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Two-dimensional electrophoretic analysis (2D-PAGE) of cell surface human DP and DR class II antigens identified a glycoprotein, designated pX, that is associated at the cell surface with DP but not DR class II antigen in activated T, B and NK lymphocytes but not in resting B lymphocytes, Raji B lymphoma cells, activated thymic epithelial cells or activated monocytes. pX is a heavily glycosylated protein with an apparent molecular mass spanning between 38 kDa and 22 kDa, that is reduced, after deglycosylation with Endo-F, to 22 kDa. The pX structure appears nonpolymorphic and independent of DP polymorphism, as suggested by 2D-PAGE migrational pattern of 125I-labelled Endo-F deglycosylated DP immunoprecipitates from T cells blasts derived from four donors with different DP allotypes. The apparent absence of polymorphism of pX is further suggested by two-dimensional peptide mapping of a single spot derived from 2D-PAGE of 125I-labelled DP deglycosylated immunoprecipitates from two donors.

IDDM patients of North East Italian region were molecularly typed for their HLA-DQB1 and DQA1 loci by using allele specific oligonucleotide probes and PCR amplified genomic DNA. IDDM status strongly correlated with DQB1 alleles carrying a non-aspartic acid residue in position 57 of DQ beta chain and DQA1 alleles with an arginine residue in position 52 of DQ alpha chain. Genotype analysis revealed that individuals with two DQB1 alleles having a non-aspartic residue in position 57 and two DQA1 alleles with an arginine residue in position 52 had the highest relative risk of disease: they constituted 41% of IDDM patients as compared to 0% of controls. Heterozygosity either at residue 57 of DQB1 or residue 52 of DQA1 was sufficient to abrogate statistical significance for disease association, although 43.6% of IDDM patients were included in these two groups as compared to 21.6% of normal controls. On the other hand the presence of two DQB1 alleles with aspartic acid in position 57 was sufficient to confer resistance to disease irrespective of the DQA1 genotype. Based on the number of possible susceptible heterodimers an individual can form, it was found that 85% of IDDM cases could form two or more heterodimers (two in cis and two in trans), but no IDDM case was found to form one susceptible heterodimer in cis. These results demonstrate that the complete HLA-DQ genotype, more than single DQB1 or DQA1 alleles or DQB1-DQA1 haplotypes, is associated with the highest risk of disease. Screening of the population for preventive purposes and/or early signs of IDDM should then take advantage of this result and "susceptible homozygous" individuals should be followed very closely and considered the first group of choice for possible new therapeutical trials.

We report on HLA-DQB1 typing in IDDM patients of north east Italian region using an enzymatic method based on the detection of hybridization reaction between PCR amplified DNA from whole blood and allele specific oligonucleotides by an antibody directed against double stranded DNA (DNA-enzyme immunoassay or DEIA). The method is reliable, simple and sensitive as the classical radioactive method with the advantage of using a universal non radioactive detection reagent. Nineteen families, each including one subject with juvenile insulin-dependent diabetes mellitus (IDDM) were analyzed. A strong association between absence of an aspartic acid (Asp) in position 57 of DQB1 beta chain in homozygous conditions and susceptibility to IDDM was found. In contrast with some previous observations, however, no significant association was found between Asp/non-Asp heterozygous genotype and IDDM. No patients were found with an homozygous Asp/Asp genotype, known to be protective in caucasoid population. Of particular interest was the DQB1 allelic distribution in our population sample. The non-Asp allele most frequently found in IDDM subjects was the DQB1 0201 allele and this finding was statistically significant (Pc value < 0.05, relative risk = 5.01). No significant association was found for any other allele including the DQB1 0302 (Pc value = not significant although with relative risk = 3.28) previously reported as the most frequent allele in IDDM caucasoid patients.

The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.

The biochemical features of a membrane antigen detected by a mouse monoclonal antibody (A1) raised against the murine thymoma cell line EL4 are described. This reagent detected a novel disulfide-linked 90,000 mol. wt dimeric membrane glycoprotein composed of two chains of approx 45,000 mol. wt. Endo-beta-N-acetylglucosaminidase F digestion generated a single 28,000 polypeptide, thus suggesting that the A1 molecule is a homodimer. No structural homology between the A1 molecule and the human T 90/44 protein (9.3 antigen) could be revealed by peptide mapping analysis. In view of the fact that three polypeptides of mol. wts 28,000-30,000, 21,000 and 15,000 respectively co-precipitated with the A1 antigen, the possible relationship of the A1 molecular complex to other known T-cell surface antigens including the antigen receptor is discussed.