**3. Conclusion**

accredited UCB banks, offering the possibility to develop an off-the-shelf therapy. Which degree of matching should be used will need further evaluation in clinical studies, especially as HLA matching is less stringent when considering UCB; trials using UCB Treg cells already suggested that it is possible to use a 4/6 match for UCB Treg cells when considering a thirdparty therapy. Furthermore, it is possible to purify Treg cells with high purity in one single step as opposite to a two-step process when isolating PB Treg cells [19]. CB Treg cells comprise

cells have a higher capacity to maintain Foxp3 expression, and a better suppressive capacity and stability after expansion [20]. UCB Treg cells may have a survival advantage over PB Treg cells as they have been shown to be more resistant to apoptosis than PB Treg cells [21]. Finally, we and others have demonstrated Treg cells from UCB to be able to inhibit the function of effector cells [19, 22, 23], while other groups have reported UCB Treg cells to have low suppressive capacity [24, 25]. The required cell dose for clinical use is crucial and dictates the

In any case, whatever the cell source considered, only very few cells can be isolated from UCB or PB; therefore, most groups have focused on developing strategy to expand UCB Treg cells to enable cell therapy [26–28]. The majority of expansion protocols seek to expand Treg cells and maintain their natural Treg (nTreg) cell phenotype. Multiple studies have reported expanding nTreg cells from both PB and UCB [14, 26]. Recently, conditions to expand Treg cells have become increasingly well defined and translated into GMP compliant protocols. The majority of groups (see **Table 1**) use anti-CD3 antibody attached to beads in combination with anti-CD28 for costimulation and supplemented with IL-2 ranging from 300 to 1000 IU/ml. When expanding from PB Treg cells, rapamycin is often added to the expansion cultures, sometimes in combination with retinoic acid both to prevent the outgrowth of effector T cells and to promote Treg cell expansion, especially in the case of multiple restimulations [29]. With UCB Treg cells, it is noticeable that rapamycin seems not so vital. Brunstein et al. used beads or an engineered cell line in order to expand UCB Treg cells [26, 27]. This recently published study used anti-CD3 antibody-loaded K562 cells modified to express the high-affinity Fc receptor CD64 and the costimulatory ligand CD86 [27]. Using this culture condition, expansion

Following expansion, Treg cells should as much as possible retain their nTreg cell phenotype

however, FOXP3 is also expressed on activated effector T cells and so in itself does not distinguish between them. However, FOXP3 expression should be high and sustained. In addition, Helios expression has been associated with natural Tregs, and its presence in expanded cells is also an additional indication that the cells have retained an nTreg phenotype. Expanded cells should be able to suppress the function of effector T cells in vitro. In addition, stability of the FOXP3 expression should also be tested. The methylation state of the FOXP3 locus is indicative of recent chromosomal remodelling and thus distinguishes between FOXP3 expression being induced over constitutive expression in Treg cells [30]. The study of Treg cell

CD62LlowCCR7+

practicality of the cell source considered and post-isolation manipulation if required.

in contrast to the adult PB Treg cells than

). FOXP3+ expression would seem vital;

). As a consequence, UCB Treg

almost entirely of a naïve T-cell phenotype CD45RA+

of up to 10,000 fold of UCB Treg cells was achieved.

FOXP3+

CD127lowCD25+

(CD3+

CD4+

have a central memory phenotype (CD62LhighCCR7highCD45RO+

174 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

Immunotherapy has been a promising option in order to improve the outcome of HSCT. UCB‐ derived immunotherapies are very promising, and future studies will help us understanding their potential better. UCB Treg cells could become an immunotherapy of choice for treating GvHD. In addition, one should also consider the use of UCB plasma that already contains proteins with the capacity to modulate the immune response in particular inflammation to treat skin GvHD as well as autoimmune diseases.
