Nanowires To Regulate Cellular Fate Trajectories of T Cells

Introduction: T cells are central to the host's defense against infection and cancer. However, immune functioning declines with age, rendering the elderly more prone to cancer, chronic inflammatory disorders, infections, and vaccination failure. With aging and associated systemic low-grade chronic inflammation, referred to as “inflammaging”, the naïve T cell subset shrinks along with the accumulation of highly differentiated memory cells. Additionally, aged CD+8 T cells exhibit impaired proliferative responses and cytokine production, reducing their ability to combat infections and tumors effectively. These changes collectively contribute to the increased susceptibility to infections and reduced vaccine efficacy observed in the elderly. Therefore, restoring T cells' functioning is crucial for developing interventions to enhance immune function in older adults. A major challenge in the field is that 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. This study builds upon our recently reported nanowire technology (Yee Mon et al, Nature Nanotechnology, 2024) to deliver miRNAs as differentiation rheostat to CD8+ T cells before T cell receptor stimulation and regulating T cells' activation threshold, controlling the balance between rapid effector cells and long-lived memory cells.

Materials and

Methods: Features of nano-dot arrays were first transferred to silicon wafers through DUV photolithography. Next, silicon nanowires were fabricated using several rounds of inductively coupled plasma (ICP) etching and deep reactive ion (DRIE) 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-mimic and/or microRNA-antagomir are incubated on PEI-functionalized nanowires for 6 hours to allow the adsorption of microRNA onto the nanowire surface. After the coating process, Pan CD8+ T cells from young ( < 35 years) or aged (>65 years) individuals are seeded on nanowires followed by gentle centrifugation (~50g). T cells were seeded for 2 days prior to removal and Dynabeads activation to allow microRNA mediated modulation of T cell dynamics. Viability, delivery efficacy, and phenotypes were then assessed using flow cytometry.

Results, Conclusions, and Discussions: Results and

Discussion:
To evaluate miRNA delivery to primary human CD8+ T cells from young donors, we compared nanowires with conventional methods: PEI complexation, Lipofectamine, lentivirus, and nucleofection. Nanowires achieved over 90% transfection efficiency, significantly outperforming other state-of-the-art methods that failed to effectively deliver miRNA to T cells in naïve state without prior activation (Fig 1.B). Both young and aged CD8+ T cells demonstrated similar delivery efficacy (>90%) using nanowires (Fig 1.C). However, analysis of transcriptional regulators and post-miRNA delivery revealed a distinct response in aged CD8+ T cells, with a lesser reduction in Eomes expression compared to younger donors (Fig 1.D).
We evaluated the functional responses of nanowire-mediated microRNA delivery to naïve young and aged T cells by activating them after miRNA delivery. As a baseline, activation of T cells in 2D cultures (96-well plate) with TCR-antibodies resulted in significant differences in the effector CD8+ T cell subpopulations between young and aged donors. Aged CD8+ T cells typically exhibited a poorer response compared to young T cells. However, specific microRNA delivery was shown to significantly improve their function with delivery of miR-150 and not miR-29. miR-150 enhanced the activation phenotype of aged T cells through increasing effector T cell differentiation (Fig 1.E), upregulating CD69 expression (Fig 1.F), and increasing and IFN-Y production (Fig 1.G), bringing aged CD8+ T cell response closer to that of young counterpart.

Conclusions:
PEI-functionalized nanowires effectively deliver miRNAs to both young and aged CD8+ T cells, modulating T-cell fitness by altering phenotypic and transcriptional dynamics. Delivering specific miRNAs to aged CD8+ T cells shows promise in addressing age-associated T cell deficiencies, and opens up the possibility of combining multiple miRNAs could further enhance T cell responses, as recently demonstrated in our published work with young T cells (Yee Mon et al, Nature Nanotechnology, 2024).

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