This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells, T cells, and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75{\%} of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7, the ocular T cell population increases to 50{\%} of CD45 + cells, leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible ({\textless} 1{\%} of CD45 + cells), the ocular B cell population increases to {\textgreater} 4{\%} by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models.

Immunotherapy is innovating clinical cancer management. Nevertheless, only a small fraction of patient's benefit from current immunotherapies. To improve clinical management of cancer immunotherapy, it is critical to develop strategies for response monitoring and prediction. In this study, we describe inducible T-cell costimulator (ICOS) as a conserved mediator of immune response across multiple therapy strategies. ICOS expression was evaluated by flow cytometry, 89Zr-DFO-ICOS mAb PET/CT imaging was performed on Lewis lung cancer models treated with different immunotherapy strategies, and the change in tumor volume was used as a read-out for therapeutic response. ImmunoPET imaging of ICOS enabled sensitive and specific detection of activated T cells and early benchmarking of immune response. A STING (stimulator of interferon genes) agonist was identified as a promising therapeutic approach in this manner. The STING agonist generated significantly stronger immune responses as measured by ICOS ImmunoPET and delayed tumor growth compared with programmed death-1 checkpoint blockade. More importantly, ICOS ImmunoPET enabled early and robust prediction of therapeutic response across multiple treatment regimens. These data show that ICOS is an indicator of T-cell-mediated immune response and suggests ICOS ImmunoPET as a promising strategy for monitoring, comparing, and predicting immunotherapy success in cancer. SIGNIFICANCE: ICOS ImmunoPET is a promising strategy to noninvasively predict and monitor immunotherapy response.See related commentary by Choyke, p. 2975.

Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However, the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain1-3. Despite progress in our understanding of the development and structure of the meningeal lymphatic system, the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here, using a mouse model of glioblastoma, we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system, resulting in uncontrolled tumour growth. However, ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes, migration of CD8 T cells into the tumour, rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore, transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours, and suggest a new therapeutic approach to treat brain tumours.

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes, including the release of proteases, phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation, or NETosis, is a specific and highly efficient process, which is induced by a variety of stimuli leading to expulsion of DNA, proteases and antimicrobial peptides to the extracellular space. However, uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here, we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly, neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF), a Panx1 channel inhibitor, decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus, we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target.

Exosomes are nanosized membranous vesicles secreted by a variety of cells. Due to their unique and pharmacologically important properties, cell-derived exosome nanoparticles have drawn significant interest for drug development. By genetically modifying exosomes with two distinct types of surface-displayed monoclonal antibodies, we have developed an exosome platform termed synthetic multivalent antibodies retargeted exosome (SMART-Exo) for controlling cellular immunity. Here, we apply this approach to human epidermal growth factor receptor 2 (HER2)-expressing breast cancer by engineering exosomes through genetic display of both anti-human CD3 and anti-human HER2 antibodies, resulting in SMART-Exos dually targeting T cell CD3 and breast cancer-associated HER2 receptors. By redirecting and activating cytotoxic T cells toward attacking HER2-expressing breast cancer cells, the designed SMART-Exos exhibited highly potent and specific anti-tumor activity both in vitro and in vivo. This work demonstrates preclinical feasibility of utilizing endogenous exosomes for targeted breast cancer immunotherapy and the SMART-Exos as a broadly applicable platform technology for the development of next-generation immuno-nanomedicines.

TFF1, a secreted protein, plays an essential role in keeping the integrity of gastric mucosa and its barrier function. Loss of TFF1 expression in the TFF1-knockout (KO) mouse leads to a pro-inflammatory phenotype with a cascade of gastric lesions that include low-grade dysplasia, high-grade dysplasia, and adenocarcinomas. In this study, we demonstrate nuclear localization of p-STATY705, with significant overexpression of several STAT3 target genes in gastric glands from the TFF1-KO mice. We also show frequent loss of TFF1 with nuclear localization of STAT3 in human gastric cancers. The reconstitution of TFF1 protein in human gastric cancer cells and 3D gastric glands organoids from TFF1-KO mice abrogates IL6-induced nuclear p-STAT3Y705 expression. Reconstitution of TFF1 inhibits IL6-induced STAT3 transcription activity, suppressing expression of its target genes. TFF1 blocks IL6R$\alpha$-GP130 complex formation through interfering with binding of IL6 to its receptor IL6R$\alpha$. These findings demonstrate a functional role of TFF1 in suppressing gastric tumorigenesis by impeding the IL6-STAT3 pro-inflammatory signaling axis.