Ben Youngblood

B7‑H3 is a promising immunotherapeutic target for pediatric brain tumors, yet the durability of B7‑H3-CAR T cell responses remains limited. Here, for the first time, we systematically evaluated whether knockout (KO) of TET2, DNMT3A, or Regnase‑1 (REG-1) enhances the antitumor activity of B7‑H3–CAR T cells. Across multiple G3MB orthotopic xenograft models spanning low to high antigen density, second‑generation B7‑H3–CAR T cells improved survival but failed to achieve curative responses, with efficacy influenced by donor‑specific variability and antigen density. While DNMT3A-KO enhanced CAR T cell persistence in vitro, neither TET2 nor DNMT3A-KO improved tumor control in vivo. In contrast, REG‑1-KO significantly augmented antitumor activity, inducing complete tumor eradication in a subset of mice and extending survival across both potent and poor‑performing donors. Single-cell transcriptomic and functional analyses show increased cytokine production, coordinated CD4–CD8 communication, and reduced exhaustion-associated programs, supporting a model in which effective responses depend on CD4-mediated immune orchestration rather than persistence or cytotoxicity alone. We also demonstrate CAR specificity: while DNMT3A and Regnase-1 disruption both enhance EphA2-targeted CAR T cells, only Regnase-1 disruption improves B7-H3 CAR T-cell efficacy, underscoring the need for context-specific engineering. Our findings challenge the prevailing assumption that persistence-enhancing strategies are central to successful CAR T cell therapies. Instead, they demonstrate that distinct engineering approaches should be considered for the specific disease and CAR target.

Maternal immune activation (MIA) during pregnancy induces autism spectrum disorder (ASD)-like phenotypes in offspring. While maternal IL-17 signaling has been implicated in prenatal brain alterations, the postnatal mechanisms sustaining this phenotype remain unknown. Here, we show that MIA in mice imprints a persistent postnatal insufficiency of regulatory T cells (Tregs) in offspring, characterized by altered DNA methylation, transcriptional dysregulation, and impaired in vivo suppressive function. This Treg insufficiency drives sustained meningeal inflammation marked by infiltration of neutrophils and IL-17-producing immune cells, resulting in region-specific alterations in brain structure and functional connectivity. Functional MRI revealed hypoconnectivity in sensory networks together with hyperconnectivity in regions involved in emotional regulation, reward processing, and interhemispheric cognitive and motor integration. Postnatal stimulation of endogenous Tregs with low-dose interleukin-2 (IL-2) in MIA offspring expanded meningeal Tregs, eliminated IL-17-producing cells, resolved meningeal inflammation, normalized brain connectivity, and reversed MIA-induced ASD-like behaviors in adulthood. Extending these findings to humans, two children with ASD associated with documented maternal immune activation received treatment with low-dose IL-2 for six months. Treatment expanded circulating Tregs and was associated with marked and progressive clinical improvement across all assessed domains - social communication, socialization, interpersonal relationships, adaptive coping, and play. To our knowledge, this represents an unprecedented pharmacological improvement of core ASD symptoms. Together, our results redefine MIA-ASD as a chronic neuroimmune disorder amenable to therapeutic intervention, and establish Treg stimulation as a clinically actionable strategy with implications extending beyond autism to other MIA-associated neuropsychiatric disorders.

Grade 3 or Higher Toxicities or Toxicities Observed in 10% or More Patients – Excluding Those Defined as Unrelated or Unlikely Related to Protocol Treatment.

A. Gating strategy applied on PBMCs samples evaluated by flow cytometry. CD8+ T cell memory was defined based on the expression of CD45RA and CCR7 (CD197). Cell surface phenotype (PD-1, CD39, and CD69) and intracellular activation (Ki-67) markers were quantified on CD8+ effector T cell (TEFF) based on indicated gates. B, CD8+ naïve and memory T cells in long-and short-survivors quantified before and after the treatment. C, Representative flow cytometry plots showing CD8+ T cells activation in PBMC samples from a short survivor. PD-1+ Ki-67+ expression on CD8+ T cells compared before and after the treatment.

Representativeness of Study Participants

List of Patients Who Experienced Immune-related Adverse Events AEs.

Specific genes sequenced in bone marrow mononuclear cells.

A& B. Frequency (A) and intensity (B) of PD-1+ CD8+ T cells by gender) before (PRE), after second treatment cycle (C2D1 or C2D8; EARLY), and after third or fourth treatment cycle (C4D1, C4D8 or C5D1; LATE). CD8+ T cell expressing CD39 (C) and CD69 (D). F: females, M: males