Effective therapy of T1D requires elimination and/or regulation of the T-cell clones that initiate and perpetuate the damage of the pancreatic β-cells. In this study, we demonstrated that the regulatory effects of gal-1 on the immune system can be employed to prevent diabetes in NOD mice and, importantly, to reverse ongoing β-cell autoimmunity at later stages of the disease in sub-clinical and overt T1D.
Previous studies have shown that gal-1 down-regulates T-cell survival, activation and proliferation (36
), suppress Th1-responses (14
) and triggers apoptosis of thymocytes and activated T-cells in humans (5
) and mice (26
). Gal-1 is expressed by Treg (39
) and immune-privileged sites (40
), is employed as a mechanism of immunoescape by neoplasms (43
) and plays an important role in feto-maternal tolerance (44
). Due to its regulatory effects on T cells, gal-1 has been used to treat T-cell-mediated diseases in murine models (12
). Although the mechanisms of the pleiotropic effects of gal-1 have not been elucidated, there is evidence that the homodimeric (bivalent) form of the lectin is critical to crosslink its ligands on the T-cell surface (9
) and to transduce the death signal (5
). T-cell apoptosis triggered by gal-1 is caspase-independent and involves rapid nuclear translocation of endonuclease G from mitochondria without release of cytochrome c (45
). Gal-1 has numerous anti-inflammatory effects, some of them at concentrations below its apoptotic threshold, including induction of IL-10 release (46
), down-regulation of secretion of TNF-α and IFN-γ (15
), and inhibition of adhesion to endothelium (48
) and trans-endothelial migration of leukocytes (49
We showed here that administration of soluble gal-1 prevents onset of T1D in female NOD mice. Interestingly, histopathologic examination of pancreata from mice treated with gal-1 showed a similar incidence of insulitis, scored simply as the percentage of islets with associated leukocytes, as the vehicle-treated group. However, the pancreatic infiltrates of gal-1-treated mice did not invade the islets and did not damage the β-cells. The non-pathogenic nature of the insulitis of the gal-1-treated mice was due likely to the qualitative difference in its cellular composition. Therapy with gal-1 shifted the balance of the ratio between Th1- and Th2-like-cells in the islet infiltrate and PLN, increasing the percentage of CD4+
T-cells with IL-4, IL-5 or IL-10 and substantially reducing the number of IFN-γ-secreting T-cells. This Th2-like bias in the T-cell response was specific to β-cell-Ag, as demonstrated in ELISPOT assays and agrees with previous studies showing that pancreatic infiltrates with inverted Th1/Th2 cell ratio cause a non-destructive peri-insulitis in NOD mice (51
). Although the critical factors that trigger leukocyte mobilization into the islets (insulitis) are unknown, the ability of gal-1 to reduce the number of IFN-γ-secreting T-cells around the islets and/or to inhibit migration of T-cells (and possibly DC and macrophages) through the extracellular matrix (47
) could account for the non-invasive nature of the infiltrates in gal-1-treated mice.
The cytokine-shift in the T-cell response against β-cell-Ag in gal-1-treated mice could be ascribed to the ability of the lectin to drive T-cell polarization at concentrations below its apoptotic-threshold and/or to trigger cell death of specific substes of effector T-cells in NOD mice. We showed that, at concentrations that do not induce T-cell apoptosis, addition of gal-1 did not alter significantly polarization of T-cells. However, in our system, gal-1 bound and triggered rapid apoptosis of effector Th1, Th17 and Tc1 cells, but spared Th2 cells and Treg, both subsets that have been proved to protect against development of T1D in NOD mice (35
). Our findings extend the results of a recent study showing that differential glycosylation of Th1, Th2 and Th17 cells regulates their resistance to gal-1-induced cell death. The susceptibility of Th1 and Th17 cells to gal-1-mediated cell death correlated directly with their high levels of core-2-O
-glycan epitopes generated by the enzyme core 2 β-1,6-N-acetyl-glucosaminyltransferase on cell surface glycoproteins, which are ligands for gal-1 (18
). In contrast, addition of α2,6-linked sialic acid to N
-glycans on Th2 cells protects these cells from gal-1-induced cell death (18
). Interestingly, although we detected that gal-1 binds to resting and (with more avidity) activated CD4+
T-cells of NOD mice, these cells were resistant to gal-1-mediated apoptosis. This finding suggests that CD4+
Treg must have a mechanism of protection from gal-1-mediated cellular suicide since they constitutively express gal-1 (39
In T1D, presentation of β-cell-Ag and activation of diabetogenic T-cells takes place initially in PLN (57
