Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity.

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept.

Lower Airway Dysbiosis Augments Lung Inflammatory Injury in Mild-to-Moderate Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.

Episodic Aspiration with Oral Commensals Induces a MyD88-dependent, Pulmonary T-Helper Cell Type 17 Response that Mitigates Susceptibility to Streptococcus pneumoniae.

Rationale: Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype.Objectives: In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model.Methods: Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds.Measurements and Main Results: 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with S. pneumoniae led to a decrease in hosts' susceptibility to this pathogen.Conclusions: Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to S. pneumoniae. Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.