Susceptibility to murine and human insulin-dependent diabetes mellitus correlates strongly with major histocompatibility complex (MHC) class II I-A or HLA-DQ alleles that lack an aspartic acid at position β57. I-A ^g7 lacks this aspartate and is the only class II allele expressed by the nonobese diabetic mouse. The crystal structure of I-A ^g7 was determined at 2.6 angstrom resolution as a complex with a high-affinity peptide from the autoantigen glutamic acid decarboxylase (GAD) 65. I-A ^g7 has a substantially wider peptide-binding groove around β57, which accounts for distinct peptide preferences compared with other MHC class II alleles. Loss of Asp ^β57 leads to an oxyanion hole in I-A ^g7 that can be filled by peptide carboxyl residues or, perhaps, through interaction with the T cell receptor.

Cholera toxin subunit B (CTB) is the nontoxic portion of cholera toxin. Its affinity to the monosialotetrahexosylganglioside (GM1) that is broadly distributed in a variety of cell types including epithelial cells of the gut and antigen presenting cells, macrophages, dendritic cells, and B cells, allows its optimal access to the immune system. CTB can easily be expressed on its own in a variety of organisms, and several approaches can be used to couple it to antigens, either by genetic fusion or by chemical manipulation, leading to strongly enhanced immune responses to the antigens. In autoimmune diseases, CTB has the capacity to evoke regulatory responses and to thereby dampen autoimmune responses, in several but not all animal models. It remains to be seen whether the latter approach translates to success in the clinic, however, the versatility of CTB to manipulate immune responses in either direction makes this protein a promising adjuvant for vaccine development.

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-A^g7, shares a common α-chain with I-A^d but has a peculiar β-chain. As with most β-chain alleles linked to diabetes susceptibility, I-A^g7 contains a nonaspartic residue at position β57. We have produced large amounts of empty I-A^g7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-A^g7. Structural information was critical in evaluating the binding studies. I-A^g7, like I-A^d, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-A^g7 over I-A^d as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-A^d and I-A^g7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the β-chain. These characteristics link I-A^g7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.

The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β-cells in type 1 diabetes. A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4⁺ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4⁺ T cell expansion. We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.

Subtle differences oppose CD4⁺ to CD8⁺ T cell physiologies that lead to different arrays of effector functions. Interestingly, this dichotomy has also unexpected practical consequences such as the inefficacy of many MHC class II tetramers in detecting specific CD4⁺ T cells. As a mean to study the CD4⁺ anti-OVA response in H-2^d and H-2^b genetic backgrounds, we developed I-A^d- and I-A^b-OVA recombinant MHC monomers and tetramers. We were able to show that in this particular system, despite normal biological activity, MHC class II tetramers failed to stain specific T cells. This failure was shown to be associated with a lack of cooperation between binding sites within the tetramer as measured by surface plasmon resonance. This limited cooperativeness translated into a low “functional avidity” and very transient binding of the tetramers to T cells. To overcome this biophysical barrier, recombinant artificial APC that display MHC molecules in a lipid bilayer were developed. The plasticity and size of the MHC-bearing fluorescent liposomes allowed binding to Ag-specific T cells and the detection of low numbers of anti-OVA T cells following immunization. The same liposomes were able, at 37°C, to induce the full reorganization of the T cell signaling molecules and the formation of an immunological synapse. Artificial APC will allow T cell detection and the dissection of the molecular events of T cell activation and will help us understand the fundamental differences between CD4⁺ and CD8⁺ T cells.