Professor Columbia University, New York, United States
Introduction:: In immunotherapy workflows, ex vivo steps of engineering immune cells provide a valuable window to prime T cells for cancer eradication. Current T cell expansion protocols rely on stiff polystyrene beads (Young’s modulus in the range of GPa) to present T cell receptor (TCR) activating and CD28 receptor costimulatory signals. However, this protocol requires T cells to sense signals on supraphysiological substrate stiffness, to which T cells have been shown to be mechanosensitive with poor proliferative outcomes (1). Additionally, solely CD28 costimulation largely understimulates T cells that have lost CD28 expression due to age or chronic illness, and CD28- effector memory T cells (2). In this study, I present alternatives to canonical T cell stimulation methods, leveraging both innate T cell mechanical sensitivity and alternative costimulation programs to improve expansion in largely untapped cell populations. The studies here highlight the costimulation of CD2 as a strongly activating and pro-proliferative alternative to CD28 costimulation, and how this synergizes with the use of soft, kilopascal range substrates over stiff substrates. The results of this study inform the design of alternative T cell expansion materials and costimulatory molecules to improve T cell immunotherapy outcomes.
Materials and Methods:: Polydimethylsiloxane elastomers were prepared from a mixture of Sylgard 184 and Sylgard 527 at varying ratios to confer activation substrates in the range of 300kPa to 2MPa. PDMS was precoated with αCD3 and αCD28 or CD58 antibody for T cell activation via the T cell receptor and costimulation, respectively. Human CD4/8+ T cells were isolated from Leukopak leukocyte isolates from healthy, adult human donors, and further depleted of CD28+ cells where noted. Cells were seeded onto activating PDMS or αCD3/28 Dynabeads for 72 hours, and maintained in long-term culture with IL-2 supplemented media. T cells were monitored over the duration of long term culture with hemocytometer cell count, CFSE proliferation dye, and surface marker analysis via flow cytometry. Short term activation was measured with cell surface capture assays for cytokine IL-2 and granule release marker LAMP2.
Results, Conclusions, and Discussions:: Our findings show that T cell activation, doublings and population shift during expansion were influenced individually by substrate stiffness and costimulatory molecule. CD2 costimulation increased the maximum doublings achieved over CD28 costimulation, and favored expansion of effector memory T cells, a valuable T cell subset in immunotherapy (Fig 1). CD2 costimulated cells demonstrate greater LAMP2 mobilization at initial hours following activation, indicating more efficient granule release, but ameliorated IL-2 secretion. In particular, CD28-CD8+ cells showed amplified LAMP2 mobilization when costimulated via CD2, which may provide a mechanistic explanation for their favored expansion. These results demonstrate that CD2 costimulation has a strong pro-proliferative population-wide effect, and in particular improves activation in valuable CD28- subsets, targeting these populations that are traditionally untapped by αCD3/28 activation protocols.
T cells also demonstrated improved expansion on softer PDMS, although interestingly this does not correlate with functional activation measurements directly following stimulation. When CD2 or CD28 costimulation is presented in solution instead of PDMS-bound alongside TCR signals, T cells perform suboptimal activation. Although the TCR is known to be a primary mechanosensitive receptor in T cells (1), these results demonstrate similar substrate stiffness sensitivity in costimulatory receptors, although to a lesser extent. Overall, these results confirm a mechanosensitive function in T cells, suggesting that initial engagement with a mechanically favorable activating substrate during the first 72 hours can initiate a more proliferative program in long term culture. Variation of PDMS stiffness was shown to capture a range of T cell expansion profiles and shift expanded T cell populations toward anti-cancer functions and other phenotypes. Thus, the utilization of tunable-stiffness PDMS substrates and CD2 costimulation during ex vivo expansion are potential modifications to improve functionality of T cells in the immunotherapy workflow.
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References (Optional): : 1. Yuan, D. J., Shi, L., & Kam, L. C. (2021). Biomaterials, 273, 120797.
2. Leitner et al. (2015). The Journal of Immunology. 195(2):477-87, 1401917.
