Shirley J. Petzold

Structural Basis of Peptide Binding and Presentation by the Type I Diabetes-Associated MHC Class II Molecule of NOD Mice

We have determined the crystal structure of I-Ag7, an integral component in murine type I diabetes development. Several features distinguish I-Ag7 from other non-autoimmune-associated MHC class II molecules, including novel peptide and heterodimer pairing interactions. The binding groove of I-Ag7 is unusual at both terminal ends, with a potentially solvent-exposed channel at the base of the P1 pocket and a widened entrance to the P9 pocket. Peptide binding studies with variants of the hen egg lysozyme I-Ag7 epitope HEL(11-25) support a comprehensive structure-based I-Ag7 binding motif. Residues critical for T cell recognition were investigated with a panel of HEL(11-25)-restricted clones, which uncovered P1 anchor-dependent structural variations. These results establish a framework for future experiments directed at understanding the role of I-Ag7 in autoimmunity.

Characterization and Quantitation of Peptide–MHC Complexes Produced from Hen Egg Lysozyme Using a Monoclonal Antibody

Here we describe generation of Aw3.18, a monoclonal antibody that recognizes peptide residues 48-62 of hen egg lysozyme (HEL) bound to the MHC class II molecule I-Ak. Epitope mapping revealed that Aw3.18 detects a change in the solvent-exposed surface of this peptide-MHC complex upon substitution of the peptide side chain at position P1. Furthermore, Aw3.18 blocked recognition by some, but not all, of the HEL 48-62-reactive T cell hybridomas tested, suggesting a heterogeneity in the T cell response toward this complex. Finally, using Aw3.18, it was possible to determine the fraction of I-Ak molecules loaded with 48-62 peptide after culture of an antigen-presenting cell in medium containing HEL.

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.

Aphidicolin inhibits eukaryotic DNA replication and repair — Implications for involvement of DNA polymerase α in both processes

Abstract Normal human lymphocytes were stimulated with phytohemagglutinin or treated with UV radiation or N-methyl-N′-nitro-N-nitroso guanidine then rendered permeable to exogenously supplied nucleotides and used to measure the replicative and repair modes of DNA synthesis. Aphidicolin, N-ethyl maleimide and several other compounds inhibited both DNA replication and repair. Since aphidicolin and N-ethyl maleimide are selective inhibitors of DNA polymerase α, these findings suggest that DNA polymerase α is involved in both the replicative and repair modes of DNA synthesis.

Register shifting of an insulin peptide–MHC complex allows diabetogenic T cells to escape thymic deletion

A single amino acid shift in TCR recognition of self peptide–MHC determines whether potentially diabetogenic CD4 T cells will be purged in the thymus or have the opportunity to undergo activation in the islets of Langerhans of mice.

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.

Synthesis of DNA and poly(adenosine diphosphate ribose) in normal and chronic lymphocytic leukemia lymphocytes.

Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL) and 12 normal control donors. The cells were assayed for synthesis of DNA and poly-(adenosine diphosphate ribose) (poly[ADPR]) immediately after isolation and on successive days following their treatment with phytohemagglutinin (PHA). Two different techniques were used to measure DNA synthesis. In the standard technique, DNA synthesis was measured by incubating intact cells with [(3)H]deoxythymidine. In the new technique, the lymphocytes were first rendered permeable to nucleotides, then DNA synthesis was measured by incubating them with [(3)H]deoxythymidine triphosphate in the presence of deoxyATP, deoxyGTP, deoxyCTP, ATP, and Mg(++). Both assays showed the anticipated rise in DNA synthesis after PHA stimulation of normal cells. PHA-stimulated lymphocytes from patients with CLL demonstrated low levels of DNA synthesis in both assay systems. The initial levels of poly(ADPR) synthesis were greater in CLL lymphocytes than in normal cells. Studies with a T-cell-depleted population of normal cells showed the same activity for poly(ADPR) synthesis that was demonstrated by the original population of normal cells. PHA stimulation produced an increase in poly(ADPR) synthesis in both the normal and CLL cells. The increase in poly(ADPR) synthesis in normal cells was coincident with the increase in DNA synthesis. The increase in poly(ADPR) synthesis in the CLL cells was dissociated from the delayed and diminished increase in DNA synthesis. Thus, CLL cells have higher than normal initial levels of poly(ADPR) synthesis. Poly(ADPR) synthesis is dissociated from DNA synthesis in CLL cells whereas it varies directly with DNA synthesis in normal lymphocytes.

