One potential path for an effective therapy to reverse type 1 diabetes would involve the restoration of homeostasis within the immune system normally controlled by Tregs. Compelling evidence exists from the NOD mouse that transfer of in vitro expanded Tregs can overcome intrinsic defects and restore tolerance in type 1 diabetes (12
). The therapeutic benefits of restoring tolerance early in type 1 diabetes would likely result in the preservation of endogenous β-cell mass and subsequent reduction in complications resulting from hyperglycemia. Treg-based cell therapies pose several advantages over cell-depleting regimens and bone marrow transplantation protocols, in that they are unlikely to compromise host immune responses to foreign pathogens.
In this study, we demonstrated that functional Tregs can be isolated from patients with type 1 diabetes and expanded in vitro to therapeutically relevant levels. Most importantly, we demonstrated that we could attain on average 1,500-fold expansion of the highly purified CD4+CD127lo/−CD25+ T-cell population in a 14-day culture period using methods that can be adapted to cGMP procedures. Moreover, cells from both type 1 diabetic patients and healthy volunteers grown under these conditions retain the hallmark characteristics of freshly isolated Tregs (i.e., they are anergic after culture, suppressive, and continue to express high levels of the transcription factor FOXP3).
Several previous studies have reported methods for isolating and expanding natural FOXP3-expressing Tregs for therapeutic applications from healthy individuals (36
). However, these alternative approaches can fall short of meeting clinical needs in terms of the initial cell yield, the feasibility of adapting protocols to cGMP levels, or the purity and stability of the expanded cell populations post-transfer, especially in patients with autoimmune diseases as highlighted below. We sought to address these concerns and meet these important therapeutic criteria.
The use of cellular therapies in individuals with type 1 diabetes raises concerns regarding the subsequent function of cells in patients presumably carrying a genetic predisposition toward autoimmunity. There is growing evidence from genetic studies in the NOD mouse and humans implicating pathways likely to influence Treg frequency and/or function. These genes include, but are not limited to, the IL-2 signaling axis (Il2
in the NOD mouse and CD25
in humans), the T-cell signaling molecule LYP (protein tyrosine phosphatase [PTPN22
]), and the negative T-cell regulator cytotoxic T-lymphocyte antigen-4 (CTLA-4
). Susceptibility alleles in these pathways may confer deficient immune activation and subsequent control by Tregs (10
). One means to overcome this inherent defect may be the use of matched allogeneic cells, such as that recently reported for bone marrow transplantation in a model of autoimmune arthritis (41
). Although this approach may be advantageous from an immunoregulatory standpoint, the safety and efficacy profile of such an approach in patients with type 1 diabetes remains unknown. These questions require initial trials with autologous cells with the expectation that in vitro activation and IL-2 exposure will augment the functional profile of Tregs (29
A major concern when proposing to expand Tregs from type 1 diabetic patients or other autoimmune subjects is the potential for outgrowth of activated Teff cells. Central to addressing this concern is our inclusion of the CD127 marker, which enables us to discriminate between activated Teff cells and bona fide Tregs (15
). As an additional constraint, we chose to isolate Tregs through a stringent FACS-based sorting approach. Using these parameters, we were able to show expansion of functional CD4+
T-cells grown in the presence of rapamycin as well as highly purified CD4+
T-cells. Surprisingly, both of these populations suppressed equally well during the in vitro suppression assay despite decreased numbers of FOXP3+
cells present in CD4+
T-cells. This may result from an unknown suppressive property of CD127lo/−
T-cells, or alternatively, from the differentiation of FOXP3−
T-cells into IL-10–or TGF-β–producing adaptive Tregs. This process has previously been reported to occur through exposure of conventional T-cells to Tregs in vitro or in vivo in a process commonly referred to as education (42
Despite the similar suppressive properties of CD4+CD127lo/− T-cells, we believe CD4+CD127lo/−CD25+ T-cells represent the optimal polyclonal Treg population for our proposed phase I clinical trial. This conclusion is based on the highly enriched nature of the starting population and the ability to expand these cells to high purity without rapamycin. While rapamycin does selectively inhibit Teff outgrowth in vitro, there is some concern that these activated cells could expand after in vivo transfer in the absence of the drug. Therefore, when considering the optimal cell population for cellular therapies, based on the parameters of stability, FOXP3 expression, and in vitro expansion potential, we have concluded that CD4+ CD127lo/−CD25+ T-cells represent the optimal population.
Despite these promising results, several key questions still remain regarding Treg cell therapy. Little is known about how long expanded Tregs will persist upon transfer, where they track, or whether they retain their suppressive phenotype in vivo. To begin to address the issue of cell stability, we assessed the cytokine production profiles of expanded Tregs. Surprisingly, analyses of intracellular cytokines from expanded human Tregs indicate FOXP3 expression, and production of IFN-γ is not mutually exclusive. In fact, we noted that several other cytokines typically associated with effector functions could also be co-expressed with FOXP3 including IL-17 and TNF-α, whereas IL-2 and IL-4 appeared more restricted to the FOXP3−
T-cell fraction (data not shown). The in vivo physiological significance of these observations in response to super-physiological stimuli is not clear, nor is the notion that these responses are detrimental. Nevertheless, these questions regarding the stability of Tregs are of great importance, and we are continuing to investigate these issues through studies with humanized mice (45
), as well as FoxP3 reporter mice that can discriminate cells that are currently expressing FoxP3, or have expressed it at any point in development (46
Animal model data support the use of Tregs to halt type 1 diabetes progression and suggest transfer of Tregs may lead to reversal of ongoing disease (7
). With that said, this may be challenging in subjects after the processes of epitope spreading and memory T-cell expansion. Here we propose the use of polyclonal Tregs as a necessary advancement in the use of Tregs for the treatment of type 1 diabetes. However, we acknowledge that overcoming the autoimmune attack in type 1 diabetes in the future may require even more challenging approaches involving combination therapies or the use of antigen-specific Tregs. To begin to address these issues, we are currently developing protocols for the generation of antigen-specific Tregs capable of recognizing diabetes autoantigens through the use of viral TCR-α and TCR-β gene transfer into CD4+
In conclusion, our data indicate functional Tregs can be isolated and expanded from recent-onset type 1 diabetic patients. These Tregs can be expanded in vitro to >1,500-fold in a 2-week period using cGMP-level materials and procedures. These cells retain potent suppressive function and FOXP3 expression without the need for rapamycin. This study outlines important tools and principles for translating Treg therapies into clinical treatments for patients with type 1 diabetes and other autoimmune disorders.