). Thus, following therapy with gal-1, apoptosis of β-cell-reactive T-cells could occur in the PLN or when the activated T-cells infiltrate the pancreas. We found that gal-1-therapy increased the percentage of apoptotic T-cells in PLN, a finding that correlated with reduction in the percentage of β-cell-reactive CD4+
T-cells specific for the IAg7
-BDC-13 complex. Although we were unable to detect an increased number of apoptotic T-cells in the islet infiltrates, we can not completely rule out the possibility that gal-1-therapy promotes T-cell death locally within the pancreas, since it is well established that apoptotic cells are removed rapidly from peripheral tissues by phagocytes (58
There is evidence that administration of in vitro-expanded CD4+
β-cell-specific Treg suppresses T1D in NOD mice (35
) and that transfer of CD4+
T-cells from gal-1-treated mice prevents onset of autoimmune uveitis in untreated recipients (17
), the latter suggesting that gal-1-therapy promotes generation/expansion of tissue-specific Treg. However, in our system, transfer of CD4+
T-cells from gal-1- treated mice did not prevent/delay onset of hyperglycemia in a transfer model of T1D. This finding suggests that, in the absence of deletion and Th2-bias of β-cell-specific T cells and probably other gal-1-mediated anti-inflammatory effects, generation of CD4+
Treg is not the main mechanism by which gal-1 prevents T1D.
Gal-1-therapy was also effective in sub-clinical stages of T1D in NOD mice. These results have clinical relevance, since prediction of early pre-diabetic stages or Ab-positive high-risk first-degree relatives has become more precise in humans (2
). Once the β-cell-reactive T-cells become activated, there is often a quite long asymptomatic period of time until development of T1D, known as “honeymoon period”, when therapy with the lectin could be beneficial.
One of the most important findings of this study is that gal-1-therapy was capable of restraining T-cell autoimmunity against β-cells even in NOD mice with declared T1D. This is of clinical importance, since most patients are diagnosed at late stages of the disease when they are overtly diabetic. Besides, remission of declared β-cell autoimmunity at a time early enough to preserve the remaining β-cell mass/precursors within the islets is still the best therapeutic approach for patients with T1D. The results indicate that once the T-cell-mediated aggression against the β-cells was abrogated by administration of gal-1, the remaining islets recuperate their capacity to maintain normoglycemia in a considerable number of the lectin-treated animals. Gal-1-therapy down-regulated β-cell autoimmunity even in those mice that remained hyperglycemic, demonstrating that the failure to restore normoglycemia was likely due to the incapacity of the β-cells to regain their function and/or recuperate the β-cell mass, leading to metabolic diabetes. This may be ascribed to the absence of viable β-cell precursors at the time of initiation of the treatment.
Since gal-1 is an endogenous lectin, it is expected to be non-toxic and non-immunogenic when injected repeatedly. In fact, administration of the lectin did not cause major side effects in NOD mice. More importantly, unlike several immunosuppressive drugs used to controls recurrence of T1D and rejection of islet cell allografts, gal-1 did not seem to be toxic for the β-cells in the mouse. The fact that gal-1 does not trigger apoptosis0 of Th2 cells could explain why, in our model, exogenous gal-1 did not affect the systemic B-cell response against a model Ag. Interestingly, although gal-1-therapy prevented T-cell autoimmunity against β-cells, it did not affect the skin CH response elicited by haptens. Since cutaneous CH is susceptible to down-modulation by local injection of cells expressing gal-1 (27
), our findings indicate that, at the dose and route employed, the concentration of exogenous gal-1 in periphery did not reach enough levels to induce generalized immunosuppression.
Gal-1 is a soluble protein with no posttranslational modifications, therefore large batches of the lectin can be produced in bacterial expression systems for therapeutic applications. However, since the subunits of the gal-1 homodimer are not covalently linked and the affinity for each other is rather low, the in vivo efficacy of gal-1 still depends on administration of relatively large amounts of the lectin. To overcome this problem, Battig et al. (59
) have generated by genetic engineering covalently linked gal-1 homodimers, 10-fold more effective than the wild type molecule, that could be employed therapeutically at much lower doses. To our knowledge, this is the first study on the use of a soluble lectin to treat T1D. Optimization of variables for therapy with gal-1, including dose and timing of administration, the use of genetically improved variants of gal-1, and its combination with other Th1 immune-regulatory galectins like gal-9 (60
), could open new possibilities for treatment of T1D.