In vivo imaging of graftversus-host disease and graftversus-leukemia - Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation - page 65

strategies to expand murine Tregs with anti-CD3 Ab or irradiated host-type
splenic stimulators, low or high IL-2, as well as the presence or absence of
TGF-β; they found that all their culture methods produced cells which could
inhibit GVHD to various degrees [56] . While the best inhibition of GVHD
was with TGF-β and low dose IL-2, this method also gave the lowest yield
of cells. Thus a better expansion of Treg was achieved with a high dose of
IL-2. This work was translated into the culture of human Treg from periph-
eral blood [57] . By preparing CD4 + CD25 + Treg with CliniMACS, followed by
flow cytometry sorting, it was demonstrated that an artificial antigen pre-
senting cell (aAPC) cell line which was approved for clinical use (KT64/84)
could achieve better expansion of Treg than anti-CD3/CD28 mAb-coated
beads. Furthermore, this study also confirmed previous reports that rapa-
mycin was necessary to maintain a suppressive phenotype of the Treg and
prevent the outgrowth of non-Tregs in culture [57] . Interestingly, Hoffmann
et al. found that by the addition of CD45RA + (in addition to CD4 + CD25 hi ) as
a marker to sort human Tregs, a FoxP3 + CD62L + CCR7 − population of sup-
pressive Tregs could be maintained at high purity for over three weeks of
culture in the absence of rapamycin [58] . Recently, Brunstein et al. have
demonstrated that ex vivo expanded Tregs are safe for clinical use [59] . By
using umbilical cord blood as a source of Tregs for culture, they demon-
strated that 3 × 10 6 /kg partially HLA-matched third-party Tregs could be
safely administered to patients and resulted in a lower risk of acute GVHD
than historical control patients.

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As immunosuppressive drugs are often used to prevent GVHD and other
autoimmune reactions, it was important to determine their effects on Tregs.
Using a major MHC-mismatched model of GVHD with bioluminescence
imaging, the effects of different immunosuppressive drugs on Tregs was
studied [60] . Surprisingly cyclosporin A (CSA) was found to suppress Treg
function (assessed by an increase in luc + Tcon proliferation) and to increase
GVHD, whereas mycophenolate mofetil (MMF) and especially rapamycin
(RAPA) did not. Further, treatment of mice with RAPA and Treg preserved
GVT against luminescent leukemia cells. The differential effects of RAPA on
Treg versus Tcon are due to different signaling pathways after IL-2 stimula-
tion. Specifically, CD4 + CD25 − conventional CD4 + T cells signal primarily via
the mTOR pathway that is inhibitable by drugs such as rapamycin, whereas
Tregs use a stat5-mediated pathway following IL-2 stimulation which is not
impacted by rapamycin [61-64] .

The differential effect of RAPA on Tregs versus Tcon, as well as the importance
of IL-2 on Treg activation and suppressive activity [65-67] , led Shin et al. to
explore combination therapy of RAPA and IL-2 to preferentially expand Tregs
in vivo following allogeneic transplantation [68] . They found that intraperi-
toneal injection of both RAPA and IL-2 had an additive effect on inhibiting
the proliferation of donor CD4 + T cells, and expanding CD4 + FoxP3 + Tregs.
When both CD25-depleted Tcon and CD4 + CD25 hi Treg were adoptively
transferred, the combination therapy of RAPA with IL-2 increased the fre-
quency of donor CD4 + FoxP3 + Tregs, resulting in approximately threefold
more Tregs in control animals. Analysis using congenic markers revealed
that the increase in Treg numbers was a result of both expansion of donor
CD4 + FoxP3 + Treg as well as increased conversion of CD4 + CD25 - donor Tcon
to a FoxP3 + phenotype. The combination therapy also produced the most