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method for the generation of cytokine-polarized human CD4
+
T cells in
rapamycin, as shown in
Figure 11.3
. This method has several potential
advantages relative to our initial method. First, T cells are generated after a
single round of costimulation, which greatly increases the feasibility of the
culture method; after only 12 days in culture, CD4
+
T cells are rendered rapa-
mycin-resistant and cytokine polarized, with an increase in Th2 relative to
Th1 composition, as defined by an approximate 5:1 ratio of T cells express-
ing GATA-3 and T-bet
[99]
(
Figure 11.3
); of interest, manufactured T-cell
expression of FOXP3 was minimal, which is a result that stands somewhat
in contrast to other observations that rapamycin can promote the expan-
sion of Treg cells
[76]
. In addition, because of the reduced time in culture
and the influence of rapamycin, manufactured T cells express a minimally
differentiated phenotype, as indicated by low levels of cytokine secretion
at completion of T-cell manufacturing. However, as shown in
Figure 11.3
,
further expansion of the T-cell product without polarizing cytokines or
rapamycin demonstrates the significant cytokine secretion potential of the
manufactured product; because both the transcription factor analysis and
the cytokine secretion profile indicate a mix of Th2 and Th1 cells, we have
used the term “T-Rapa” cells to describe the manufactured product.
238
The
ex vivo
manufacturing of mixed Th2/Th1 cells in rapamycin carries the
feasibility disadvantage of limited T-cell expansion during culture, with a
typical culture yielding only a few-fold increase in CD4 cell number relative
to input cell number. Nonetheless, CD4 cell harvest from a 5- to 10-liter
apheresis product will allow T-Rapa cell therapy at the intermediate dose
established in our initial clinical trial, 2.5 × 10
7
manufactured cells/kg. This
second-generation clinical trial (NCT00074490), in addition to evaluating a
new method of T-cell manufacturing, is also evaluating a new post-trans-
plant GVHD chemoprophylaxis regimen consisting of cyclosporin plus
a short course of Sirolimus (through day 14 post-transplant) (
Figure 11.3
schema). In our murine studies
[78]
, we found that rapamycin-resistant T
cells did not necessarily exhibit resistance to rapamycin
in vivo;
we specu-
late that these observations may relate to the inhibitory effect of rapamy-
cin on host or donor APCs
[76]
(see Chapter 9) that drive T-cell expansion
in vivo.
In addition, this immune-suppression strategy places dual-agent
suppression on the unmanipulated T cells contained in the hematopoietic
cell allograft and single-agent cyclosporin suppression on the manufac-
tured product, which is administered at day 14 post-transplant as a preemp-
tive donor lymphocyte infusion (DLI), thereby creating a selective pressure
favoring immune responses generated from the T-Rapa cells.
In addition, as outlined in
Figure 11.3
, we have evaluated the day 14 post-
transplant T-Rapa cell DLI in the context of a low-intensity chemotherapy
regimen that uses a cyclophosphamide dose that is 75% reduced relative
to our first-generation clinical trial
[97]
. Use of this low-intensity regimen
offers both potential therapeutic advantages (reduced toxicity in heavily
pretreated patients with comorbid conditions and chemotherapy refractory
disease) and a new opportunity to evaluate the biologic activity of T-Rapa
cells (ability to convert mixed chimerism). Indeed, using this platform, we
found that each of 40 transplant recipients had mixed donor/host chime-
rism in both T-cell and myeloid cell lineages at day 14 post-transplant prior
to T-Rapa cell infusion
[100]
. Then, within 2 weeks of T-Rapa cell infusion,
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