Jonathan P. Schneck

Hypoxia-inducible factor 1 (HIF-1) orchestrates the transcriptional regulation of thousands of genes involved in breast cancer (BC) progression. Here, we identified protein phosphatase 2A (PP2A) methylesterase 1 ( PPME1 ) as a critical HIF-1 target gene that drives oncogenic signaling under hypoxic conditions. In BC cells, HIF-1–dependent PPME1 expression caused inhibition of the PP2A catalytic subunit (PP2Ac), thereby diminishing PP2A activity, which led to AKT activation, phosphorylation of β-catenin, and its nuclear translocation. Nuclear β-catenin cooperates with HIF-1 to promote BC stem cell specification by activating transcription of the NANOG and KLF4 genes, which encode pluripotency factors, and to drive immune evasion by activating transcription of VEGFA , which recruits and polarizes immunosuppressive tumor-associated macrophages and ISG20 , which represses STAT1/IRF1-dependent expression of CXCL10 , thereby impairing CD8 ⁺ T cell recruitment. In vivo, PPME1 knockdown altered the tumor immune microenvironment, enhanced antitumor immunity, and synergized with anti–CTLA-4 immunotherapy to enable complete tumor eradication. These findings establish PPME1 as a critical regulator linking hypoxia signaling, stemness, and immune evasion and highlight its potential as a BC therapeutic target in combination with immune checkpoint blockade.

Messenger RNA lipid nanoparticles are promising vaccine carriers, yet their movement through the body after intramuscular injection and the resulting tissue-specific immune effects remain poorly defined. Here we show that lipid nanoparticle trafficking from the injection site into systemic organs is a key determinant of localized immunity, particularly the formation of tissue-resident cytotoxic T cells. We compared multiple nanoparticle formulations that differed in lipid composition, including ionizable lipids and helper lipids commonly used in clinical vaccines. These formulations exhibited distinct trafficking behaviours: some remained largely at the injection site, whereas others entered the bloodstream and preferentially accumulated in organs such as the liver or lungs. Organ-specific accumulation resulted in corresponding differences in antigen expression and local cytotoxic T cell responses. Nanoparticles that more efficiently reached the liver elicited stronger liver-resident T cell immunity and improved control of liver tumours in mice. Our findings demonstrate that tuning lipid composition can direct the systemic movement and organ targeting of messenger RNA nanoparticles, enabling the design of vaccines that promote durable, tissue-specific protection for diseases where local immunity is essential.

While chimeric antigen receptor (CAR) T cell therapies have demonstrated therapeutic efficacy against B cell malignancies, widespread implementation of these therapies is hindered by a cumbersome, ex vivo manufacturing process. Delivery of CAR-encoding messenger RNA (mRNA) to endogenous T cells can generate these therapeutic cells in vivo and streamline this manufacturing workflow. To accomplish this, T cell–activating ligands were conjugated to a biodegradable polymeric mRNA nanoparticle to form T cell–targeted particles. By conjugating multiple activating ligands, T cell transfection and stimulation in vitro was increased, and greater T cell transfection and selectivity in vivo was achieved compared to an untargeted particle. These nanoparticles can flexibly encapsulate mRNA cargos and were used to deliver anti-CD19 CAR mRNA in vivo, enabling depletion of 95% of B cells in the peripheral blood and 50% depletion of splenic B cells in healthy mice. These findings regarding nanoparticle tropism and their potential therapeutic efficacy highlight the importance of this nonviral, polymeric platform to address key limitations associated with current CAR T practices.

CD4⁺ T cells are critical central regulators of adaptive immunity and have emerged as promising candidates for engineered cellular immunotherapies. While current nonviral approaches enable pan-T cell targeting, strategies for selective genetic modulation of antigen-specific CD4⁺ T cells remain undeveloped. Here, we present a proof-of-concept platform employing lipid nanoparticles (LNPs) decorated with cognate peptide-MHC class II (pMHC-II) ligands (MHC-II LNPs) for antigen-specific mRNA delivery. Ex vivo, MHC-II LNPs achieved highly precise transfection of naïve antigen-specific CD4⁺ T cells without overt activation or toxicity, demonstrating comparable efficiency as pMHC-I based targeting, despite the lower affinity of pMHC-II:TCR interactions. In vivo administration in TCR-transgenic mice resulted in selective mRNA delivery to cognate CD4⁺ T cells in the spleen, liver, and blood. Furthermore, the delivery of CD19-directed CAR mRNA produced functional antigen-specific CD4⁺ CAR-T cells that mediated peripheral B cell depletion. These findings establish that pMHC-II ligands can be exploited for selective, nonviral genetic programming of CD4⁺ T cells. This strategy provides a foundational approach for precision engineering of antigen-specific CD4⁺ T subsets with potential applications in cancer, autoimmunity, and infectious disease.