Association of poly(ADP-rib) synthesis with cessation of DNA synthesis and DNA fragmentation

CHO cells and cs-4-D3 cells were used to investigate the association between poly(ADP-rib) synthesis and the cessation of DNA synthesis and DNA fragmentation. The cs4-D3 cells are cold-sensitive DNA synthesis arrest mutants of CHO cells. Upon incubation at 33 degrees C, DNA synthesis in the cs4-D3 cells stops and the cells enter a prolonged G1 or G0 phase. The events that occurred when cs4 cells were incubated at 33 degrees C were similar to those that occurred when wild-type CHO cells grew to high density. (1) In both cases, DNA synthesis and cell growth stopped. (2) The NAD+ concentration/cell was 20-25% lower in growth-arrested cells than in logarithmically growing cells. (3) Poly(ADP-rib) synthesis was 3-4 fold higher in growth-arrested cells than in logarithmically growing cells. (4) The growth-inhibited cells developed DNA strand breaks which resulted in large percentages of their DNA appearing in the low molecular weight range of alkaline sucrose gradients. (5) Both the increased rate of poly(ADP-rib) synthesis and the development of DNA strand breaks appears to be characteristic of the G1 phase of the cell cycle. (6) When growth-inhibited cells were restored to conditions favorable for DNA synthesis and cell growth, the DNA strand breaks were repaired. (7) Prolonged incubation under growth-restrictive conditions resulted in the accumulation of more DNA strand breaks than the cells could repair. This was followed by cell death when the cells were restored to conditions favorable for cell growth.

Cutting edge: H-2DM is responsible for the large differences in presentation among peptides selected by I-Ak during antigen processing

We quantitated the amounts of peptides from hen egg-white lysozyme presented by I-Ak molecules in APC lines. The large chemical gradient of presentation of the four hen egg-white lysozyme epitopes observed in cell lines expressing HLA-DM or H-2DM (referred to in this study as DM) was significantly diminished in the T2.Ak line lacking DM. Differences in levels of presentation between wild-type and DM-deficient APC were observed for all four epitopes, but differences were most evident for the highest affinity epitope. As a result of these quantitative differences in display, presentation of all four epitopes to T cells was impaired in the line lacking DM. The binding affinity of the pool of naturally processed peptides from DM-expressing lines was higher than that from the DM-deficient line. Thus, using a direct biochemical approach in APC, we demonstrate that DM influences the selection of peptides bound to MHC class II by favoring high affinity peptides.

Relation of poly(adenosine diphosphoribose) synthesis to DNA synthesis and cell growth

A permeable cell technique has been used to measure the synthesis of DNA and poly(adenosine diphosphoribose) (poly(ADPR)) in mouse L cells subjected to different perturbations of cell growth. Cells leaving log phase growth and entering plateau phase, showed a decrease in DNA synthesis and an associated increase in intrinsic poly(ADPR) synthesis. In contrast to the variations in intrinsic poly(ADPR) synthesis, the total poly(ADPR) synthesis activity, measured in the presence of added DNAase, remained relatively constant during the fluctuations in cell growth status. Cells subjected to acute glucose deficiency also demonstrated a decrease in DNA synthesis and an associated increase in intrinsic poly(ADPR) synthesis. Similarly, cells infected with vaccinia virus demonstrated an abrupt cessation of DNA synthesis associated with an increase in poly(ADPR) synthesis. Treatment of cells with cytosine arabinoside, inhibited cellular DNA synthesis. This was also associated with an increase in the intrinsic activity of poly(ADPR) synthesis. However, in this case, the increase in poly(ADPR) synthesis was associated with an increase in activity of the DNA synthesis complex, despite the overall inhibition of cell DNA synthesis. These studies demonstrate, that in mouse L cells, suppression of DNA synthesis by multiple different physiologic mechanisms is always associated with an increase in intrinsic activity of poly(ADPR) synthesis.