Regulatory T cells (Treg) are critical players of immune tolerance that develop in the thymus via two distinct developmental pathways involving CD25+Foxp3- and CD25-Foxp3lo precursors. However, the mechanisms regulating the recently identified Foxp3lo precursor pathway remain unclear. Here, we find that the membrane-bound lymphotoxin α1β2 (LTα1β2) heterocomplex is upregulated during Treg development upon TCR/CD28 and IL-2 stimulation. We show that Lta expression limits the maturational development of Treg from Foxp3lo precursors by regulating their proliferation, survival, and metabolic profile. Transgenic reporter mice and transcriptomic analyses further reveal that medullary thymic epithelial cells (mTEC) constitute an unexpected source of IL-4. We demonstrate that LTα1β2-lymphotoxin β receptor-mediated interactions with mTEC limit Treg development by down-regulating IL-4 expression in mTEC. Collectively, our findings identify the lymphotoxin axis as the first inhibitory checkpoint of thymic Treg development that fine-tunes the Foxp3lo Treg precursor pathway by limiting IL-4 availability.

Eosinophils are involved in host protection against multicellular organisms. However, their recruitment to the mesenteric lymph node (mLN) during type 2 immunity is understudied. Our results demonstrate that eosinophil association with lymphoid stromal niches constructed by fibroblastic reticular cells (FRCs) and lymphatic endothelial cells is diminished in mice selectively lacking interleukin (IL)-4Rα or lymphotoxin-β (LTβ) expression on B cells. Furthermore, eosinophil survival, activation, and enhanced Il1rl1 receptor expression are driven by stromal cell and B cell dialogue. The ligation of lymphotoxin-β receptor (LTβR) on FRCs improves eosinophil survival and significantly augments IL-33 expression and eosinophil homing to the mLN, thus confirming the significance of lymphotoxin signaling for granulocyte recruitment. Eosinophil-deficient ΔdblGATA-1 mice show diminished mLN expansion, reduced interfollicular region (IFR) alarmin expression, and delayed helminth clearance, elucidating their importance in type 2 immunity. These findings provide insight into dialogue between stromal cells and B cells, which govern mLN eosinophilia, and the relevance of these mechanisms during type 2 immunity.

Solid organ transplantation remains the life-saving treatment for end-stage organ failure, but chronic rejection remains a major obstacle to long-term allograft outcomes and has not improved substantially. Tertiary lymphoid organs (TLOs) are ectopic lymphoid structures that form under conditions of chronic inflammation, and evidence from human transplantation suggests that TLOs regularly form in allografts undergoing chronic rejection. In this study, we utilized a mouse renal transplantation model and manipulation of the lymphotoxin αβ/lymphotoxin β receptor (LTαβ/LTβR) pathway, which is essential for TLO formation, to define the role of TLOs in transplantation. We showed that intragraft TLOs are sufficient to activate the alloimmune response and mediate graft rejection in a model where the only lymphoid organs are TLOs in the allograft. When transplanted to recipients with a normal set of secondary lymphoid organs, the presence of graft TLOs or LTα overexpression accelerated rejection. If the LTβR pathway was disrupted in the donor graft, TLO formation was abrogated, and graft survival was prolonged. Intravital microscopy of renal TLOs demonstrated that local T and B cell activation in TLOs is similar to that observed in secondary lymphoid organs. In summary, we demonstrated that immune activation in TLOs contributes to local immune responses, leading to earlier allograft failure. TLOs and the LTαβ/LTβR pathway are therefore prime targets to limit local immune responses and prevent allograft rejection. These findings are applicable to other diseases, such as autoimmune diseases or tumors, where either limiting or boosting local immune responses is beneficial and improves disease outcomes.

