One of the obstacles in the implementation of clinical protocols using Tregs is their low frequency, 1–3% of total peripheral blood CD4+ T cells, and data (from animal models) which suggest that, for these cells to suppress immune responses, high doses of Tregs in relation to effectors is required [52, 53]. This means that for cellular therapy, it will almost certainly be necessary to use a polyclonal stimulus to expand Tregs in vitro. In this regard, the large-scale ex-vivo expansion of human Tregs by stimulation with anti-CD3 and anti-CD28 monoclonal antibody-coated beads and high-dose NVP-BGJ398 IL-2 has been demonstrated successfully [54]. However, effectors have the potential to proliferate

vigorously under such conditions, so that even a trace of effectors in the starting population can be expanded in high numbers. The injection of such cells would, therefore, be detrimental to the patient and may lead to rejection. Thus, it is essential to either initiate the expansion culture with highly purified Tregs (a challenge in view of the absence of a Treg-specific cell surface marker) or create culture conditions that favour Treg cell growth. Two different RG7420 purchase combinations of markers appear to be promising

for Treg isolation. The first seeks to isolate CD4+CD25hi Tregs, but with the addition of an antibody to select for CD45RA+ cells and so eliminate antigen-experienced or memory T cells [16]. The second combination also uses the CD4+CD25hi phenotype, but includes CD127 expression. The rationale for using CD127 as a marker for Treg isolation (as explained in earlier sections) is on the basis that in human Tregs there is a reciprocal expression of CD127 and FoxP3, and thus CD127 provides a sortable surrogate marker for FoxP3+ Tregs [24]. Moreover, the so-called ‘naive’ Treg population based on the co-expression of CD4 and CD45RA yield Tregs with a greater suppressive capacity than total CD25hi cells [55]. The reason for this became clear when Miyara et al. [22] noted the subpopulations of human FoxP3+ T cells and discovered that the CD25+CD45RA-FoxP3hi

cells contain many Th17 precursors. Furthermore, after 3 weeks of in-vitro expansion the CD45RA+-expanded Rolziracetam Tregs remained demethylated (compared to the CD127– Tregs that became methylated) at the Treg-specific demethylation region (TSDR), which is a conserved region upstream of exon 1 within the FoxP3 locus [completely demethylated in natural Tregs but methylated fully in induced Tregs and effector T cells (Teff)] [55, 56]. Such studies, therefore, support the isolation of Tregs based on CD45RA+ expression, bearing in mind that they are the most stable population for expansion and have the greatest expansion potential [16]. Despite such studies, one drawback is that the number of naive Tregs declines in the peripheral blood with age [57], and hence isolation based on CD127 expression may still be a practical approach.