Such studies have important implications for the design of future clinical studies. The search for further surface markers to aid the isolation of purer or more potent Treg populations led to studies investigating markers such as CD121a/CD121b, TGF-β/ latency associated peptide (LAP) [59] and CD39 [60]. However, all these proteins are expressed only on activated Tregs and

would be of use only to re-isolate Tregs after expansion. This may not be feasible, in view of the costs of re-isolating billions of Tregs on a per-patient basis. Other studies complicate the story even further. Ito et al. [61] showed that FoxP3+ Tregs could be grouped into two subsets based on the expression of the inducible T cell co-stimulator (ICOS). They showed that while ICOS–FoxP3+ Tregs mediate their suppressive function via TGF-β, Inhibitor Library Protein Tyrosine Kinase inhibitor ICOS+FoxP3+ Tregs additionally secrete IL-10. Therefore, depending on the type of immune response to be suppressed, it may be useful to isolate subsets of Tregs which have specific

mechanisms of action. Moreover, a recent study by Ukena et al. [62] compared different Treg isolation strategies in order to define the most promising Treg target cell population for cellular therapy. They compared CD4+CD25hi enrichment, CD4+CD25hi enrichment and depletion of CD127+, enrichment of CD4+CD25hiCD45RA T cells, depletion of CD49d+ (a marker of proinflammatory cytokine-producing effector T cells) and CD127+ T cells and enrichment of CD4+CD25hi ICOS+ and ICOS– Tregs. They concluded that while CD4+CD25hiCD127– and CD4+CD25hiICOS+

Tregs are the most promising Tregs for fresh cell infusions in clinical trials with respect to cell yield, phenotype, function and stability, the CD4+CD25+ Tregs qualify as the best candidate for in-vitro expansion. Such studies, therefore, paint a complicated picture that when choosing the Treg marker for cell isolation we should also bear in mind Mirabegron other factors other than simply purity, i.e. isolating potent cells with a mechanism of action to suppress the immune response of interest and cells with the desired expansion profiles. Despite this, however, what limits choice when devising a clinically applicable protocol is that isolation techniques need to be good manufacturing practice (GMP)-compliant, and GMP purification reagents for all the various markers outlined above are not yet available. The clinical Treg selection protocols used to date in the United Kingdom have used a combination of depletion and positive selection steps, with the isolation tools involving mainly the automated CliniMACS plus system (Miltenyi Biotec, Bisley, UK). This enables GMP-compliant cell selection by magnetic bead activated cell sorting [63].