Of mice and lymphoid aggregates: modeling tertiary lymphoid structures in cancer.

Tertiary lymphoid structures (TLS) are lymph node-like aggregates that can form in association with chronic inflammation or cancer. Mature TLS are organized into B and T cell zones, and are not encapsulated but include all cell types necessary for eliciting an adaptive immune response. TLS have been observed in various cancer types and are generally associated with a positive prognosis as well as increased sensitivity to cancer immunotherapy. However, a comprehensive understanding of the roles of TLS in eliciting anti-tumor immunity as well as the mechanisms involved in their formation and function is still lacking. Further studies in orthotopic, immunocompetent cancer models are necessary to evaluate the influence of TLS on cancer therapies, and to develop new treatments that promote their formation in cancer. Here, we review key insights obtained from functional murine studies, discuss appropriate models that can be used to study cancer-associated TLS, and suggest guidelines on how to identify TLS and distinguish them from other antigen-presenting niches.

Screening the medicine for malaria venture's Pandemic Response Box to identify novel inhibitors of Candida albicans and Candida auris biofilm formation.

Candida spp. are opportunistic yeasts capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. We screened the Pandemic Response Box containing 400 diverse drug-like molecules active against bacteria, viruses or fungi, for inhibitors of Candida albicans and Candida auris biofilm formation. Initial hits were identified based on the demonstration of >70% inhibitory activity. Dose-response assays were used to confirm the antifungal activity of initial hits and establish their potency. The spectrum of antifungal activity of the leading compounds was determined against a panel of medically important fungi, and the in vivo activity of the leading repositionable agent was evaluated in murine models of C. albicans and C. auris systemic candidiasis. The primary screening identified 20 hit compounds, and their antifungal activity and potency against C. albicans and C. auris were validated using dose-response measurements. From these experiments, the rapalog everolimus, emerged as the leading repositionable candidate. Everolimus displayed potent antifungal activity against different Candida spp., but more moderate levels of activity against filamentous fungi. Treatment with everolimus increased survival of mice infected with C. albicans, but not those with C. auris. The screening of the Pandemic Response Box resulted in the identification of several drugs with novel antifungal activity, with everolimus emerging as the main repositionable candidate. Further in vitro and in vivo studies are needed to confirm its potential therapeutic use.

Tailoring vascular phenotype through AAV therapy promotes anti-tumor immunity in glioma.

Glioblastomas are aggressive brain tumors that are largely immunotherapy resistant. This is associated with immunosuppression and a dysfunctional tumor vasculature, which hinder T cell infiltration. LIGHT/TNFSF14 can induce high endothelial venules (HEVs) and tertiary lymphoid structures (TLS), suggesting that its therapeutic expression could promote T cell recruitment. Here, we use a brain endothelial cell-targeted adeno-associated viral (AAV) vector to express LIGHT in the glioma vasculature (AAV-LIGHT). We found that systemic AAV-LIGHT treatment induces tumor-associated HEVs and T cell-rich TLS, prolonging survival in αPD-1-resistant murine glioma. AAV-LIGHT treatment reduces T cell exhaustion and promotes TCF1+CD8+ stem-like T cells, which reside in TLS and intratumoral antigen-presenting niches. Tumor regression upon AAV-LIGHT therapy correlates with tumor-specific cytotoxic/memory T cell responses. Our work reveals that altering vascular phenotype through vessel-targeted expression of LIGHT promotes efficient anti-tumor T cell responses and prolongs survival in glioma. These findings have broader implications for treatment of other immunotherapy-resistant cancers.

DNA sensor-associated type I interferon signaling is increased in ulcerative colitis and induces JAK-dependent inflammatory cell death in colonic organoids.

