Discussion
Adoptive Treg transfer with the aim to improve the control of immune
responses and re-establish peripheral tolerance holds therapeutic
promises in transplantation, GVHD, and autoimmune diseases. In a future
perspective, antigen expanded as well as TCR engineered or CAR Treg will
rapidly become available alternatives to polyclonal Treg. The clinical
experience however has shown some issues that may impact the therapeutic
value of this approach. Specifically, the need of a high number of Treg
for adoptive therapy has been fulfilled with robust in vitro expansion
protocols, but expanded Treg have shown fragilities and a limited
capacity to persist in patients. Moreover, there is a need of measurable
biological parameters that, beside the mere cell yield, can predict the
capacity of expanded Treg to adapt to an in vivo environment in which
competition with other cells for growth factors and nutrients generate a
selective pressure. We report that Treg can be expanded using a
combination of IL-7 and IL-2 with several advantages in term of
resistance to apoptosis and stress and maintenance of a poorly
differentiated phenotype. These findings are relevant to the
improvements of Treg expansion protocols for adoptive Treg therapy and
to overcome some issues related to the long-term persistence of Treg
once infused in patients.
In vitro expansion of Treg for adoptive transfer traditionally rely on
the use of high doses of IL-2 (17) (18) (19). The final Treg product is
composed by a large majority of Treg with a short life-span in vivo and
a long-lived subset that can persist for over a year (8). Based on
studies performed mainly on conventional T cell we hypothesized the use
of homeostatic cytokines for Treg expansion. Homeostatic cytokines such
as IL-7 induces conventional T cell expansion (20) and provide
anti-apoptotic signals (21). However, their use in Treg expansion has
not been fully explored based on the low expression of the IL-7 receptor
alpha chain, and assuming that Treg do not respond to IL-7.
Treg are phenotypically defined as
CD4+CD25highFOXP3+T cells and sorted as
CD4+CD25highCD127lowT cells for in vitro expansion (14). Compared to circulating B cells
that lack the expression of CD127 (15) we found a low but significant
expression of CD127 that is sufficient to trigger significant STAT5
phosphorylation upon stimulation with 10ng/ml of IL-7. We previously
reported that Treg are responsive to IL-7, even though at higher
concentrations than conventional T cells (12). At physiological IL-7
concentrations of 2-8 pg/ml (22) Treg are not likely to receive
significant signals, but when IL-7 is used at high concentration for in
vitro expansion can elicit a robust response to IL-7. Importantly, we
reported that in patients experiencing lymphopenia in which IL-7 is
present at supra-physiological concentrations it may contribute to their
homeostasis and proliferation to reconstitute the depleted Treg
compartment (13). The response to IL-7 was higher in Treg with a naïve
or memory stem T cell phenotype and decline with the progression of
differentiation into central-memory and effector-memory subsets. The
responsiveness was not strictly related to changes in the expression of
CD127, suggesting that other unidentified factors may regulate STAT-5
phosphorylation beside the mere expression of the receptor. High IL-7
responsiveness of CD45RA+CD62L+ Treg
was one reason why we decided to sort
CD45RA+CD62L+ for expansion. The
other reason was to obtain a Treg product with an immature phenotype
that apparently are the cells that survive longer in patients (8). Since
IL-7 per se was not sufficient to trigger significant Treg expansion we
combined IL-7 to IL-2. Surprisingly, we did not observed an additive or
synergistic effect but instead the presence of IL-7 reduced the
proliferation rate induced by IL-2. We experimentally proved that in the
presence of IL-7 the formation of the high affinity IL-7 receptor engage
a significant amount of the common-γ chain (CD132), reducing the ability
of IL-2 to form the high affinity IL-2 receptor with CD25, CD122 and
CD132. We have previously shown that competition of CD127 and CD25 for
CD132 can occur in conventional T cells (16). Accordingly, expansion of
CD45RA+CD62L+ Treg with the IL-7M
resulted in a reduced final cell yield as well as a different surface
phenotype in which a significant higher proportion of Treg display a
CD45RA+CD62L+CD95+ phenotype. This
phenotype is reminiscent of that of conventional memory stem T cells
(23) and, to the best of our knowledge Treg with a stem cell memory
