Modulation of T Cell Phenotype Through Presentation of Multiple Co-Stimulatory Ligands on a Functional Hydrogel

Introduction:: T cell activation is dependent on stimulation with three signals between the T cell and the antigen presenting cell (APC): signal 1, interaction between the T cell receptor and cognate peptide-MHC (pMHC), signal 2, co-stimulation mediated by several different receptor-ligand interactions, and signal 3, cytokine-mediated stimulation. In the context of adoptive cell therapies (ACT), T cells are often stimulated by biomimetic platforms that present these signals. Recently, our lab at Johns Hopkins has developed a T cell-stimulating hydrogel comprised of hyaluronic acid (HA) microparticles that are functionalized with either anti-CD3 or a pMHC molecule (S1), anti-CD28 (S2), and bound or exogenous IL2 (S3) [1]. Although this hydrogel has demonstrated robust antigen-specific T cell expansion and activation in vitro and in vivo, there is potential for further modulation of T cell phenotype. The first iteration of this platform used anti-CD28 as signal 2, as CD28 is the canonical co-stimulatory molecule. However, the T cell-APC interaction is highly complex, and providing co-stimulation through additional signal 2 molecules, such as CD27, 41BB, OX40, and CD40L, may lead to alterations of the resulting T cell phenotype [2]. By functionalizing this HA hydrogel with multiple different co-stimulatory molecules at varying ratios, we hope to better recapitulate endogenous T cell stimulation, which in turn enables modulation of cell expansion, activation, function, and memory phenotype. This ability to modulate phenotype and activity with different signal 2s can improve ACT manufacturing by enabling better control of T cell function.


Materials and Methods:: T cells were stimulated with HA microparticles conjugated with aCD3 (S1) and varying signal 2 molecules, either alone or in combination at a 50:50 ratio. Signal 2 molecules investigated here include anti-CD28 (aCD28), aCD27, a41BB, aOX40, and aCD40L. Hydrogel microparticles were added to CD8+ T cells isolated from a B6 mouse and cultured for 7 days with exogenous IL2 (S3). On D7, cell count, surface markers, and cytokine release were analyzed to evaluate expansion, activation/memory phenotype, and function, respectively.

Hydrogels microparticles were first formulated by mixing aCD3 and the different signal 2 molecules at varying ratios. All functional molecules were DTT-modified to enable binding to the hydrogel. The immunologically relevant signals were then complexed with hyaluronic acid and a crosslinker to form a hydrogel. Finally, this hydrogel was passed through a steel mesh with a pore size of 150 um to form functionalized hydrogel microparticles. An overview of this fabrication process can be seen in Figure 1a.


Results, Conclusions, and Discussions:: First, the effect of stimulating T cells in vitro with hydrogels expressing aCD3 and either aCD28, aCD27, a41BB, aOX40, and aCD40L as the signal 2 was examined. All conditions achieved robust expansion (50-100x). Interestingly, the untreated (no hydrogel) condition also demonstrated ~40x expansion, most likely because exogenous IL2 was added. Analysis of memory phenotype and activation revealed that stimulation with aCD28 produced a higher ratio of effector to memory cells and higher CD25 (activation) expression than any other S2. Analysis of cytokine release did not reveal any significant differences.

This experiment highlights the efficacy of alternative signal 2s to expand T cells. However, given that aCD28 produced the highest effector/activation phenotype and is the canonical co-stimulatory signal, it was selected for further characterization in the context of presenting multiple signals with the hydrogel.

Stimulation of T cells with both aCD28 and another signal 2 in a 50:50 ratio was examined, with the amount of total signal 2 protein kept the same as in previous experiments. All conditions achieved robust expansion (50-100x) over 7 days (Figure 1b). Co-stimulation with CD28+CD27 and CD28+OX40 produced a higher ratio of effector to memory cells (~2), while CD28+41BB produced more memory cells (~0.5 ratio) and CD28+CD40L produced an approximately a 1:1 ratio (Figure 1c). Analysis of activation phenotype (Figure 1d) showed that CD28+CD27 and CD28+OX40 produced higher CD25 (activation) and CD127 (differentiation/development) than other conditions. Analysis of functionality (Figure 1e) confirmed that all groups produced IL2, TNFa, and IFNy, but did not reveal any differences.

This data demonstrates the potential to achieve expansion/modulation of T cell phenotype with alternative co-stimulation in a functionalized hydrogel platform. Moreover, co-stimulation with both aCD28 and another signal 2 allows for modulation of memory and activation phenotypes while maintaining robust expansion and functionality in vitro. Ongoing experiments are being conducted to study this co-stimulatory hydrogel in vivo. While ACT has conventionally relied on manufacture of effector CD8+ cells, recent literature suggests that memory T cell subsets are also therapeutically relevant [3]. As such, the expansion platform we have developed provides a framework for modulating cell phenotype towards different desired subsets.






Acknowledgements (Optional): :

References (Optional): : [1] Hickey JW, Dong Y, Chung JW, et al. Engineering an artificial t‐cell stimulating matrix for immunotherapy. Adv Mater. 2019;31(23):1807359. doi:10.1002/adma.201807359

[2] Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013;13(4):227-242. doi:10.1038/nri3405

[3] Gattinoni L, Klebanoff CA, Restifo NP. Paths to stemness: building the ultimate antitumour T cell. Nat Rev Cancer. 2012;12(10):671-684. doi:10.1038/nrc3322