NOVEL IMMUNOTHERAPIES IN TYPE 1 DIABETES: EXPLORING ANTIGEN-SPECIFIC AND NON-SPECIFIC APPROACHES

Type 1 Diabetes (T1D) is a chronic autoimmune disease characterized by loss of tolerance towards beta-cell antigens (Ags). The complete pathogenesis of the disease is still not fully understood but what is known is that the clinical onset of the disease is preceded by a silent asymptomatic phase in which beta-cells are progressively destroyed by auto-reactive effector T (Teff) cells, finally culminating in chronic hyperglycemia and diagnosis of the disease. There is currently no cure for T1D and that represents a major predicament for the public health care system as the rates and incidences of this disease increase. Therefore, discovering therapies which target the etiology of the disease and not only treat the clinical symptoms of T1D becomes imperative. That is not simple as T1D is a complex disease triggered by the interaction of multiple factors such as genetic predisposition, environmental triggers and relevant antigenic exposure. As such, due to this multitude of causative factors it is difficult to select therapies which will target such a broad range of variables, without for example, causing generalized immunosuppression. Furthermore, because T1D is the result of loss of tolerance, one should also consider Ag-specific therapies that will re-establish tolerance towards disease relevant auto-Ags. Thus, the main purpose of this work was to establish different therapeutic strategies that would prevent or revert T1D in the NOD mice which spontaneously develop diabetes. Several therapeutic protocols using both Ag-specific as well as Ag-non-specific mono-therapies have been successful in preventing or reverting T1D in animal models. Up-to-date, however, mono-therapies have done quite poorly in the clinical setting. There are several possible explanations as to the reasons for these failures. For one, the complexity of the disease itself does not facilitate the choice of a therapeutic agent and to add difficulty to this conundrum, pre-clinical studies are in their majority employed in a genetically identical group of animals and thus success in these settings does not mean they will translate into humans that possess a much broader pool and variability of genes. Thus, it is reasonable to rationalize that CT which bring together different therapeutic agents and target different arms of the pathogenic attack may be more efficient at combatting disease. Hence, we have taken this hypothesis into consideration and set up a CT using systemic low dose anti-CD3 together with orally administered GM L.lactis secreting PINS and IL10 (LL-PINS+IL10). We have shown in Chapters 4 and 5 that CT was able to stably reverse diabetes in 59% of recently diagnosed NOD mice by reinduction of tolerance to PINS. Initial beta-cell mass at start of therapy seems to be an important factor that determines the success rate of CT, reversal of diabetes in mice with starting glycemia below 350 mg/dL was significantly higher compared to mice with starting glycemia above 350 mg/dL, indicatives of higher and lower remaining beta-cell mass at start of therapy. Furthermore, we demonstrated that the addition of Anakinra treatment on top of CT enhanced diabetes reversal in mice with starting glycemia below 350 mg/dL, all mice that received triple therapy normalized whereas in CT alone 70% of mice normalized. In addition to these observations, our data indicate that the main mechanism by which CT controls disease is by increasing the frequencies of CD4+CD25+Foxp3+ Tregs in the pancreatic draining lymph nodes (PLN) and pancreas of CT-cured mice. Importantly, Tregs responded specifically to PINS and not to other irrelevant Ags such as ovalbumin (OVA). These cells also appear to be crucial for the maintenance of tolerance by CT, as depletion of the Foxp3+ population results in diabetes recurrence.In another set of experiments described in Chapter 6, we wanted to establish if different doses of regular vitamin D supplementation given at different periods in life were able to prevent diabetes. Data from epidemiological studies have largely indicated that vitamin D supplementation during early life correlates to protection from T1D while deficiency is associated with higher risk for disease. Most of these studies however emphasize the importance of vitamin D supplementation in at-risk individuals who are vitamin D deficient or insufficient, but do not clarify the effects of increasing 25(OH)D levels in subjects who are already vitamin D sufficient. The age at which vitamin D supplements should be administered for most effectiveness is also not clear, as clinical data yield contradictory results. For example, vitamin D insufficiency is more prevalent in pregnant/lactating women and while some studies point that vitamin D supplementation during this period decreases the risk of T1D in offspring, other reports observe no effect. An even more fundamental issue that generates intense debate is what should be considered as a vitamin D sufficient status and what should be the guidelines for vitamin D supplementation. Thus taking all of these issues into consideration and to address some of these questions, we first performed a dose titration test in which NOD mice received from 2,000 IU up to 8,000 IU of vitamin D3 daily, to evaluate whether these doses presented any toxic or calcemic effects. We observed that all the doses above 2,000 IU induced severe weight loss and 25(OH)D levels that were clearly toxic. Therefore, we selected lower doses of 1,250 IU and 800 IU/daily given at different time-points; during intra-utero/neonatal life, short-term (from 3 wks of age until 14 wks of age) and long-term (from 3wks of age until 35 wks of age). Our data show that only long-term vitamin D3 supplementation (with both the 800 IU as well as the 1,250 IU doses) can significantly prevent diabetes. Of interest, control mice received a vitamin D3 sufficient diet of 4 IU/daily and presented normal 25(OH) levels of a median of 71 ng/dL which increased to 116 ng/mL and 124ng/mL, when mice received 800 IU and 1,250 IU of vitamin D3 respectively. These increased levels of serum 25(OH) correlated with protection from disease. Moreover, we also verified that long-term vitamin D supplementation did not cause noticeable calcemic effects, no vascular calcifications were observed in kidney nor in heart and bone parameters were normal and even improved in vitamin D supplemented mice. Important changes in the immunological profiles were also observed, a significant decrease in IFN-g+CD8+Tcells while inversely an increase in CD4+Foxp3+ Tregs occurred. In summary, our data set should be of added value in the elaboration of future clinical trials as well as establishment of guidelines for vitamin D supplementation in the prevention of T1D.As a general conclusion, the current data presented in this thesis show promising therapeutic approaches for the prevention and intervention of T1D. It also indicates that different therapeutics approaches should be employed considering the time-point of intervention. We have shown for the first time that oral regular vitamin D3 supplementation can prevent diabetes when given long-term and that this does not seem to induce harmful side-effects. A more drastic approach seems necessary once disease has already been established. Here, (CT may bring new hope to this field. We have in fact shown that our CT approach which combines an Ag-specific together with a systemic mmunomodulator can re-introduce beta-cell specific tolerance and establish long lasting normoglycemia in cured animals. In the near future, we hope to fine-tune these findings in order to move on to clinical studies.nrpages: 154status: publishe