Abstract
Adoptive transfer of regulatory T cells (Treg) is a promising new therapeutic option to treat detrimental inflammatory conditions after transplantation and during autoimmune disease. To reach sufficient cell yield for treatment, ex vivo isolated autologous or allogenic Tregs need to be expanded extensively in vitro during manufacturing of the Treg product. However, repetitive cycles of restimulation and prolonged culture have been shown to impact T cell phenotypes, functionality and fitness. It is therefore critical to scrutinize the molecular changes which occur during T cell product generation, and reexamine current manufacturing practices. We performed genome-wide DNA methylation profiling of cells throughout the manufacturing process of a polyclonal Treg product that has proven safety and hints of therapeutic efficacy in kidney transplant patients. We found progressive DNA methylation changes over the duration of culture, which were donor-independent and reproducible between manufacturing runs. Differentially methylated regions (DMRs) in the final products were significantly enriched at promoters and enhancers of genes implicated in T cell activation. Additionally, significant hypomethylation did also occur in promoters of genes implicated in functional exhaustion in conventional T cells, some of which, however, have been reported to strengthen immunosuppressive effector function in Tregs. At the same time, a set of reported Treg-specific demethylated regions increased methylation levels with culture, indicating a possible destabilization of Treg identity during manufacturing, which was independent of the purity of the starting material. Together, our results indicate that the repetitive TCR-mediated stimulation lead to epigenetic changes that might impact functionality of Treg products in multiple ways, by possibly shifting to an effector Treg phenotype with enhanced functional activity or by risking destabilization of Treg identity and impaired TCR activation. Our analyses also illustrate the value of epigenetic profiling for the evaluation of T cell product manufacturing pipelines, which might open new avenues for the improvement of current adoptive Treg therapies with relevance for conventional effector T cell products.
Highlights
T Lymphocytes are one of the most promising effectors for adoptive cellular therapy (ACT) by facilitating targetspecific immune interventions, as has recently been impressively demonstrated in clinical practice (Guedan et al, 2019)
Treg products from three healthy donors were generated twice by two indepenent manufacturing runs. These first generation (1st gen.) Treg products are generated from peripheral blood-derived starting material which is depleted of CD8+ cells and enriched for CD25-expressing cells using the CliniMACS system, yielding a starting populations enriched for CD8− CD4+ CD25+ Tregs
As current options for in vitro functional testing and phenotype validation are limited, the quality control measures are highly variable between manufacturing sites (Fuchs et al, 2017)
Summary
T Lymphocytes are one of the most promising effectors for adoptive cellular therapy (ACT) by facilitating targetspecific immune interventions, as has recently been impressively demonstrated in clinical practice (Guedan et al, 2019). Extensive TCR stimulationdriven in vitro expansion of the starting material is required, which poses risks for the outgrowth of contaminating proinflammatory T cells (Battaglia et al, 2005, 2006), possible loss of Treg identity (Marek et al, 2011; Bailey-Bucktrout et al, 2013), terminal differentiation, and perhaps functional exhaustion or senescence as reported for conventional T cells (Chou and Effros, 2013; Wherry and Kurachi, 2015; Okuda et al, 2019). Many different protocols for Treg product manufacturing have been developed and tested (Duggleby et al, 2018; Fraser et al, 2018; MacDonald et al, 2019; Alzhrani et al, 2020), but are difficult to compare due to the lack of a standardized quality control procedure (Fuchs et al, 2017). It is of great importance to assess functional or molecular alterations which occur during Treg product generation in order to identify current therapeutic limitations and develop strategies for improvement
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