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technologies if a full leukapheresis product is available
[149,150]
. Since the
coadministration of Treg and Tconv is intended in this clinical situation, Treg
purity is not a major issue as products containing more than 50% Treg can
be generated consistently. Yet, for the use of cord blood Treg or for potential
therapeutic applications of peripheral blood donor Treg, effective
in vitro
expansion systems are required. Because of their hypoproliferative behavior
after TCR stimulation, Treg expansion was initially considered impossible.
Yet, the combined stimulation of Treg with cross-linked anti-CD3 and anti-
CD28 antibodies, either by artificial beads or by feeder cells, together with
high-dose IL-2 permits the efficient
in vitro
expansion of Treg
[50,151,152]
.
Yet, even when highly FACS-purified starting populations expressing >95%
FOXP3 are used, a variable fraction of Treg lose FOXP3 expression upon
expansion
[50]
. A detailed examination of the cells maintaining FOXP3 in
culture revealed that those cells largely maintain the expression of CD62L
and CCR7 despite their extensive proliferation. These lymph node homing
receptors are usually expressed on naïve or central memory-type T cells.
The further assessment of the origin of CD62L/CCR7-positive expanded
Treg revealed that Treg in peripheral blood do not consist solely of CD45RO
+
cells, but that a variable fraction of Treg also expresses CD45RA, a marker
usually associated with naïve T cells
[50,153,154]
. When such CD45RA
+
Treg
were expanded, they homogeneously maintained FOXP3 expression over a
3-week culture period; they did not secrete proinflammatory cytokines and
they maintained potent suppressive activity. In contrast, the CD45RO
+
sub-
population of natural Treg downregulated FOXP3 to variable degrees dur-
ing expansion
[155]
, started to secrete predominantly Th2-like cytokines
[156]
, and showed diminished suppression
in vitro.
Furthermore, the TSDR
of the FOXP3 locus is increasingly methylated during culture of CD45RO
+
Treg, further confirming the instability of this subpopulation
[43,155]
. Since
the TSDR has an important enhancer function and its demethylated state
is required for stable FOXP3 expression, these findings suggest that the
CD45RA
+
subpopulation of natural Treg is an ideal starting population for
the generation of homogeneous Treg cell products
[50,64,155]
. Yet, for rou-
tine clinical settings such strategies are cumbersome, since the already low
frequency of Treg in leukapheresis products is further reduced (depending
on donor age, only 10-50% of Treg are CD45RA
+
) and good manufacturing
practice-compatible isolation strategies for this subpopulation are not yet
available, though are currently under development. Potential advantages of the
expansion of CD45RA
+
Treg are the minimal
in vitro
manipulation of Treg,
the high purity of FOXP3 expression after culture, and the high proliferative
capacity of such starting populations.
260
An alternative and technically much simpler approach is the
in vitro
expan-
sion of Treg in the presence of rapamycin
[157,158]
. Here, simple CD25-bead
enrichment strategies can be used, as rapamycin has been shown to impair
the survival and expansion of Tconv cells
in vitro,
while Treg cells are com-
parably resistant to mTOR inhibition because they do not require the phos-
phatidylinositol 3-kinase/AKT pathway upon TCR stimulation
[159-161]
.
Thus, Treg preferentially expand in rapamycin-containing medium and
rapamycin has even been suggested to induce Treg from Tconv cells
[162]
.
The finding that rapamycin also seems to spare Treg
in vivo
[161,163,164]
makes this straightforward approach highly attractive. Potential hazards
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