DNA sensor pathways can initiate inflammasome, cell death, and type I interferon (IFN) signaling in immune-mediated inflammatory diseases (IMIDs), including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC) by analyzing the expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control (n = 31), inactive UC (n = 31), active UC (n = 33), and a UC single-cell RNA-Seq dataset. The effects of type I IFN (IFN-ß), IFN-γ, and TNF-α on gene expression, cytokine production, and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLR family pyrin domain-containing 3 (NLRP3), caspase, or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1ß/IL-18/CXCL10. The expression of DNA sensor pathway genes-PYHIN family members [absent in melanoma 2 (AIM2), IFI16, myeloid cell nuclear differentiation antigen (MNDA), and pyrin and HIN domain family member 1 (PYHIN1)- as well as Z-DNA-binding protein 1 (ZBP1), cyclic GMP-AMP synthase (cGAS), and DDX41 was increased in active UC and expressed in a cell type-restricted pattern. Inflammasome genes (CASP1, IL1B, and IL18), type I IFN inducers [stimulator of interferon response cGAMP interactor 1 (STING), TBK1, and IRF3), IFNB1, and type I IFN biomarker genes (OAS2, IFIT2, and MX2) were also increased in active UC. Cotreatment of organoids with IFN-ß or IFN-γ in combination with TNFα increased expression of IFI16, ZBP1, CASP1, cGAS, and STING induced cell death and IL-1ß/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC.NEW & NOTEWORTHY This study found that patients with active UC have significantly increased colonic gene expression of cytosolic DNA sensor, inflammasome, STING, and type I IFN signaling pathways. The type I IFN, IFN-ß, in combination with TNF-α induced JAK-dependent but NLRP3 and inflammasome-independent inflammatory cell death of colonic organoids. This novel inflammatory cell death phenotype is relevant to UC immunopathology and may partially explain the efficacy of the JAKinibs tofacitinib and upadacitinib in patients with UC.

Tertiary Lymphoid Structures in the Central Nervous System: Implications for Glioblastoma.

Glioblastoma is the most common and aggressive brain tumor, which is uniformly lethal due to its extreme invasiveness and the absence of curative therapies. Immune checkpoint inhibitors have not yet proven efficacious for glioblastoma patients, due in part to the low prevalence of tumor-reactive T cells within the tumor microenvironment. The priming of tumor antigen-directed T cells in the cervical lymph nodes is complicated by the shortage of dendritic cells and lack of appropriate lymphatic vessels within the brain parenchyma. However, recent data suggest that naive T cells may also be primed within brain tumor-associated tertiary lymphoid structures. Here, we review the current understanding of the formation of these structures within the central nervous system, and hypothesize that promotion of tertiary lymphoid structures could enhance priming of tumor antigen-targeted T cells and sensitize glioblastomas to cancer immunotherapy.

Agonistic CD40 therapy induces tertiary lymphoid structures but impairs responses to checkpoint blockade in glioma.

Gliomas are brain tumors characterized by an immunosuppressive microenvironment. Immunostimulatory agonistic CD40 antibodies (αCD40) are in clinical development for solid tumors, but are yet to be evaluated for glioma. Here, we demonstrate that systemic delivery of αCD40 in preclinical glioma models induces the formation of tertiary lymphoid structures (TLS) in proximity of meningeal tissue. In treatment-naïve glioma patients, the presence of TLS correlates with increased T cell infiltration. However, systemic delivery of αCD40 induces hypofunctional T cells and impairs the response to immune checkpoint inhibitors in pre-clinical glioma models. This is associated with a systemic induction of suppressive CD11b+ B cells post-αCD40 treatment, which accumulate in the tumor microenvironment. Our work unveils the pleiotropic effects of αCD40 therapy in glioma and reveals that immunotherapies can modulate TLS formation in the brain, opening up for future opportunities to regulate the immune response.

Platelet-Specific PDGFB Ablation Impairs Tumor Vessel Integrity and Promotes Metastasis.

Platelet-derived growth factor B (PDGFB) plays a crucial role in recruitment of PDGF receptor ß-positive pericytes to blood vessels. The endothelium is an essential source of PDGFB in this process. Platelets constitute a major reservoir of PDGFB and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. Here, we show that tumor vascular function, as well as pericyte coverage is significantly impaired in mice with conditional knockout of PDGFB in platelets. A lack of PDGFB in platelets led to enhanced hypoxia and epithelial-to-mesenchymal transition in the primary tumors, elevated levels of circulating tumor cells, and increased spontaneous metastasis to the liver or lungs in two mouse models. These findings establish a previously unknown role for platelet-derived PDGFB, whereby it promotes and maintains vascular integrity in the tumor microenvironment by contributing to the recruitment of pericytes. SIGNIFICANCE: Conditional knockout of PDGFB in platelets demonstrates its previously unknown role in the maintenance of tumor vascular integrity and host protection against metastasis.