(588e) Characterization and Modeling of Regulatory T Cell Enrichment through Immunophenotypic Modulation

Introduction: Regulatory T cells (Treg) are a subset of T cells that suppress aberrant activation of self-reactive effector lymphocytes and are widely regarded as the primary mediators of peripheral tolerance. Cell-based therapy using Tregs effectively treats autoimmune diseases in pre-clinical models and clinical trials are underway to evaluate efficacy in humans. However, sourcing Treg from leukapheresis is inefficient as they circulate at a low frequency in the blood and therefore ex vivo expansion is required to enhance their numbers. Current ex vivo Treg expansion techniques rely on labor intensive purification and diminish Treg function. These requirements for expanding and isolating Tregs ex vivo limit their use for cell therapy. We hypothesized that metabolic modulation of a heterogenous T cell population using β-cylodextrin-rapamycin complexes (CRCs) would efficiently promote the preferential expansion of functional Tregs. Here, we characterize the immunomodulatory properties of CRCs and use our results to develop a kinetic model for manufacturing Treg as an immune cell-based therapy.

Materials and Methods: To test the hypothesis, we first characterized the encapsulation of rapamycin (Rapa) in Mono-(6-amino-6-deoxy)-beta-cyclodextrin (βCD) using spectrophotometry and measured the bioactivity of Rapa encapsulated in CRCs, by phenotypic and metabolic modulation of mouse and human T cells. Metabolic modulation was quantified using (i) cell counts to quantify suppression of T cells stimulated with Dynabeads and (ii) flow cytometry was used to quantify the phenotypic and functional changes of splenic CD4 T cells. To further enrich Treg, we tested combinations of CRC with immunomodulatory growth factor transforming growth factor-beta 1 (TGF-β1) and measured changes in phenotype and cytokine profile using flow cytometry. To validate the findings and model for manufacturing human Treg, we repeated assays with human T cells. To explain the observed effects between TGF-β1 and CRCs, we developed and optimized a kinetic growth model describing cell differentiation and expansion and fit key model parameters using experimental data from mouse and human T cell experiments.

Results and Discussion: CRCs enhanced the solubility of Rapa in water 154 -fold and maintained bioactivity of Rapamycin for at least 30 days in solution. CRCs enriched the fraction by 5-fold of Tregs in vitro from both PBMC-isolated human T cells and mouse splenocyte-isolated T cells. CRCs synergize with TGF-β1 to enhance the enrichment of murine Tregs. The combination also enhanced the fraction of human Tregs over single factor treatments. CRCs reduced interferon-gamma (IFN-γ) expression by T cells and increased the fraction of T cells that expressed tumor necrosis factor-α (TNF-α) without IFN-γ. In human T cells, combining CRC and TGF-β1 further enhanced the enrichment of IFN-γ- TNF-α+ T cells, while in mouse T cells the addition of TGF-β1 increased the fraction of IFN-γ+ T cells, both alone and in combination with CRCs. Computational modeling of T cell expansion and differentiation revealed that CRCs mediate Treg enrichment primarily through suppression of effector T cell expansion, while TGF-β1 does so through promoting Treg differentiation. Strikingly, the model accurately predicts the synergistic effect of CRCs and TGF-β1, and predicts the distribution of T cells by phenotypic subsets, depending on the starting cell population.

Conclusions: The findings from our experiments collectively suggest that the βCD encapsulated Rapa enhances functional Treg in vitro. The increased bioavailability of Rapa with this complex is potentially due to strong interactions with βCD, which was confirmed via spectrophotometry. Compared with DMSO, the CRC could serve to (i) significantly improve the solubility of Rapa without inducing T cell toxicity, (ii) shield Rapa from degradation, thereby increasing half-life and uptake by T cells, (iii) synergize with TGF-β1 to permit preferential expansion of Treg. Additionally, CRCs can act to significantly decrease the production of IFN-γ by effector T cells in culture that may cause naïve T cells to acquire a Th1 phenotype. The enhancement in TNF-α expression may act to prime the Tregs and enhance functionality. Furthermore, the kinetic model predicts the synergism between the CRCs and TGF-β1 in modulating the kinetics of Treg expansion in vitro and provides a basis for applying quality and process control to manufacturing Treg for clinical therapy.