Thymic-derived Tregs: A potential novel source for suppressive and efficacious Treg-EVs for clinical use
Jordan Bazoer
School of Medicine and Biosciences
Supervisors:
Dr Lesley Smyth
School of Medicine and Biosciences
Dr Bernadine Idowu
School of Medicine and Biosciences
Regulatory T cell-derived extracellular vesicles (Treg-EVs) offer an interesting therapeutic solution for several settings including transplantation, where cellular therapies, due to their instability and poor persistence in vivo, are limited. Current strategies to separate functional doses of Treg-EVs are largely stifled by the need for ex vivo expansion of the parent Treg, typically derived from peripheral blood (bTreg). Treg expansion requires repeated activation which can result in weakened functionality, reduced proliferation and the expression of inhibitory molecules; a phenomenon termed ‘exhaustion’. Thus, expansion of Tregs for therapeutic use are halted at three stimulations. Recent reports have suggested that thymic-Tregs (tTregs) are less susceptible exhaustion; and can be stimulated up to five times, without compromising functionality. However, to our knowledge, no one has shown that tTreg produce EVs, that can inhibit immune responses similar to bTreg EVs. Hence, in this study we aim to demonstrate that tTregs present a better source for the separation of functional Treg-EVs fit for therapeutic use. We hypothesised that tTregs will produce EVs capable of inhibiting the immune system and that repeated stimulation of tTregs will result in negligible difference in suppressive function of their EVs.
Following 10 days of expansion, both tTreg and bTreg cell lines were assessed for suppressive function against HLA-mismatched T effector cells (Teffs). As excepted both tTregs and bTregs inhibited T cell activation. Next, EV production of tTregs was assessed by stimulating cells with anti-CD3 and CD28 antibodies for 24 hours and culture supernatants collected. EVs were separated using precipitation and their presence confirmed by Nanoparticle Tracking Analysis (NTA) and expression of CD81 and CD63.
The use of tTregs as the source of Treg-EV takes this research area one step closer to the generation of GMP protocols and ultimately therapeutic use in the clinic in a range of settings, including transplantation.