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-Ag7, shares a common α-chain with I-Ad but has a peculiar β-chain. As with most β-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position β57. We have produced large amounts of empty I-Ag7 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-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, 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-Ag7 over I-Ad 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-Ad and I-Ag7 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-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.
CD4 T cells are pivotal for effective immunity, yet their initial differentiation into effector subsets after infection remains poorly defined. We examined CD4 T cells specific for the immunodominant Leishmania major LACK antigen using MHC/peptide tetramers and IL-4 reporter mice. Comprising approximately 15 cells/lymph node in naive mice, LACK-specific T cells expanded over 100-fold, and 70% acquired IL-4 expression by 96 hr. Despite their pathogenic role in susceptible mice, LACK-specific precursor frequency, expansion, and IL-4 expression were comparable between susceptible and resistant mice. When injected with unrelated antigen, Leishmania efficiently activated IL-4 expression from naive antigen-specific T cells. CD4 subset polarization in this highly characterized model occurs independently from IL-4 expression by naive T cells, which is activated indiscriminately after parasitism.
To detect and characterize autoreactive T cells in diabetes-prone NOD mice, we have developed a multimeric MHC reagent with high affinity for the BDC-2.5 T cell receptor, which is reactive against a pancreatic autoantigen. A distinct population of T cells is detected in NOD mice that recognizes the same MHC/peptide target. These T cells are positively selected in the thymus at a surprisingly high frequency and exported to the periphery. They are activated specifically in the pancreatic LNs, demonstrating an autoimmune specificity that recapitulates that of the BDC-2.5 cell. These phenomena are also observed in mouse lines that share with NOD the H-2 g7 MHC haplotype but carry diabetes-resistance background genes. Thus, a susceptible haplotype at the MHC seems to be the only element required for the selection and emergence of autoreactive T cells, without requiring other diabetogenic loci from the NOD genome.
To detect and characterize autoreactive T cells in diabetes-prone NOD mice, we have developed a multimeric MHC reagent with high affinity for the BDC-2.5 T cell receptor, which is reactive against a pancreatic autoantigen. A distinct population of T cells is detected in NOD mice that recognizes the same MHC/peptide target. These T cells are positively selected in the thymus at a surprisingly high frequency and exported to the periphery. They are activated specifically in the pancreatic LNs, demonstrating an autoimmune specificity that recapitulates that of the BDC-2.5 cell. These phenomena are also observed in mouse lines that share with NOD the H-2 g7 MHC haplotype but carry diabetes-resistance background genes. Thus, a susceptible haplotype at the MHC seems to be the only element required for the selection and emergence of autoreactive T cells, without requiring other diabetogenic loci from the NOD genome.
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-2d and H-2b genetic backgrounds, we developed I-Ad- and I-Ab-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.
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