Introduction
CD4+CD25+FOXP3+regulatory T cells (Treg) are a T cell subset specialized in the
regulation of the immune response and maintenance of immune tolerance
(1)(2). These properties underscored their potential for adoptive
immunotherapy to control transplant rejection (3), graft vs host disease
(4), and autoimmunity (5), including type 1 diabetes (T1D) in more than
50 active or completed clinical trials (6). While polyclonal Treg are
still the cells more used in clinical adoptive immunotherapy, CAR or TCR
transgenic Treg are expected to become widely available in the near
future (7). Adoptive immunotherapy with polyclonal Treg involves their
isolation from peripheral blood and the use of in vitro expansion
protocols to obtain a final Treg product of therapeutic value in terms
of cell yield, stability and suppressive function. In patients with T1D,
the “first-in-man” clinical trial with polyclonal Treg proved feasible
and safe but showed some limitations in terms of long-term survival of
adoptively transferred Treg (8). Specifically, it showed a bi-phasic
exponential decay kinetic with a short-lived Treg subset (75-90%) with
a half-life of few days, and a long-lived Treg subset (10-25%)
detectable up to one year after infusion. Notably, while Treg after
expansion displayed a
CCR7+CD45RO+CD45RA-central-memory phenotype, Treg that survive longer in patients displayed
a
CCR7+CD45RA+CD45RO-/+phenotype similar to that of less differentiated naïve or memory stem T
cells. Treg are traditionally isolated in bulk as
CD4+CD25highCD127low (9) and
expanded using anti CD3/CD38 microbeads in combination with high doses
IL-2 which act as a potent mitogen but also pro-differentiating cytokine
leading to the generation of memory subsets (10). T cells proliferate
also in response to homeostatic cytokines such as IL-7 and IL-15 that
are permissive for expansion but preserves a poorly differentiated
phenotype. Studies conduced on conventional T cells showed the capacity
of IL-7 (7) or combination of IL-7 and IL-15 (11) to generate memory
stem T cells from naïve precursors after expansion. Treg express low
levels of the IL-7Ralpha (CD127) (9). However, we have previously showed
the capacity of human naïve Treg to respond to and proliferate in the
presence of IL-7 in vitro (12). Notably, in conditions of Treg
depletion, IL-7 contributes to the reconstitution of the Treg
compartment also in vivo in patients treated with the anti CD25
monoclonal antibody basiliximab (13).
We hypothesized that modifications to the in vitro expansion protocol
that are permissive for Treg expansion, while preserving a poorly
differentiated CD45RA+ phenotype can be associated to an increased
resistance of Treg to stress signals and therefore improve the long-term
survival of Treg once infused in patients in adoptive transfer
protocols. To test our hypothesis we sorted Treg
(CD4+CD25highCD127low)
with a naïve
CD62L+CD45RA+CD95-phenotype and added IL-7 to the culture medium during the expansion. Our
findings suggest that expansion in the presence of IL-7 generated a Treg
product with an immature phenotype and improved resistance to stress and
apoptosis.