In response to growing clinical demands for more targeted and effective immunotherapies to treat cancer, biomaterial-based strategies have emerged as powerful tools for locally regulating immune responses. Among these, hydrogels, a class of biocompatible and tunable polymeric networks, are increasingly being leveraged for their high versatility and adaptability for creating tailored immune environments. By enabling controlled delivery of immune cues and direct cellular engineering, hydrogels utilized in vivo can precisely regulate both innate and adaptive immune responses while minimizing systemic toxicity. In this review, we outline essential hydrogel design features necessary for in vivo functionality including injectability, degradation kinetics, and immune-specific functionalization. Building on these principles, we explore how hydrogels have been employed to enhance T cell activation and dendritic cell maturation and guide macrophage reprogramming. Beyond cellular modulation, we further examine the use of hydrogels for cytokine and immunoregulatory agent delivery, tumor microenvironment remodeling, and the creation of tertiary-like lymphoid structures. Finally, we review recently completed and ongoing clinical trials of hydrogels in the cancer immunotherapy space. Together, these insights underscore the growing potential of in vivo hydrogel systems as immuno-interactive platforms capable of reshaping immune responses across diverse disease contexts.

Hydrogel materials have emerged as versatile platforms for various biomedical applications. Notably, the engineered nanofiber-hydrogel composite (NHC) has proven effective in mimicking the soft tissue extracellular matrix, facilitating substantial recruitment of host immune cells and the formation of a local immunostimulatory microenvironment. Leveraging this feature, here we report an mRNA lipid nanoparticle (LNP)-incorporated NHC microgel matrix, termed LiNx, by incorporating LNPs loaded with mRNA encoding tumour antigens. Harnessing the high transfection efficiency of LNPs in antigen-presenting cells, LiNx demonstrates substantial levels of immune cell recruitment, antigen expression and presentation, and cellular interaction. These attributes collectively create an immunostimulating microenvironment and yield a potent immune response with a single dose at a level comparable to the conventional three-dose LNP immunization protocol. Further investigation reveals that the LiNx generates not only high levels of Th1 and Th2 responses, but also a distinct Type 17 T helper cell response critical for bolstering antitumour efficacy. Our findings elucidate the mechanism underlying LiNx’s role in potentiating antigen-specific immune responses, presenting a strategy for cancer immunotherapy.

The ability to characterize and separate cells based on their surface marker expression profiles using flow cytometry revolutionized our understanding of the immune system at the level of single cells. However, the use of surface protein quantification to functionally evaluate T cells following stimulation fails to capture important outcomes of T cell activation, most prominently, cytokine secretion. This key limitation hinders elucidation of the mechanistic correlates of T cell function, which in turn limits our ability to design effective cell therapies for diseases such as cancer and autoimmune disorders. Here, we employ a single-cell microparticle-based technology (termed nanovials) to assess both the cell surface expression profiles and the protein secretion behavior of stimulated T cells. Using protein-conjugated nanovials, we demonstrated the capacity to stimulate ovalbumin-specific primary mouse CD8⁺ T cells, both nonspecifically by coating anti-CD3 and anti-CD28 antibodies on the particle, and specifically by coating ovalbumin peptide-loaded major histocompatibility complexes and anti-CD28 antibody on the particle. We further showed that nanovials could simultaneously capture secretions of multiple cytokines from single cells and that viable T cells contained within the nanovials could be sorted based on cytokine secretion levels. We applied this platform to characterize the phenotypic profile of resting T cells that would go on to secrete the interferon gamma (IFNγ) cytokine following activation on nanovials by staining cells for surface marker expression prior to on-particle stimulation. Overall, the nanovial platform and assays developed herein help elucidate the functional properties of T cells, which will inform the engineering of future cell therapies.

The mRNA lipid nanoparticles (LNPs) represent a new generation of vaccine carriers designed to elicit potent immune responses against infectious diseases and cancer. Despite the clinical success and rapid advancements in mRNA LNP technologies, the trafficking patterns of LNPs after intramuscular (i.m.) administration and the subsequent tissue-specific immunological effects have not been systematically characterized. Here, we report that trafficking of mRNA LNPs to different organs following i.m. injection is crucial for the induction of tissue-specific immunity beyond systemic immune response, particularly in tissue-resident CD8 ⁺ T cell generation, which is important for localized defense. By fine-tuning the composition of mRNA LNPs, trafficking patterns to systemic organs can be modulated, which can alter the resulting tissue-specific immune response. Formulations with a greater ability to enter the bloodstream can preferentially localize and transfect cells in specific organs like the liver, elicit stronger tissue-specific CD8 ⁺ T cell immune responses, and achieve enhanced efficacy in a liver tumor model. These findings highlight the potential to tailor mRNA LNP compositions to modulate trafficking following i.m. injection, thereby providing novel strategies for designing tissue-specific vaccines. Such strategies are particularly valuable for organ-specific diseases like cancer and infectious diseases, where tissue targeting and long-lasting immunity are essential for therapeutic success.