Poster CC15 - Programming Naïve Primary T Cells for Enhanced Immunotherapy

Introduction: Naive CD8+ T cells, upon encountering an infection, undergo clonal expansion and differentiate into effector and memory T cells, playing a critical role in pathogen clearance and long-term immunity. The adoptive transfer of CD8+ T cells, especially those with an enhanced effector phenotype, is a promising immunotherapy strategy. However, variable response rates in adoptive T cell therapy are often linked to increased T cell exhaustion and suboptimal T cell phenotypes. Recent studies indicate that effector CD8+ T cells derived from naive subsets outperform those from memory subsets in adoptive immunotherapy. Current ex vivo T cell engineering methods involve pre-activating T cells with CD3/CD28 antibodies and cytokines, resulting in the loss of the naive state. This pre-activation limits T cells' ability to expand, persist, and effectively treat diseases, while also inducing a differentiation program prone to exhaustion. To address this, it is crucial to program naive CD8+ T cells without pre-activation to induce more durable effector responses. Silicon nanowires (NWs) represent a novel delivery system that can administer bio-cargos directly into cells without activation, potentially modulating CD8+ T cell differentiation and achieving therapeutic responses in vivo without pre-activation. This study, based on our recent work in Nature Nanotechnology (1), explores the use of cationic-polymer-functionalized nanowires to pre-program naive CD8+ T cells by delivering microRNAs (miRNAs) without pre-activation, aiming to enhance their therapeutic potential by maintaining their naive state and improving their fitness, phenotypic profiles, and effector functions.

Materials and

Methods: Silicon nanowires were fabricated using several rounds of inductively coupled plasma etching and deep reactive ion etching to achieve vertical arrays with a height of ~3.5 μm and tip diameters of ~20 nm. Nanowires were then functionalized with polyethyleneimine (PEI) for improved nucleic acid adsorption. Suspensions of microRNA-29-antagomir and microRNA-130-mimic are incubated on PEI-functionalized nanowires, followed by seeding and gentle centrifugation (~50g) of primary naive mouse and human CD8+ T cells on nanowires for microRNA delivery. Viability, delivery efficacy, and effector-associated phenotypes were then assessed using flow cytometry. C57BL/6 gBT-I mice were used for in vivo experiments. Nanowire-modified mouse naïve CD8+ T cells were transferred into recipient mice, and their proliferation and cytokine secretion were measured post-infection. Effector and memory T cell phenotypes were analyzed in spleen and lung tissues following influenza infection.

Results, Conclusions, and Discussions: Results and

Discussion: The nanowire is found to be able to effectively penetrate membranes of T cells (Fig 1.B) and achieve over 95% microRNA delivery efficacy to inactivated, naïve CD8+ T cells (Fig 1.C) while the commercial lentiviral system can only achieve ~10%. Programming of naïve T cells using microRNA delivered through nanowires significantly increased the proliferation capacity of T cells where a 50-fold increase in cell count is found on Day 8 post Dynabeads activation, compared to only 10-fold in 2D cultures (Fig 1.D). In addition, the programming of CD8+ T cells using nanowire-delivered co-delivery of miRNAs for suppression and overexpression of miR-29 and miR-130 resulted in the modulation of T cell fitness by altering activation-associated phenotypes of T cells (Fig 1.E). Furthermore, the in vivo adoptive transfer of pre-programmed naïve CD8+ T cells provided enhanced immune protection against influenza challenge through directing T cells less toward precursor central memory T cells (1) and more toward short-lived effector cells (SLEC) or precursor effector memory T cells (pre-Tem) (Fig 1.F).

Conclusions: PEI-functionalized nanowires are found to effectively deliver microRNAs to naïve CD8+ T cells and subsequently modulate T-cell fitness by altering the T-cell proliferation, phenotypic, and transcriptional dynamics both in vitro and in vivo. This technology can potentially explore the distinct ways that T-cell effector heterogeneity is regulated as short-lived versus memory precursor and how this mixed pool can be altered to avoid exhaustion. The nanowire’s ability to toggle the effector and memory states of naïve T cells also opens new avenues of immunological research in neonatal, adult, and aged T cells, as well as generating CAR-T cells from naïve T cells.

References: 1. Yee Mon, et al. Nature Nanotechnology, 2024

Acknowledgements (Optional):