The Time is Crucial for Ex Vivo Expansion of T Regulatory Cells for Therapy

Abstract

Ex vivo expanded CD4+ CD25 high CD127 − T regulatory cells (Tregs) are recognized as a promising candidate for immunosuppressive therapy in humans. However, due to the plasticity of Tregs lineage and artificial environment present during ex vivo expansion, Tregs easily lose suppressive activity. Here, we followed expanding CD4 + CD25 high CD127 − Tregs and their naive (CD45RA + ) and memory-like (CD45RA − ) subsets in order to establish the best conditions of the expansion. We found that, regardless of the phenotype sorted, expanding Tregs were undergoing changes resembling homeostatic proliferation and transformed into effector memory-like cells which produced not only suppressive interleukin-10 (IL-10) but also IL-6, IL-17, and interferon-γ (IFN-γ). With the time ex vivo, Tregs were losing the expression of FoxP3 and suppressive activity both when stimulated and when at rest. The only variable that helped preserve suppressive abilities of Tregs was the limitation of the time of ex vivo cultures to 2 weeks only. According to our study, the highest number of highly suppressive Tregs could be yielded with CD4 + CD25 high CD127 − Tregs cultured no longer than 2 weeks. Thorough quality check, preferentially with the assessment of FoxP3 expression and IFN-γ suppression assay, should be applied to assess suppressive activity of the cells.

Factors affecting long-term efficacy of T regulatory cell-based therapy in type 1 diabetes

“…The patients administered with two doses of Tregs preserved proportions closest to the baseline ratio. This temporary change was probably mostly caused by the content of the administered expanded Tregs, which are usually Tcm FoxP3 high Tregs [9]. The fact that they reverted to the baseline ratio in treated subjects suggests a possibility that the level and proportions of Tregs, like other lymphocytes, are homeostatically regulated to some ‘baseline’ levels.…”

“…The expansion was performed under GMP conditions and according to our previously described protocol using anti-CD3/anti-CD28 beads, interleukin 2 (IL-2) and autologous serum. The final product on release kept the FoxP3 expression above 90% [median (min.–max) = 91% (90–97)] [9]. …”

“…The gate CD25 high CD127 − from the later population was also used to assess the content of FoxP3 + T-cells in order to estimate an overlap between the two phenotypes of Tregs. Finally, CD3 + CD4 + FoxP3 + T-cells were subdivided into naive CD3 + CD4 + FoxP3 + CD62L + CD45RA + (Tn) Tregs, central memory, CD3 + CD4 + FoxP3 + CD62L + CD45RA − (Tcm) Tregs, and effector memory, CD3 + CD4 + FoxP3 + CD62L − CD45RA − (Tem) Tregs [9]. …”

Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells

“…However, there is evidence that Treg that remain in ex vivo culture for too long begin losing their characteristic phenotype and function (36). Similarly, it has been shown that too many restimulations decrease Treg suppressive function despite continued expression of Foxp3 (14).…”

Utilization of leukapheresis and CD4 positive selection in Treg isolation and the ex-vivo expansion for a clinical application in transplantation and autoimmune disorders

“…Donor age is only one example of many factors which can affect fold of expansion [10, 25, 26]. Even despite optimized expansion protocol (culture conditions and time), lower starting with numbers of Tregs requires higher fold increase during expansion to generate sufficient final number of Tregs for clinical application, which leads to higher risk of failure [26]. As increasing blood drawing volume to obtain higher starting Treg number is impractical, we decided to utilize leukapheresis for initial Treg cell retrieval.…”