Lucy H. Maynard

BACKGROUND: Understanding the mechanisms of cardiomyocyte development is critical for fulfilling the potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Although myocyte development is known to depend on internal and external mechanical cues, further investigation is required to understand the contributions of different signals and how they are integrated together to generate an adult cardiomyocyte. Here, we address this gap by examining the role of calcium-activated contractility in sarcomere formation and maturation and its influence on the iPSC-CM response to nanopatterns. METHODS: We generated iPSCs with homozygous D65A cTnC (cardiac troponin C) substitutions. This engineered cTnC cannot bind to calcium at site II, resulting in tropomyosin blocking strong myosin binding to the thin filament and inhibiting sarcomere contraction. The iPSCs were differentiated into cardiomyocytes and matured in culture over 60 days. Cells were characterized via imaging, metabolic assays, and calcium transient analysis. Proteomes were examined using mass spectrometry throughout differentiation and maturation. We also replated partially matured cardiomyocytes onto nanopatterned surfaces to investigate how external mechanical signals affect maturation in contractile versus noncontractile cells. RESULTS: Surprisingly, we found that sarcomeres formed in the D65A cTnC cardiomyocytes, though these sarcomeres were underdeveloped and disorganized. The D65A cardiomyocytes also exhibited significant proteomic maturation defects and abnormal calcium transients. Replating the noncontractile cardiomyocytes onto nanopatterns improved several structural and proteomic maturation metrics. In contrast, wildtype maturation did not benefit from the introduction of nanopatterns. CONCLUSIONS: Calcium-activated contractility is dispensable for sarcomerogenesis but critical for cardiomyocyte maturation. In noncontractile, D65A cTnC cardiomyocytes, nanopatterns enhanced maturation, suggesting that external mechanical cues may partially compensate for defective contractility. However, nanopatterns did not facilitate wildtype maturation, suggesting that maturity may reduce the efficacy of nanopatterns. In addition to these novel findings, these mass spectrometry data sets cataloging iPSC-CM maturation represent a useful resource for the cardiovascular research community.

Chimeric antigen receptor T (CAR T)-cell therapy has demonstrated curative potential in B-cell malignancies; yet, translating this success to chronic infections such as HIV remains a major challenge. In people living with HIV who are receiving suppressive antiretroviral therapy (ART), low-antigen levels limit CAR T-cell expansion and persistence. We previously reported data from a pilot study suggesting that HIV-targeted CD4CAR T cells could overcome this barrier through exogenous antigen supplementation, leading to robust in vivo expansion. Here we sought to comprehensively confirm and expand on those findings. We tested a broad array of strategies to enhance CD4CAR T-cell efficacy, including CRISPR-Cas9-mediated gene editing of immune checkpoint and HIV-associated genes, single and pooled competitive infusions of engineered CAR T cells, distinct CAR constructs incorporating either CD28 or 4-1BB costimulatory domains, and exogenous antigen boosting. We also developed highly sensitive droplet digital polymerase chain reaction assays to quantify CAR T-cell frequency and to corroborate the flow cytometry–based quantification of CD4CAR T-cell expansion. We evaluated these new approaches across multiple nonhuman primate (NHP) models of HIV, including both simian immunodeficiency virus– and simian-human immunodeficiency virus–infected, ART-suppressed NHPs. Although CD4CAR T-cell products exhibited antigen-specific proliferation and cytotoxicity ex vivo, they failed to expand, persist, or control viremia in vivo. We were also unable to confirm the previously observed CD4CAR T-cell expansions from our earlier studies, which have been retracted. Together, these data highlight the need for alternative strategies to potentiate anti-HIV CD4CAR T cells in the immunocompetent setting.

Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of B-cell malignancies; however, >60% of patients relapse within 1 year, often due to insufficient CAR-T persistence. Although mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted, to our knowledge, the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. After ex vivo characterization, we administered pooled autologous CAR-T arrays to 3 NHPs and tracked CAR abundance longitudinally using a novel digital droplet polymerase chain reaction assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral CAR-T cells and significantly outperforming canonical CD28- and 4-1BB–based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study end point, unlike other CARs, which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T activity.

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized treatment for B-cell malignancies, yet over 60% of patients relapse within one year, often due to insufficient CAR-T persistence. While mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. Following ex vivo characterization, we administered pooled autologous CAR-T arrays to three NHPs and tracked CAR abundance longitudinally using a novel digital droplet PCR assay. Ex vivo , CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo , MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study endpoint, unlike other CARs which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T cell activity. Key points: We developed the first pooled CAR-T screening platform in an immunocompetent nonhuman primate model to directly compare CAR designs. We identified MyD88-CD40 costimulatory domain as vastly superior to conventional domains in proliferation, trafficking and persistence.