells. We aim at highlighting the complex and sometimes diverging roles of the five NF-κB subunits in health and disease.

PDK1 Is Required for Maintenance of CD4+ Foxp3+ Regulatory T Cell Function.

Regulatory T (Treg) cells have an essential role in maintaining immune homeostasis, in part by suppressing effector T cell functions. Phosphoinositide-dependent kinase 1 (PDK1) is a pleiotropic kinase that acts as a key effector downstream of PI3K in many cell types. In T cells, PDK1 has been shown to be critical for activation of NF-κB and AKT signaling upon TCR ligation and is therefore essential for effector T cell activation, proliferation, and cytokine production. Using Treg cell-specific conditional deletion, we now demonstrate that PDK1 is also essential for Treg cell suppressive activity in vivo. Ablation of Pdk1 specifically in Treg cells led to systemic, lethal, scurfy-like inflammation in mice. Genome-wide analysis confirmed that PDK1 is essential for the regulation of key Treg cell signature gene expression and, further, suggested that PDK1 acts primarily to control Treg cell gene expression through regulation of the canonical NF-κB pathway. Consistent with these results, the scurfy-like phenotype of mice lacking PDK1 in Treg cells was rescued by enforced activation of NF-κB downstream of PDK1. Therefore, PDK1-mediated activation of the NF-κB signaling pathway is essential for regulation of Treg cell signature gene expression and suppressor function.

Tissue-restricted control of established central nervous system autoimmunity by TNF receptor 2-expressing Treg cells.

CD4+Foxp3+ regulatory T (Treg) cells are central modulators of autoimmune diseases. However, the timing and location of Treg cell-mediated suppression of tissue-specific autoimmunity remain undefined. Here, we addressed these questions by investigating the role of tumor necrosis factor (TNF) receptor 2 (TNFR2) signaling in Treg cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We found that TNFR2-expressing Treg cells were critical to suppress EAE at peak disease in the central nervous system but had no impact on T cell priming in lymphoid tissues at disease onset. Mechanistically, TNFR2 signaling maintained functional Treg cells with sustained expression of CTLA-4 and Blimp-1, allowing active suppression of pathogenic T cells in the inflamed central nervous system. This late effect of Treg cells was further confirmed by treating mice with TNF and TNFR2 agonists and antagonists. Our findings show that endogenous Treg cells specifically suppress an autoimmune disease by acting in the target tissue during overt inflammation. Moreover, they bring a mechanistic insight to some of the adverse effects of anti-TNF therapy in patients.

NF-κB in Cancer Immunity: Friend or Foe?

The emergence of immunotherapies has definitely proven the tight relationship between malignant and immune cells, its impact on cancer outcome and its therapeutic potential. In this context, it is undoubtedly critical to decipher the transcriptional regulation of these complex interactions. Following early observations demonstrating the roles of NF-κB in cancer initiation and progression, a series of studies converge to establish NF-κB as a master regulator of immune responses to cancer. Importantly, NF-κB is a family of transcriptional activators and repressors that can act at different stages of cancer immunity. In this review, we provide an overview of the selective cell-intrinsic contributions of NF-κB to the distinct cell types that compose the tumor immune environment. We also propose a new view of NF-κB targeting drugs as a new class of immunotherapies for cancer.

Transcriptional Control of Regulatory T Cells in Cancer: Toward Therapeutic Targeting?

Extensive research in the past decades has highlighted the tight link between immunity and cancer, leading to the development of immunotherapies that have revolutionized cancer care. However, only a fraction of patients display durable responses to these treatments, and a deeper understanding of the cellular and mechanisms orchestrating immune responses to tumors is mandatory for the discovery of novel therapeutic targets. Among the most scrutinized immune cells, Forkhead Box Protein P3 (Foxp3)+ Regulatory T cells (Treg cells) are central inhibitors of protective anti-tumor immunity. These tumor-promoting functions render Treg cells attractive immunotherapy targets, and multiple strategies are being developed to inhibit their recruitment, survival, and function in the tumor microenvironment. In this context, it is critical to decipher the complex and multi-layered molecular mechanisms that shape and stabilize the Treg cell transcriptome. Here, we provide a global view of the transcription factors, and their upstream signaling pathways, involved in the programming of Treg cell homeostasis and functions in cancer. We also evaluate the feasibility and safety of novel therapeutic approaches aiming at targeting specific transcriptional regulators.

Tumor necrosis factor receptor family costimulation increases regulatory T-cell activation and function via NF-κB.

Several drugs targeting members of the TNF superfamily or TNF receptor superfamily (TNFRSF) are widely used in medicine or are currently being tested in therapeutic trials. However, their mechanism of action remains poorly understood. Here, we explored the effects of TNFRSF co-stimulation on murine Foxp3+ regulatory T cell (Treg) biology, as they are pivotal modulators of immune responses. We show that engagement of TNFR2, 4-1BB, GITR, and DR3, but not OX40, increases Treg proliferation and survival. Triggering these TNFRSF in Tregs induces similar changes in gene expression patterns, suggesting that they engage common signal transduction pathways. Among them, we identified a major role of canonical NF-κB. Importantly, TNFRSF co-stimulation improves the ability of Tregs to suppress colitis. Our data demonstrate that stimulation of discrete TNFRSF members enhances Treg activation and function through a shared mechanism. Consequently, therapeutic effects of drugs targeting TNFRSF or their ligands may be mediated by their effect on Tregs.

