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.