We herein demonstrate the first 96-well plate platform to screen effects of micro- and nanotopographies on cell growth and proliferation. Existing high-throughput platforms test a limited number of factors and are not fully compatible with multiple types of testing and assays. This platform is compatible with high-throughput liquid handling, high-resolution imaging, and all multiwell plate-based instrumentation. We use the platform to screen for topographies and drug-topography combinations that have short- and long-term effects on T cell activation and proliferation. We coated nanofabricated “trench-grid” surfaces with anti-CD3 and anti-CD28 antibodies to activate T cells and assayed for interleukin 2 (IL-2) cytokine production. IL-2 secretion was enhanced at 200 nm trench width and >2.3 μm grating pitch; however, the secretion was suppressed at 100 nm width and <0.5 μm pitch. The enhancement on 200 nm grid trench was further amplified with the addition of blebbistatin to reduce contractility. The 200 nm grid pattern was found to triple the number of T cells in long-term expansion, a result with direct clinical applicability in adoptive immunotherapy.

Practical deployment of cellular therapies requires effective platforms for producing clinically relevant numbers of high-quality cells. This report introduces a materials-based approach to improving activation and expansion of T cells, which are rapidly emerging as an agent for treating cancer and a range of other diseases. Electrospinning is used to create a mesh of poly(ε-caprolactone) fibers, which is used to present activating ligands to CD3 and CD28, which activate T cells for expansion. Incorporation of poly(dimethyl siloxane) elastomer into the fibers reduces substrate rigidity and enhances expansion of mixed populations of human CD4+ and CD8+ T cells. Intriguingly, this platform also rescues expansion of T cells isolated from CLL patients, which often show limited responsiveness and other features resembling exhaustion. By simplifying the process of cell expansion, compared to current bead-based platforms, and improving T cell expansion, the system introduced here may accelerate development of cellular immunotherapy.

The immune system maintains a balance between protection and tolerance. Regulatory T cells (Tregs) function as a vital tolerance mechanism in the immune system to suppress effector immune cells. Additionally, Tregs can be utilized as a form of immunotherapy for autoimmune disorders. As T cells have previously been shown to exhibit sensitivity to the rigidity of an activating substrate upon activation via IL-2 secretion, we herein explore the previously unknown effect of substrate rigidity on the induction of Tregs from conventional naïve mouse CD4+ T cells. Substrates with modulatable rigidities ranging from a hundred kilopascals to a few megapascals were fabricated via poly(dimethylsiloxane). We found that there was a significant increase in Treg induction at lower substrate rigidities (i.e., E ~ 100 kPa) compared to higher rigidity levels (i.e., E ~ 3 MPa). To confirm that this significant difference in induction rate was truly related to T cell mechanosensing, we administered compound Y-27632 to inhibit myosin contractility. In the presence of Y-27632, the myosin-based contractility was disrupted and, as a result, the difference in Treg induction caused by the substrate rigidity was abrogated. This study demonstrates that mechanosensing is involved in Treg induction and raises questions about the underlying molecular mechanisms involved in this process.

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