Cutaneous p38 mitogen-activated protein kinase activation triggers psoriatic dermatitis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by IL-17-mediated immune responses. p38 is known to be highly activated in the psoriatic epidermis; however, whether p38 is involved in the development of psoriasis is unclear. OBJECTIVE: We sought to demonstrate that activation of p38 mitogen-activated protein kinase is sufficient to induce psoriatic inflammation in mice and that cutaneous p38 activities are the topical therapeutic targets for psoriasis. METHODS: A p38 activator, anisomycin, was applied daily to murine skin. Transcriptomic analyses were performed to evaluate the similarities of the skin responses to those in human psoriasis and the existing animal model. BIRB796, a small-molecule inhibitor targeting p38 activities, was applied to the murine psoriatic models topically or to human psoriatic skin specimens ex vivo. RESULTS: Topical treatment with anisomycin induced key signatures in psoriasis, such as epidermal thickening, neutrophil infiltration, and gene expression of Il1a, Il1b, Il6, Il24, Cxcl1, Il23a, and Il17a, in treated murine skin. These responses were fully abrogated by topical treatment with BIRB796, and were reduced in IL-17A-deficient mice. Transcriptomic analyses demonstrated the similarities of anisomycin-induced dermatitis to human psoriasis and imiquimod-induced murine psoriatic dermatitis. Furthermore, BIRB796 targeting of p38 activities reduced expression of psoriasis-related genes in both human keratinocytes stimulated with recombinant IL-17A in vitro and psoriatic skin specimens ex vivo. CONCLUSION: Therefore our findings suggest that cutaneous p38 activation can be a key event in patients with psoriasis and a potential topical therapeutic target of a small molecule.

The Alternative NF-κB Pathway in Regulatory T Cell Homeostasis and Suppressive Function.

CD4+Foxp3+ regulatory T cells (Tregs) are essential regulators of immune responses. Perturbation of Treg homeostasis or function can lead to uncontrolled inflammation and autoimmunity. Therefore, understanding the molecular mechanisms involved in Treg biology remains an active area of investigation. It has been shown previously that the NF-κB family of transcription factors, in particular, the canonical pathway subunits, c-Rel and p65, are crucial for the development, maintenance, and function of Tregs. However, the role of the alternative NF-κB pathway components, p100 and RelB, in Treg biology remains unclear. In this article, we show that conditional deletion of the p100 gene, nfkb2, in Tregs, resulted in massive inflammation because of impaired suppressive function of nfkb2-deficient Tregs. Surprisingly, mice lacking RelB in Tregs did not exhibit the same phenotype. Instead, deletion of both relb and nfkb2 rescued the inflammatory phenotype, demonstrating an essential role for p100 as an inhibitor of RelB in Tregs. Our data therefore illustrate a new role for the alternative NF-κB signaling pathway in Tregs that has implications for the understanding of molecular pathways driving tolerance and immunity.

NF-κB c-Rel Is Crucial for the Regulatory T Cell Immune Checkpoint in Cancer.

Regulatory T cells (Tregs) play a pivotal role in the inhibition of anti-tumor immune responses. Understanding the mechanisms governing Treg homeostasis may therefore be important for development of effective tumor immunotherapy. We have recently demonstrated a key role for the canonical nuclear factor κB (NF-κB) subunits, p65 and c-Rel, in Treg identity and function. In this report, we show that NF-κB c-Rel ablation specifically impairs the generation and maintenance of the activated Treg (aTreg) subset, which is known to be enriched at sites of tumors. Using mouse models, we demonstrate that melanoma growth is drastically reduced in mice lacking c-Rel, but not p65, in Tregs. Moreover, chemical inhibition of c-Rel function delayed melanoma growth by impairing aTreg-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. Our studies therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.

An NF-κB Transcription-Factor-Dependent Lineage-Specific Transcriptional Program Promotes Regulatory T Cell Identity and Function.

Both conventional T (Tconv) cells and regulatory T (Treg) cells are activated through ligation of the T cell receptor (TCR) complex, leading to the induction of the transcription factor NF-κB. In Tconv cells, NF-κB regulates expression of genes essential for T cell activation, proliferation, and function. However the role of NF-κB in Treg function remains unclear. We conditionally deleted canonical NF-κB members p65 and c-Rel in developing and mature Treg cells and found they have unique but partially redundant roles. c-Rel was critical for thymic Treg development while p65 was essential for mature Treg identity and maintenance of immune tolerance. Transcriptome and NF-κB p65 binding analyses demonstrated a lineage specific, NF-κB-dependent transcriptional program, enabled by enhanced chromatin accessibility. These dual roles of canonical NF-κB in Tconv and Treg cells highlight the functional plasticity of the NF-κB signaling pathway and underscores the need for more selective strategies to therapeutically target NF-κB.

A Novel Link between Inflammation and Cancer.

Immune checkpoint-blockade treatments targeting PD-1/PD-L1 have revolutionized cancer therapy. Hence, understanding the regulation of PD-L1 expression has major clinical relevance. In this issue of Cancer Cell, Lim et al. report that inflammation-induced and NF-κB-driven expression of deubiquitinating enzyme CSN5 leads to PD-L1 stabilization and immune suppression in tumors.