New hydrogels for T cell immunotherapy

Cancer appears from the own cells of an organism and creates a tumor microenvironment with immune evasion and immunosuppression signals capable of avoiding the immune response [1]. Immunotherapy is a medical strategy applied to the treatment of cancer based on harnessing the immune system of patients. With the development of new technologies, T cells can be genetically modified by engineering their T-cell receptors (TCRs) or making them express chimeric antigen receptors (CARs) to recognize and eliminate cancer cells more effectively, allowing their use in adoptive cell therapy (ACT) [2]. These therapies are technically challenging, as modified T cells are difficult on manufacture and the efficiency of the ex vivo cell expansion to achieve relevant clinical doses is a major limiting factor to translate the therapy to clinics [3].

Ex vivo T cell expansion is usually achieved in suspension using artificial antigen presenting cells (APCs) in the form of artificial beads decorated with α-CD3 and α-CD28 antibodies, which activates the TCR complex and the costimulatory receptor CD28, thus mimicking the immunological synapse. In our body, secondary lymphoid organs (SLOs) and, specially, lymph nodes (LNs), constitute the places where APCs activate T cells through its TCR. Within the LNs the homing signals are provided by the cytokine CCL21, which is immobilized in the tissue and interacts with naïve T cells and APCs, thus enhancing T cell proliferation and promoting random migration [4, 5]. 

T cell expansion in a CCL21-loaded PEG-Hep hydrogel during the adoptive cell therapy process. Pérez Del Río E, et al. CCL21-loaded 3D hydrogels for T cell expansion and differentiation. Biomaterials. 2020 Nov; 259:120313. doi: 10.1016/j.biomaterials.2020.120313.

How can we mimic the environment of immunological tissues to promote T cell expansion and survival?

One of the main strategies to recreate the natural environment of cells is based on the use of hydrogels, which are three-dimensional (3D) natural or synthetic structures often used to mimic the extracellular matrix (ECM) [6] that offer more realistic conditions than hard planar substrates, also improving standard cell culture techniques [7]. Poly (ethylene glycol) (PEG) hydrogels have been extensively studied because of their interesting physicochemical properties. They are biologically inert due to their hydrophilic character, which prevents unspecific attachment to proteins, but can also be easily engineered to present different functionalities such as cell adhesive motifs [8], and can be manufactured under mild and cyto compatible conditions with high reproducibility [9]. 

Recently, in a research paper published in Biomaterials [10], Judith Guasch et al. developed and further characterized a PEG heparin (PEG-Hep) hydrogel loaded with CCL21 in order to imitate the conditions of the ECM of the LNs with the objective to increase primary human T cell proliferation and tune their differentiation for ACT. They also combined PEG-Hep-CCL21 3D structures with artificial APCs in the form of Dynabeads, improving the state-of-the-art suspension systems. 

Normalized replication, expansion and proliferation indexes of CD4+ T cells stimulated with Dynabeads 5 days after seeding in unloaded PEG-Hep hydrogels. Pérez Del Río E, et al. CCL21-loaded 3D hydrogels for T cell expansion and differentiation. Biomaterials. 2020 Nov; 259:120313. doi: 10.1016/j.biomaterials.2020.120313.

The effect of immobilizing CCL21 compared to having it in solution on CD4+ T cell cultures was first confirmed using planar surfaces to reduce the complexity of the system given by the 3D structure. Different concentrations of CCL21 were seeded in suspension and fixed on planar gold (Au) surfaces and CD4+ T cell proliferation was evaluated by flow cytometry through CFSE staining by calculating three indexes: expansion and replication indexes, which determine the fold-expansion of the overall culture and that of the responding cells, respectively, and the proliferation index, which is the average number of divisions that all responding cells have undergone since the beginning of the cell culture. Authors found that there were no significant differences among the different CCL21 concentrations in suspension, whereas immobilizing CCL21 resulted in a significant increment of the proliferation and expansion indexes, and a slight increase of the replication index.

Next, 3%wt PEG-Hep hydrogels with or without CCL21 were used to culture primary human CD4+ T cells activated with Dynabeads, which enabled cell infiltration and resembled a truly 3D culture. Cell proliferation was assessed through the expansion, replication and proliferation indexes. PEG-Hep hydrogels without CCL21 showed statistically significant increases of the three indexes compared with the positive controls, being the most significant the replication index, which indicates that the responding cells that get activated in the synthetic hydrogels proliferate more than the activated cells in suspension. When the hydrogels were loaded with CCL21, a 29% increase of the replication index on day 6 was obtained in comparison with the positive control. Proliferation and expansion indexes showed less pronounced increases. 

Furthermore, to determine the phenotype of T cells after proliferation, differentiation assays were performed 5 days after seeding and stimulation by identifying naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4+ T cells by flow cytometry. Unstimulated cells (negative control) mainly showed a Tn phenotype (53%), whereas the Tcm and Tem phonotypes were found un smaller percentages (12% and 32%, respectively). After stimulation, the median value of Tn cells decreased to 4% in suspension (positive control), 14% in CCL21 unloaded and 11% in CCL21 loaded hydrogels. Consequently, the Tcm and Tem increased in comparison with the negative control. The median values for the Tcm phenotype were lower when using hydrogels, whereas the median values for the Tem phenotype were higher compared to the positive control. These results suggest that the cytokine-loaded hydrogels promoted an increased proportion of effector cells, pointing to the use of PEG-Hep hydrogels to modify the resulting phenotype of T cells and the use of different chemical stimuli as a promising strategy to achieve diverse differentiation pathways. 

The fabrication of an artificial matrix capable of mimicking the ECM of SLOs is expected to be a powerful tool to promote immune cell expansion as well as preserve and augment T cell cytotoxicity towards cancer cells. They can also be useful to overcome the current limitations during the culture and expansion of T cells ex vivo for cellular immunotherapies, as 3D PEG Hep hydrogels showed interconnected porous structures with adequate rheological properties to mimic the physical structure of the SLOs, thus favoring CD4+ T cell migration through the matrix, their interaction with other cells and their proliferation and survival.

As 3D PEG hydrogels can be further functionalized with other biomolecules such as adhesion molecules, cytokines or chemokines, they constitute a powerful tool for the generation of artificial interaction models for immune system cells, allowing researchers to further study their biology and function. 

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