Efficacy of clonal deletion vs. anergy of self-reactive CD4 T-cells for the prevention and reversal of autoimmune diabetes

Abstract

The self-reactive CD4 T-cells play an essential role in triggering and sustaining organ-specific autoimmune diseases. Silencing or elimination of these cells can prevent and reverse an autoimmune process. We have previously showed that a single dose-administration of a soluble dimeric MHC II–peptide chimera (DEF) in double-transgenic mice delayed the onset autoimmune diabetes, and restored the euglycemia in already diabetic mice for a period of 1 week. DEF dimer protection relied on induction of anergy of diabetogenic CD4 T-cells in spleen, and stimulation of IL-10-secreting T regulatory type 1 cells in pancreas. Herein, we show that an octameric form of DEF has doubled the period of protection and reversal of disease by clonal deletion of diabetogenic CD4 T-cells in both the thymic and peripheral compartments. Deletion occurred by activation-induced cell death subsequent to repartitioning and signaling of FAS–FADD apoptotic module in the plasma membrane lipid rafts. Our previous and present data indicated first, that DEF valence translates into various effects on the antigen-specific CD4 T-cells, i.e., Th2 immune deviation, anergy, and apoptosis. Second, the present findings argue for a better efficacy of clonal deletion than anergy of diabetogenic CD4 T-cells for the protection and reversal of autoimmune diabetes.

Introduction

Autoimmune diabetes (Type 1 Diabetes, T1D; insulin-dependent diabetes mellitus, IDDM) is a chronic autoimmune disease resulting from the T-cell-mediated destruction of pancreatic β-cells, and loss of insulin secretion [1], [2]. The role of CD4 T-cells in pathogenesis of autoimmune diabetes has been overtly demonstrated in various animal models [3], [4], [5].

Anti-mitotic agents like Cyclosporin A, anti-inflammatory cytokines, or antibodies directed against inflammatory cytokines or T-cell markers were shown to delay the onset of type 1 diabetes in animal models, but the risk for systemic immune suppression makes their use questionable in asymptomatic humans. Thus, targeting preferentially the self-reactive (antigen-specific) T-cells appears to be an attractive therapeutic approach [6]. Among the immune specific therapeutics, the synthetic peptides were shown to prevent or ameliorate the disease in animal models [7], but their short life in vivo requires recurrent administration at considerable high doses to reach the therapeutic effect. We have recently shown that a new class of antigen-specific ligands, namely soluble MHC/peptide complexes genetically engineered to express a self-peptide, (i) have longer half-life in vivo [8], (ii) are able to engage both the TCR and CD4 co-receptor on T-cells in an antigen-specific manner [9], and (iii) their modulatory potency on T-cell function can be detected at 1000-fold lower amounts on a molar basis than that of synthetic peptides [9]. In contrast to the synthetic peptide HA110-120, at low TCR/CD4 occupancy the soluble DEF reagent induces antigen-specific Th2 polarization by a negative regulation of STAT4 [9], whereas at high receptor occupancy induces anergy by a blockade of TCR signaling [10]. In a TCR-HA/RIP-HA double-transgenic (dTg) mouse model for autoimmune diabetes, the DEF dimer prevented diabetes when administrated in the prediabetic stage, and restored euglycemia in the recent-onset diabetic mice by induction of IL-10 secreting T regulatory cells (TR-1) in pancreas and anergy of diabetogenic CD4 T-cells in spleen [11]. However, the protection by DEF dimer required repeated administrations every 5–7 days, which indicated a transient immunomodulatory effect of this reagent. We have enzymatically engineered highly multimerized DEF reagents by cross-linking the carbohydrate moieties of DEF dimer at the galactose sites via diaminated polyethylene glycol NH₂-PEG₂₅₀₀-NH₂ [8].

Herein, we present evidence that the octameric form of DEF chimera provides a prolonged protection and better survival of TCR-HA/RIP-HA dTg prediabetic mice than its dimeric form, when administered at the early onset of hyperglycemia. The mechanism by which DEF octamer protected against diabetes relied mainly on the clonal deletion of HA-specific (diabetogenic) T-cell precursors in thymus subsequent to activation-induced cell death (AICD) of these cells through the reorganization and signaling of Fas apoptotic receptor and FADD adapter in plasma membrane lipid rafts.