Bhagirath Singh

Immunization With the Larger Isoform of Mouse Glutamic Acid Decarboxylase (GAD67) Prevents Autoimmune Diabetes in NOD Mice

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) has been implicated in autoimmune diabetes in NOD mice, but the role of the 67-kDa GAD isoform (GAD67) is less clear. We found that immunization of 4-week-old NOD mice with purified recombinant mouse GAD67 prevented or significantly delayed the onset of diabetes. To further explore this phenomenon, we characterized anti-GAD67 immune responses in naive and GAD-immunized NOD mice. Anti-GAD67 antibodies titers were relatively low in naive mice at all ages, but a single immunization with GAD67 at 4 weeks induced high titers of anti-GAD antibodies by 6 weeks of age. In both 4-week-old and diabetic NOD mice, there were significant endogenous T-cell proliferative responses against purified recombinant mouse GAD67. These T-cell proliferative responses were blocked by anti-I-ANOD and anti-CD4 antibodies. To characterize the anti-GAD T-cell responses in the NOD mice, we established T-cell lines and T-cell clones which recognized GAD67, and we used recombinant subfragments of GAD to localize the predominant T-cell epitopes in GAD67. T-cells from naive NOD mice proliferated in response to all GAD subfragments, whereas T-cells from diabetic mice responded primarily to the COOH-terminal 83 amino acids of GAD67. These results suggest that GAD67 is an autoantigen in IDDM and immunization of prediabetic NOD mice with GAD67 can prevent the onset of diabetes.

Quantitation of the capacity of the mouse placenta to absorb monoclonal anti-fetal H-2K antibody

Abstract Previous results have indicated that the murine placenta can serve as an immunoabsorbent for anti-H-2 antibodies in the maternal circulation, including monoclonal anti-H-2K. In this study we have used double-reciprocal plots of binding curves obtained in vivo by injection of graded amounts of monoclonal anti-H-2K to obtain an estimate of the number of H-2K antigenic determinants available for binding in the placenta. This number is 1.1 × 10 13 /g placenta. Competitive inhibition studies carried out both in vitro and in vivo indicate that the specificity of the antibody was not altered by radiolabelling and purification procedures.

Epitopes of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins recognized by antibodies in the sera of HIV-1-infected individuals☆

Sera from human immunodeficiency virus (HIV)-infected study subjects and controls were analyzed by enzyme-linked immunosorbent assay using 10 synthetic peptides to identify epitopes of HIV envelope glycoproteins (ENVgp) that were recognized by antibodies. Two epitopes of HIV ENVgp, ENVP466 (amino acids 466-481) and ENVP497 (amino acids 497-509), were recognized by antibodies in the sera of most HIV-infected individuals. The frequency of individuals with detectable serum antibodies to these two epitopes was not associated with the stage of HIV disease. Purified antibodies to ENV497 had only very weak neutralizing activity against infectious HIV. These data suggest that a particular dominant type of antibody response to HIVs ENVgp has minimal protective effects. These and other studies to identify and stimulate immune responses to selected epitopes of HIV antigens may be useful in the design of vaccines to prevent or treat HIV infections.

A New "Marker" Protein for Astrocytes

A monoclonal antibody (Mab J1-31) has been produced by using human brain homogenate as immunogen in mouse. Double-label immunofluorescence microscopy on cryostat sections of human, rabbit and rat brain, reveals staining of cells that are also stained with antiserum to glial fibrillary acidic protein (GFAP, a commonly used marker protein for astrocytes). However, there is no decrease in staining due to Mab J1-31 in sections incubated in antiserum to GFAP prior to incubation with the J1-31 ascites fluid. Immunoprecipitation of aqueous and detergent extracts of brain homogenate gives a single band at 30K by SDS PAGE followed by autoradiography. Immunoelectron microscopy shows that the J1-31 antigen is associated with the cytoskeleton. Thus, the Mab J1-31 recognizes a new protein present in GFAP positive cells (astrocytes) in the brain.

Theoretical studies of protein structures: prediction of antigenic determinants

Abstract The determination of stable conformations of proteins by means of a 1/R formulation, developed previously at this laboratory for the study of molecular interactions, is discussed. The details of the method (and of the corresponding program) are presented and its practical applicability is examined on the basis of the results obtained for some chosen systems. This method may be used as an auxiliary tool, in conjunction with the results obtained from hydrophilicity-recognition profiles, for the theoretical prediction of the antigenic determinants of proteins.

Unusually diverse T cell response to a repeating tripeptide epitope

The immune system utilizes a diverse T cell repertoire for the recognition of foreign antigens in the context of self MHC gene products. We have examined the potential diversity of the T cell response directed to a immunodominant repeating tripeptide epitope (EYA)5. This peptide represents one of the two T cell epitopes on the synthetic alpha-helical polypeptide antigen Poly 18, Poly EYK(EYA)5 in H-2d mice and does not require antigen processing prior to presentation to Poly 18-specific T cell hybridomas. The T cell response directed to the repeating tripeptide epitope (EYA)5 is extremely heterogenous even though the epitope has a relatively simple amino acid sequence. We have analyzed the fine specificity of 21 randomly chosen Poly 18-reactive, (EYA)5-specific and H-2d-restricted T cell hybridomas derived from H-2d, H-2bxd, and H-2b----H-2bxd Poly 18-responding mice to determine the number of unique antigen reactivity patterns represented by this T cell population. We used alanine- and/or lysine-substituted (EYA)5 peptides and a panel of haplotype-varied splenocytes and observed a great deal of microheterogeneity in response. We find that 13 of the 21 hybridomas have a distinct fine antigen specificity and T cell receptors. The binding of (EYA)5 to the antigen-binding groove of I-Ad appears to generate a highly diversified T cell response. Therefore, (EYA)5-I-Ad complex allows the activation of unrelated T cell clonotypes with the same overall antigen specificity and MHC restriction, but with distinct microheterogeneity in response and receptor usage.