phenotype have never been described before. Further studies are needed
to clarify whether a subset with memory stem cell function exist also in
the Treg compartment with characteristics of self-renewal and the
capacity to generate the full phenotypic diversity of Treg subsets. An
increased expression of CD95 was found in expanded Treg from cord-blood
as compared to expanded Treg from peripheral blood (24) that also
contain an increased proportion of
CD45RA+CD62L+ Treg. Such
phenotypically immature Treg population obtained with the IL-7M also
showed relevant differences in terms of metabolic machinery and survival
capacity. An improved metabolic fitness in terms of mitochondrial mass
and capacity to uptake glucose can advantage Treg expanded with the
IL-7M once infused in patients. Compared to conventional T cells, Treg
cells are less reliant on glycolysis and use mitochondrial metabolism
and oxidative phosphorylation (OXPHOS) for energy production (25). In
vitro studies revealed that Foxp3 is directly responsible in
reprograming T cell metabolism by suppressing glycolysis and enhancing
OXPHOS (26)(27). Elevated glycolysis may be detrimental to Treg cell
induction and suppressive function and deletion of HIF-1a, a
transcription factor that can promote glycolysis, leads to increased
Foxp3 induction (28). However, also Treg needs glycolysis to support
some processes such as proliferation (29) and migration (30) when a high
amount of ATP and metabolic intermediates are needed. It is reasonable
to speculate that Treg cells precisely balance cellular glucose
consumption, when glycolysis increases Treg cell proliferation and
expansion, but this activity is balanced OXPHOS to maintain lineage
stability and suppressive activity. Treg obtained with the IL-7M showed
signs of both increased glycolysis (2NBDG uptake and lactate production)
but also an increased mitochondrial mass. Reduction of glycolysis after
a few days of resting corresponded, in our study, to a recovery of the
Treg suppressive capacity. An important issue is also that Treg adoptive
transfer implicates moving Treg from in vitro culture conditions with
high glucose, oxygen and intense cytokine and TCR/CD28 signaling to an
in vivo environment where these factors are reduced or completely
missing. Therefore, a improved metabolic machinery can be helpful to
Treg to adapt to changing conditions and survive in vivo overcoming
substrate and grow factor signaling restrictions. We showed indeed and
increased survival capacity of Treg both after growth factor deprivation
but also in response to direct apoptotic stimuli possibly. Naïve Treg do
not express CD95 and become susceptible to fas ligand mediated apoptosis
only when start expressing CD95 upon stimulation (31). Increased
resistance to fas ligand mediated apoptosis in Treg expanded with the
IL-7M can be due to the increased expression of the anti-apoptotic
molecule Bcl-2, which is presumably induced by the presence of IL-7
(21). We also observed a reduced telomere shortening in Treg expanded
with the IL-7M. Telomere length is regulated by telomere erosion during
cell division, and by the activity of telomerase which is negligible is
T cells (32). Preservation of telomere length during IL-7 mediated T
cell expansion has been reported (32), however in our Treg expansion
model this could also be due to the reduced rate of expansion of Treg
with the IL-7M. Preliminary data indicate an increased capacity of Treg
expanded with the IL-7M to survive in NSG mice as well as a distribution
pattern that includes migration to the bone marrow. While these
observations require confirmation in relevant disease models to
determine whether the increased persistence is associated to an improved
therapeutic effect, our data prompted us to further characterized Treg
expanded with the IL-7M. It has to be determined whether a lower number
of phenotypically immature Treg but also with better performances can
increased the therapeutic value as compared to a higher number of
phenotypically mature Treg with fragilities in terms of resistance to
stress and apoptosis.
We therefore suggest that addition of IL-7 during expansion improves the
performances of Treg despite a lower final cell yield and a (reversible)
reduction of suppressive function. Our expansion model needs to be
further explored as a potential improvement of current Treg expansion
protocols based on IL-2. The fragility of Treg used for adoptive
transfer represents an issue that need to be resolved in order to
increase the efficacy or this promising immune-therapy.