The intricate immune mechanisms governing mucosal healing following intestinal damage induced by cytotoxic drugs remain poorly understood. The goal of this study was to investigate the role of lymphotoxin beta receptor (LTβR) signaling in chemotherapy-induced intestinal damage. LTβR deficient mice exhibited heightened body weight loss, exacerbated intestinal pathology, increased proinflammatory cytokine expression, reduced IL-22 expression, and proliferation of intestinal epithelial cells following methotrexate (MTX) treatment. Furthermore, LTβR-/-IL-22-/- mice succumbed to MTX treatment, suggesting that LTβR- and IL-22- dependent pathways jointly promote mucosal repair. Although both LTβR ligands LIGHT and LTβ were upregulated in the intestine early after MTX treatment, LIGHT-/- mice, but not LTβ-/- mice, displayed exacerbated disease. Further, we revealed the critical role of T cells in mucosal repair as T cell-deficient mice failed to upregulate intestinal LIGHT expression and exhibited increased body weight loss and intestinal pathology. Analysis of mice with conditional inactivation of LTβR revealed that LTβR signaling in intestinal epithelial cells, but not in Lgr5+ intestinal stem cells, macrophages or dendritic cells was critical for mucosal repair. Furthermore, inactivation of the non-canonical NF-kB pathway member RelB in intestinal epithelial cells promoted MTX-induced disease. Based on these results, we propose a model wherein LIGHT produced by T cells activates LTβR-RelB signaling in intestinal epithelial cells to facilitate mucosal repair following chemotherapy treatment.

Psoriasis (PS) and atopic dermatitis (AD) are common skin inflammatory diseases characterized by hyper-responsive keratinocytes. Although, some cytokines have been suggested to be specific for each disease, other cytokines might be central to both diseases. Here, we show that Tumor necrosis factor superfamily member 14 (TNFSF14), known as LIGHT, is required for experimental PS, similar to its requirement in experimental AD. Mice devoid of LIGHT, or deletion of either of its receptors, lymphotoxin β receptor (LTβR) and herpesvirus entry mediator (HVEM), in keratinocytes, were protected from developing imiquimod-induced psoriatic features, including epidermal thickening and hyperplasia, and expression of PS-related genes. Correspondingly, in single cell RNA-seq analysis of PS patient biopsies, LTβR transcripts were found strongly expressed with HVEM in keratinocytes, and LIGHT was upregulated in T cells. Similar transcript expression profiles were also seen in AD biopsies, and LTβR deletion in keratinocytes also protected mice from allergen-induced AD features. Moreover, in vitro, LIGHT upregulated a broad spectrum of genes in human keratinocytes that are clinical features of both PS and AD skin lesions. Our data suggest that agents blocking LIGHT activity might be useful for therapeutic intervention in PS as well as in AD.

Lymphotoxin beta receptor (LTBR) is a positive T cell proliferation regulator gene. It is closely associated with the tumor immune microenvironment. However, its role in cancer and immunotherapy is unclear. Firstly, the expression level and prognostic value of LTBR were analyzed. Secondly, the expression of LTBR in clinical stages, immune subtypes, and molecular subtypes was analyzed. The correlation between LTBR and immune regulatory genes, immune checkpoint genes, and RNA modification genes was then analyzed. Correlations between LTBR and immune cells, scores, cancer-related functional status, tumor stemness index, mismatch repair (MMR) genes, and DNA methyltransferase were also analyzed. In addition, we analyzed the role of LTBR in DNA methylation, mutational status, tumor mutation burden (TMB), and microsatellite instability (MSI). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were used to explore the role of LTBR in pan-cancer. Finally, the drugs associated with LTBR were analyzed. The expression of LTBR was confirmed using quantitative real-time PCR and Western blot. LTBR is significantly overexpressed in most cancers and is associated with low patient survival. In addition, LTBR expression was strongly correlated with immune cells, score, cancer-related functional status, tumor stemness index, MMR genes, DNA methyltransferase, DNA methylation, mutational status, TMB, and MSI. Enrichment analysis revealed that LTBR was associated with apoptosis, necroptosis, and immune-related pathways. Finally, multiple drugs targeting LTBR were identified. LTBR is overexpressed in several tumors and is associated with a poor prognosis. It is related to immune-related genes and immune cell infiltration.