Gene conversion may be responsible for the generation of the alloreactive repertoire

Meiotic gene conversion in the major histocompatibility complex (MHC) appears to be involved in the generation of MHC polymorphism. Here the authors suggest that mitotic gene conversion of MHC may generate somatic variants which sensitize the immune system. This could lead to an MHC-skewed repertoire with a high background alloreactive potential crossreacting with foreign antigens in the context of self MHC. Allodeterminant-bearing autologous cells may be deleted by the immune cells, thereby maintaining the observed genotype/phenotype correlation.

Role of the first external domain of I-Aβ chain in immune responses and diabetes in non-obese diabetic (NOD) mice

Diabetes in the non-obese diabetic (NOD) mouse is a multigenic autoimmune disease and is possibly controlled by three recessive loci, including one that is linked to the major histocompatibility complex (MHC). The first external domain of the Class II MHC I-A beta chain in these mice is unique and has been suggested as being responsible for autoimmunity. The I-A alpha chain in these mice is I-A alpha d, and they lack the expression of I-E molecules. We have investigated immune responses to various Ir gene control antigens in NOD mice to determine the influence of the NOD Ia and particularly the I-A beta chain. We find that sheep insulin is highly immunogenic while other insulins are weakly immunogenic in these mice. Hen egg lysozyme, pigeon cytochrome C and the synthetic polypeptide Poly 18, Poly EYK(EYA)5 antigen produce good antibody responses. Apart from H-2d, NOD are the only mice where Poly 18 antigen is immunogenic. In these mice Poly 18 induced good T-cell proliferative response, which was inhibited by anti-Ia antibody, and the mice were able to respond to tyrosine-containing polypeptide Poly EYA but not to the phenylalanine-containing antigen Poly EFA. We also found that synthetic peptide 48-60 of the NOD I-A beta chain is highly immunogenic in syngeneic NOD mice both for T cells and B cells. Using an I-A beta chain-specific monoclonal antibody, we are able to prevent induction of diabetes when the antibody was administrated in prediabetic, young mice. Our results suggest that the immune response to various antigens and autoimmune diabetes in NOD mice is directly influenced by the I-A beta chain.

Characterization of agretopes and epitopes involved in the presentation of beef insulin to T cells

Beef insulin-specific I-Ad-restricted T cell hybridomas were derived from the fusion of antigen-primed (BALB/c X B6)F1 T cells with BW5147 thymoma. Specificity analysis revealed that the A-chain loop region is involved in antigen recognition. Hybridoma A20.2.15 is specific for beef insulin and cross-reacted with sheep insulin, but not with pork insulin. Using synthetic peptides we showed that the A-chain loop containing peptide A1-A14 jointed to the B7-B15 peptide by a disulfide bond can activate this hybridoma. Fragments generated by enzyme digest further suggest that the peptide recognized on beef insulin appears to involve A-chain loop residues A5-A12 and B-chain residues B7-B13 that are linked by the A7-B7 disulfide bridge. We found that beef insulin needs to be processed prior to T cell activation. Glutaraldehyde fixation and chloroquine treatment of presenting cells abolished their capacity to present insulin. Beef insulin denatured by pH changes cannot activate, thus suggesting that simple denaturation is not sufficient for presentation by antigen presenting cells. Finally, the agretope on beef insulin is comprised of two functional regions B7-B13 on the B chain and the A-chain loop in the A-chain, while residues A8 and A10 are probably involved in interaction with the T cell receptor.

Theoretical studies of transplantation antigens: Predicted conformation and structure-function relationship of the murine MHC class i antigen H-2Kb

Abstract Despite the elucidation of the primary sequences of a number of transplantation antigens from human and murine sources, no tertiary structure has yet been determined. In an attempt to determine a theoretically-stable spatial conformation for the heavy chain and the β2-microglobulin, which constitute the murine class I transplantation antigen H-2Kb, a computer program has been used. This program, developed by us, searches for the preferred conformations of proteins, under an energy criterion, through variation of the dihedral angles Φ,Ψ,χ,1. A stable conformation of the complex formed by both chains was then obtained, also under an energy criterion, by means of a molecular interactions program developed in this laboratory. The information obtained from the resulting structure (spatial distribution of the residues and helical content), together with complementary information from the hydrophilicity and recognition profiles of the heavy chain, suggest that the sequence regions 63–72 and 129–139 constitute the alloantigenic determinants of H2Kb. The fact that the 63–72 region is an alloantigenic determinant has been confirmed by the use of a synthetic peptide 61–69, which elicits specific T and B cell immune responses against the H-2Kb molecule.