Matteo G. Levisetti

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function.

Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes

In addition to the genetic framework, there are two other critical requirements for the development of tissue-specific autoimmune disease. First, autoreactive T cells need to escape thymic negative selection. Second, they need to find suitable conditions for autoantigen presentation and activation in the target tissue. We show here that these two conditions are fulfilled in diabetic mice of the nonobese diabetic (NOD) strain. A set of autoreactive CD4+ T cells specific for an insulin peptide, with the noteworthy feature of not recognizing the insulin protein when processed by antigen-presenting cells (APCs), escaped thymic control, participated in diabetes and caused disease. Moreover, APCs in close contact with beta cells in the islets of Langerhans bore vesicles with the antigenic insulin peptides and activated peptide-specific T cells. Our findings may be relevant for other cases of endocrine autoimmunity.Besides the genetic framework, there are two critical requirements for the development of tissue-specific autoimmune diseases. First, autoreactive T cells need to escape thymic negative selection. Second, they need to find suitable conditions for autoantigen presentation and activation in the target tissue. We show here that these two conditions are fulfilled in diabetic NOD mice. A set of autoreactive CD4+ T cells specific for an insulin peptide, with the noteworthy feature of not recognizing the insulin protein when processed by the antigen presenting cells (APC) escape thymic control, participate in diabetes and can cause disease. We also find that APCs situated in close contact with the beta cells in the islets of Langerhans bear vesicles with the antigenic insulin peptides and activate the peptide-specific T cells. These findings may be relevant for other cases of endocrine autoimmunity.

The Insulin-Specific T Cells of Nonobese Diabetic Mice Recognize a Weak MHC-Binding Segment in More Than One Form

Several naturally occurring anti-insulin CD4 T cells were isolated from islet infiltrates of NOD mice. In accordance with the results of others, these T cells recognized the segment of the β-chain from residues 9–23. Peptides encompassing the B:(9–23) sequence bound weakly to I-Ag7 in two main contiguous registers in which two residues at the carboxyl end, P20Gly and P21Glu, influenced binding and T cell reactivity. Naturally occurring insulin-reactive T cells exhibited differing reactivities with the carboxyl-terminal amino acids, although various single residue changes in either the flanks or the core segments affected T cell responses. The insulin peptides represent another example of a weak MHC-binding ligand that is highly immunogenic, giving rise to distinct populations of autoimmune T cells.

Weak Proinsulin Peptide–Major Histocompatibility Complexes Are Targeted in Autoimmune Diabetes in Mice

OBJECTIVE—Weak major histocompatibility complex (MHC) binding of self-peptides has been proposed as a mechanism that may contribute to autoimmunity by allowing for escape of autoreactive T-cells from the thymus. We examined the relationship between the MHC-binding characteristics of a β-cell antigen epitope and T-cell autoreactivity in a model of autoimmune diabetes. RESEARCH DESIGN AND METHODS—The binding of a proinsulin epitope, proinsulin-1(47–64) (PI-1[47–64]), to the MHC class II molecules I-Ag7 and I-Ak was measured using purified class II molecules. T-cell reactivity to the proinsulin epitope was examined in I-Ag7+ and I-Ak+ mice. RESULTS—C-peptide epitopes bound very weakly to I-Ag7 molecules. However, C-peptide–reactive T-cells were induced after immunization in I-Ag7–bearing mice (NOD and B6.g7) but not in I-Ak–bearing mice (B10.BR and NOD.h4). T-cells reactive with the PI-1(47–64) peptide were found spontaneously in the peripancreatic lymph nodes of pre-diabetic NOD mice. These T-cells were activated by freshly isolated β-cells in the presence of antigen-presenting cells and caused diabetes when transferred into NOD.scid mice. CONCLUSIONS—These data demonstrate an inverse relationship between self-peptide–MHC binding and T-cell autoreactivity for the PI-1(47–64) epitope in autoimmune diabetes.

Identification of naturally processed peptides bound to the class I MHC molecule H‐2Kd of normal and TAP‐deficient cells

This report details the biochemical features of natural peptides selected by the H‐2Kd class I MHC molecule. In normal cell lines, the length of the naturally processed peptides ranged from 8 to 18 amino acids, although the majority were 9‐mers (16% were longer than nine residues). The binding motif for the 9‐mer peptides was dominated by the presence of a tyrosine at P2 and an isoleucine/leucine at the P9 position. The P2 residue contributed most towards binding; and the short peptides bound better and formed longer‐lived cell surface complexes than the long peptides, which bound poorly and dissociated rapidly. The longer peptides did not exhibit this strictly defined motif. Trimming the long peptides to their shorter forms did not enhance binding and conversely, extending the 9‐mer peptides did not decrease binding. The long peptides were present on the cell‐surface bound to H‐2Kd (Kd) and were not intermediate products of the class I MHC processing pathway. Finally, in two different TAP‐deficient cells the long peptides were the dominant species, which suggested that TAP‐independent pathways selected for long peptides by class I MHC molecules.

Antigen presentation : Lysoyme, autoimmune diabetes, and listeria-what do they have in common?

We discuss three areas of antigen presentation and macrophage biology being investigated in the laboratory. Using hen egg-white lysozyme as a protein antigen, all the segments of the molecules selected by the class II histocompatibility molecule I-Ak were identified and characterized. The display of each family of peptides was explained biochemically and quantitated. Conformational isomers of a peptide-major histocompatibility complex (MHC) complex were identified. The relationship between the amounts of peptide-MHC displayed by the antigen-presenting cells and two biologic responses, central thymic selection and T-cell responses after immunization in adjuvant, were examined. The class II MHC molecule of the nonobese diabetic I-Ag7 is being examined for its properties of peptide selection. The objective is to identify the diabetogenic peptides, as well as the repertoire of protein antigens from β-cells that trigger autoantibodies. The I-Ag7 molecule selects peptides that show very distinctive sequence motifs: one or more acidic residues at the carboxy terminus that interact at the P9 pocket of the binding groove. Finally, the investigations in listeriosis examined the early events in immune induction. More important, we found that Listeria causes marked apoptosis of lymphocytes around infective foci resulting from the apoptogenic properties of the poreforming molecule Listeriolysin O.