OBJECTIVE: HCC is the most common primary liver tumor, with an increasing incidence worldwide. HCC is a heterogeneous malignancy and usually develops in a chronically injured liver. The NF-κB signaling network consists of a canonical and a noncanonical branch. Activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of noncanonical NF-κB and its downstream effectors is not established.
RESULTS: Four human HCC cohorts (total n = 1462) and 4 mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro , NF-κB signaling, proliferation, and cell death were measured, proving a pro-proliferative role of v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) activated by means of NF-κB-inducing kinase. In vivo , lymphotoxin beta was identified as the predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico , RELB activity and RELB-directed transcriptomics were validated on the The Cancer Genome Atlas HCC cohort using inferred protein activity and Gene Set Enrichment Analysis. In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to v-rel avian reticuloendotheliosis viral oncogene homolog A, nuclear enrichment of noncanonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence.
CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent, and cross-species consistent activation of a lymphotoxin beta/LTβR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation.

Despite over a decade of clinical trials combining inhibition of emerging checkpoints with a PD-1/L1 inhibitor backbone, meaningful survival benefits have not been shown in PD-1/L1 inhibitor resistant or refractory solid tumours, particularly tumours dominated by a myelosuppressive microenvironment. Achieving durable anti-tumour immunity will therefore likely require combination of adaptive and innate immune stimulation, myeloid repolarisation, enhanced APC activation and antigen processing/presentation, lifting of the CD47/SIRPα (Cluster of Differentiation 47/signal regulatory protein alpha) \'do not eat me\' signal, provision of an apoptotic \'pro-eat me\' or \'find me\' signal, and blockade of immune checkpoints. The importance of effectively targeting mLILRB2 and SIRPAyeloid cells to achieve improved response rates has recently been emphasised, given myeloid cells are abundant in the tumour microenvironment of most solid tumours. TNFSF14, or LIGHT, is a tumour necrosis superfamily ligand with a broad range of adaptive and innate immune activities, including (1) myeloid cell activation through Lymphotoxin Beta Receptor (LTβR), (2) T/NK (T cell and natural killer cell) induced anti-tumour immune activity through Herpes virus entry mediator (HVEM), (3) potentiation of proinflammatory cytokine/chemokine secretion through LTβR on tumour stromal cells, (4) direct induction of tumour cell apoptosis in vitro, and (5) the reorganisation of lymphatic tissue architecture, including within the tumour microenvironment (TME), by promoting high endothelial venule (HEV) formation and induction of tertiary lymphoid structures. LTBR (Lymphotoxin beta receptor) and HVEM rank highly amongst a range of costimulatory receptors in solid tumours, which raises interest in considering how LIGHT-mediated costimulation may be distinct from a growing list of immunotherapy targets which have failed to provide survival benefit as monotherapy or in combination with PD-1 inhibitors, particularly in the checkpoint acquired resistant setting.

Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Using mouse models of ALL and AML, here we show that leukemic cells turn off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin α1β2 and activate lymphotoxin beta receptor (LTβR) signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin α1β2 expression in leukemic cells. Genetic or pharmacological disruption of LTβR signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage.

Estrogen is a disease-modifying factor in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) via estrogen receptor alpha (ERα). However, the mechanisms by which ERα signaling contributes to changes in disease pathogenesis have not been completely elucidated. Here, we demonstrate that ERα deletion in dendritic cells (DCs) of mice induces severe neurodegeneration in the central nervous system in a mouse EAE model and resistance to interferon beta (IFNβ), a first-line MS treatment. Estrogen synthesized by extragonadal sources is crucial for controlling disease phenotypes. Mechanistically, activated ERα directly interacts with TRAF3, a TLR4 downstream signaling molecule, to degrade TRAF3 via ubiquitination, resulting in reduced IRF3 nuclear translocation and transcription of membrane lymphotoxin (mLT) and IFNβ components. Diminished ERα signaling in DCs generates neurotoxic effector CD4+ T cells via mLT-lymphotoxin beta receptor (LTβR) signaling. Lymphotoxin beta receptor antagonist abolished EAE disease symptoms in the DC-specific ERα-deficient mice. These findings indicate that estrogen derived from extragonadal sources, such as lymph nodes, controls TRAF3-mediated cytokine production in DCs to modulate the EAE disease phenotype.