Intervention after onset of human T1D using a clinically acceptable dose of a single systemic immunomodulatory compound, such as anti-CD3 mAb in the Protégé study (teplizumab) (10
) and the DEFEND-1 study (otelixizumab) (9
), or an Ag-specific monotherapy (e.g., GAD65 immunization; ref. 13
) has not lived up to the expectations raised by the preclinical successes in animal models or the initial phase II studies (6
). For several years now, preclinical research evidence has prompted experts to propose the combination of systemic immune modulation and Ag-specific interventions as the best therapy to revert T1D (1
In this study, a novel Ag-based vaccination approach was found to reverse autoimmune diabetes in mice. GM L. lactis
bacteria target PINS and a biologically active immunomodulatory cytokine, IL-10, to the intestinal mucosa and, in combination with low doses of anti-CD3 mAb, reset the immune system toward β cell–specific tolerance. Such a therapeutic approach is expected to boost the induction of autoAg-specific immune tolerance for several reasons. First, the gastrointestinal tract is the preferred and most attractive route for the safe delivery of Ags aimed at inducing and/or restoring immune tolerance. Second, in the gut, IL-10 especially can act locally on intestinal epithelial cells, thereby enhancing Ag capture, and on APCs by influencing Ag presentation through modulation of MHC class II. This can lead to preferential priming of naive T cells to generate Tregs and/or directly act on Tregs to maintain Foxp3 expression and their suppressive function (27
). Third, the observations by Peakman’s group suggest that misfolded and misprocessed PINS-derived peptides are crucial and early autoAgs in T1D (29
). Fourth, anti-CD3 mAb treatment induces migration of human Tregs to the intestine (30
) and thus promotes Treg interaction with the Ags delivered in the intestine. Additionally, anti-CD3 can temporarily and dose-dependently halt C-peptide decline in newly diagnosed patients as demonstrated in the studies using high doses of anti-CD3 mAbs, pointing toward the potential of this intervention in altering the course of human T1D (5
). However, side effects, although manageable, remain a concern. There is thus a need for other strategies, including short-term application of lower doses of anti-CD3 mAb such as in the CT presented here.
Our intervention effectively reverted diabetes in newly diagnosed diabetic NOD mice, with a success rate seldom approached in NOD mice. The therapy allowed us to exploit lower doses of anti-CD3 (ideally to circumvent side effects and undesired reactions) in combination with other interventions to enhance therapeutic efficacy. In particular, our CT using L. lactis
expressing PINS and hIL10 and low-dose anti-CD3 preserved functional β cell mass, resolved severe insulitis, and increased the frequencies of functional CD4+
Tregs. In vitro, Tregs from CT-cured mice were more potent than Tregs from anti-CD3–cured mice in suppressing CD4+
T cell responses. Tregs from CT-treated mice also transferred protection in vivo, in line with the concept that PINS is indeed a crucial autoAg (31
). Nevertheless, the observed production of IL-10 by these Tregs in an in vitro suppression assay and the stable reversal of diabetes suggest bystander suppression. We further demonstrated that these Tregs accumulate in draining PLNs and pancreatic islets and respond in an Ag-specific manner as evidenced by their ability to effectively suppress T cell responses in the presence of the autoAg PINS. It is conceivable that the Ag-specificity of the Tregs in CT-cured mice contributed to their increased pancreatic accumulation and observed proliferation. Although the results are suggestive, we cannot conclude from our studies whether the relationship between Treg induction and clinical outcome is truly causal or merely associative.
Interestingly, these immune effects, in particular the induction of Ag-specific Tregs, were present independent of whether the treated mice were cured, but subtle differences in numbers and characteristics of Tregs induced by the CT were observed between cured and non-cured mice. Thus, CT-non-cured mice had lower numbers of Tregs in the pancreas, and Tregs from cured mice were more efficient in secreting IL-10 and suppressing IFN-γ production by responder T cells. These small differences suggest that other factors, such as remaining functional β cell mass at diagnosis, also contributed to clinical outcome. This mass, as measured by insulin content and histology, increased under therapy and remained higher than at diabetes onset throughout the study. The CT could even cure mice with very high glycemic levels (>350 mg/dl) at onset. We did not detect β cell proliferation in histological samples, suggesting that therapy caused re-granulation or reactivation of β cells silenced by immune inflammation, but could only be clinically effective when a sufficient number of β cells were present at treatment initiation. These observations further strengthen the argument that early diagnosis of T1D and the rapid initiation of immunomodulatory therapies will be necessary for efficacy (34
In 2006, Bresson et al (35
) suggested a potential synergy between Ag-specific modalities and anti-CD3 therapy in reverting recent-onset T1D. In that study, intranasally administered human PINS along with a 5-day course of systemically administered anti-CD3 F(abι)2
fragments reverted recent-onset diabetes in two different experimental T1D models, and, as in our study, therapeutic efficacy was associated with the emergence of PINS-responsive Tregs. Of note, compared with our group, Bresson et al. used higher doses of Ab but in an Fc-non-binding format. Importantly, the use of free Ag may be extremely time consuming when translated to human disease because of its critical dependence on factors including purity, source, dose, and mode of Ag presentation to the mucosal immune system. Here, active intestinal synthesis and delivery of an Ag by GM L. lactis
obviated the need for large-scale purification of human (auto)Ags. Furthermore, in other diseases, oral application of GM L. lactis
bacteria repeatedly proved more effective than administration of free purified proteins alone, highlighting not only the efficacy of topical immunomodulation, but also the importance of the mode of therapeutic protein presentation (36
). Finally, we can state that the outcome of our therapy did not result from the direct exposure to microbial products, known to prevent diabetes in NOD mice (38
), because the L. lactis
empty vector control LL-pT1NX did not revert diabetes.
The introduction of the GM L. lactis
may prove a major step forward in Ag-based tolerance regimens for T1D as it can be rapidly translated to clinical use. L. lactis
is a Gram-positive bacterium that has been used since ancient times in preparation of food products, and it is widely recognized as being completely safe for human consumption. When delivered to cells in the gastrointestinal tract, GM L. lactis
bacteria reside there for 8–48 hours, during which time they continuously release therapeutic amounts of biologically active proteins. Such a release profile more closely mimics the way in which natural biomolecules are released in the body, in contrast to the pharmacokinetic profile of many conventional drug products. These lactic acid bacteria can be genetically modified to efficiently express any desired protein or peptide without disturbing their biological activity. New prototypes of GM L. lactis
strains expressing single as well as multiple human β cell–specific Ag(s) are currently under investigation. Safety is guaranteed by the non-colonizing, noninvasive, and food-grade nature of the bacterium. Also, in terms of deliberate release, an elegant strategy for environmental containment of GM bacteria was developed and positively evaluated by several health authorities and biosafety committees. First-in-human trials have been performed in colitis (39
) and are in progress in oral mucositis (ClinicalTrials.gov identifier NCT00938080).
In conclusion, we have demonstrated that orally administered L. lactis modified to concurrently secrete whole human PINS and hIL10 can stably reinstates normoglycemia in newly diagnosed diabetic NOD mice when introduced during the window of opportunity for tolerance created by low-dose systemic anti-CD3 mAb. Combination-treated NOD mice had higher β cell mass and augmented levels of functional autoAg-responsive Foxp3+ Tregs, which resided and proliferated locally in the pancreas. Experience with this bacterial delivery tool in other human diseases and its acceptance by the regulatory authorities should allow rapid translation into the T1D clinical setting.