Modulating T cell phenotype and expansion via alternating co-stimulatory molecule ratio on a functional hydrogel

Introduction: Adoptive T cell therapy relies on ex vivo expansion of therapeutic T cells. During this ex vivo expansion, T cells can often get exhausted and lose expression of clinically favorable phenotypes. To address this concern, our lab at Johns Hopkins has developed a biomimetic hydrogel functionalized with anti-CD3, anti-CD28, and IL-2. This hydrogel has shown significant T cell expansion and activation in vitro and in vivo. Additionally, we have shown that T cell co-stimulation with multiple ligands, in addition to anti-CD28, has a significant effect on modulating cell phenotype and function. However, the relative importance of each co-stimulatory molecule in driving T cell phenotype and function remains unclear. To address this gap, different ratios of co-stimulatory molecules were conjugated to the hydrogel. We endeavor to investigate the effects of varying the ratio of these co-stimulatory molecules on T cell memory subsets and functional phenotypes, including how the synergistic impact of the combination can be enhanced.

Materials and

Methods: Hydrogel microparticles were first synthesized by mixing pre-thiolated hyaluronic acid with pre-thiolated antibodies. All hydrogels included aCD3 and aCD28. Additionally, another signal 2 molecule (S2a) was conjugated to the gel at varying ratios of CD28:S2a of 1:1, 1:2, 1:4, 2:1, and 4:1, along with soluble IL-2. The microparticles were then repeatedly passed back and forth through a steel mesh device with a screen pore size of 150 μm to form the functionalized particles, which are schematically illustrated in Figure 1A. The functionalized hydrogel microparticles were plated with CD8+ T cells isolated from a B6 mouse and cultured for seven days. The cell's activation or memory phenotype, surface markers, and fold expansion were evaluated on day 7 using flow cytometry.

Results, Conclusions, and Discussions: The impact of combining anti-CD28 and another signal 2 (S2a) molecule at different ratios was initially explored. The greatest expansion, approximately 120-fold, occurred with a 4:1 ratio of aCD28 to S2a, while the lowest, around 30-fold, was observed with a 1:4 ratio (Figure 1B). Increasing the amount of aCD28 enhanced expansion and favored an effector T cell phenotype, as shown in Figure 1C. Conversely, higher levels of S2a promoted central memory (CM) T cells, as evidenced by an increasing CM/Effector T cell ratio detailed in Figure 1C. Flow cytometry plots, especially the CD62L versus CD44 plot, highlighted how shifts from a 4:1 to a 1:4 ratio altered the distribution of effector T cells across different quadrants (Figure 1D). These results were then validated by exploring the effect of combining CD28 with another co-stimulatory molecule, S2b. Similar trends in functional memory profiles and expansion were observed, confirming our understanding of the relative importance of these different co-stimulatory molecules. This data shows how T cell phenotypes can be modified by combining co-stimulatory molecules in varying amounts in a specific hydrogel platform. Simultaneous co-stimulation of aCD28 with additional S2 molecules in varying amounts allows for the adjustment of memory and effector features while maintaining strong proliferation and performance in a controlled setting. Additional experiments will be carried out to explore how these combinations, especially with varying quantities, operate in a unique manner and result in distinct phenotypes by monitoring mTOR signal activity.

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