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Mutations underlying disease in tuberous sclerosis complex (TSC) give rise to tumors with biallelic mutations in TSC1 or TSC2 and hyperactive mammalian target of rapamycin complex 1 (mTORC1). Benign tumors might exhibit de novo expression of immunogens, targetable by immunotherapy. As tumors may rely on ganglioside D3 (GD3) expression for mTORC1 activation and growth, we compared GD3 expression in tissues from patients with TSC and controls. GD3 was overexpressed in affected tissues from patients with TSC and also in aging Tsc2+/- mice. As GD3 overexpression was not accompanied by marked natural immune responses to the target molecule, we performed preclinical studies with GD3 chimeric antigen receptor (CAR) T cells. Polyfunctional CAR T cells were cytotoxic toward GD3-overexpressing targets. In mice challenged with Tsc2-/- tumor cells, CAR T cells substantially and durably reduced the tumor burden, correlating with increased T cell infiltration. We also treated aged Tsc2+/- heterozygous (>60 weeks) mice that carry spontaneous Tsc2-/- tumors with GD3 CAR or untransduced T cells and evaluated them at endpoint. Following CAR T cell treatment, the majority of mice were tumor free while all control animals carried tumors. The outcomes demonstrate a strong treatment effect and suggest that targeting GD3 can be successful in TSC.

Previous studies have demonstrated the presence of microbial DNA in the fetal environment. However, it remains unclear whether this DNA represents viable bacteria and how it relates to the maternal microbiota across body sites. We studied the microbiota of human and mouse dyads to understand these relationships, localize bacteria in the fetus, and demonstrate bacterial viability. In human preterm and full-term mother-infant dyads at the time of cesarean delivery, the oral cavity and meconium of newborn infants born as early as 24 weeks of gestation contained a microbiota that was predicted to originate from in utero sources, including the placenta. Using operative deliveries of pregnant mice under highly controlled, sterile conditions in the laboratory, composition, visualization, and viability of bacteria in the in utero compartment and fetal intestine were demonstrated by 16S rRNA gene sequencing, fluorescence in situ hybridization, and bacterial culture. The composition and predicted source of the fetal gut microbiota shifted between mid- and late gestation. Cultivatable bacteria in the fetal intestine were found during mid-gestation but not late gestation. Our results demonstrate a dynamic, viable mammalian fetal microbiota during in utero development.