High-throughput antibody generation and characterization

A phage display library has been constructed containing over 1010 human antibodies, allowing the large-scale generation of antibodies. Over 38,000 recombinant antibodies against 292 antigens were selected, screened and sequenced, and 4,400 resultant unique clones characterized further.

Autoimmune gastritis is a CD4+ T cell-mediated disease induced in genetically susceptible mice by thymectomy on the third day after birth. Previous linkage analysis indicated that Gasa1 and Gasa2, the major susceptibility loci for gastritis, are located on mouse chromosome 4. Here we verified these linkage data by showing that BALB.B6 congenic mice, in which the distal approximately 40 Mb of chromosome 4 was replaced by C57BL/6 DNA, were resistant to autoimmune gastritis. Analysis of further BALB.B6 congenic strains demonstrated that Gasa1 and Gasa2 can act independently to cause full expression of susceptibility to autoimmune disease. Gasa1 and Gasa2 are located between D4Mit352-D4Mit204 and D4Mit343-telomere, respectively. Numerical differences in Foxp3+ regulatory T cells were apparent between the BALB/c and congenic strains, but it is unlikely that this phenotype accounted for differences in autoimmune susceptibility. The positions of Gasa1 and Gasa2 correspond closely to the positions of Idd11 and Idd9, two autoimmune diabetes susceptibility loci in nonobese diabetic (NOD), mice and this prompted us to examine autoimmune gastritis in NOD mice. After neonatal thymectomy, NOD mice developed autoimmune gastritis, albeit at a slightly lower incidence and severity of disease than in BALB/c mice. Diabetes-resistant congenic NOD.B6 mice, harbouring a B6-derived interval encompassing the Gasa1/2-Idd9/11 loci, demonstrated a slight reduction in the incidence of autoimmune gastritis. This reduction was not significant compared with the reduction observed in BALB.B6 congenic mice, suggesting a difference in the genetic aetiology of autoimmune gastritis in NOD and BALB mice.

BALB/c mice thymectomized on their third day of life develop a high incidence of experimental autoimmune gastritis (EAG) which closely resembles human chronic atrophic (type A, autoimmune) gastritis. Linkage analysis of (BALB/cCrSlcxC57BL/6)F2 mice previously demonstrated that the Gasa1 and Gasa2 genes on distal Chromosome (Chr) 4 have major effects on the development of EAG in this murine model, while other loci displayed a trend towards linkage. Here, we implemented partitioned chi(2)-analysis in order to develop a better understanding of the genotypes contributing to susceptibility and resistance at each linkage region. This approach revealed that linkage of Gasa1 and Gasa2 to EAG was due to codominant and recessive BALB/cCrSlc alleles, respectively. To identify additional EAG susceptibility genes, separate linkage studies were performed on Gasa1 heterozygotes and Gasa2 C57BL/6 homozygotes plus heterozygotes so as to minimize the effects of these disease genes. The enhanced sensitivity of these analyses confirmed the existence of a third EAG susceptibility gene (designated Gasa3) on Chr 6. Epistatic interactions between the Gasa2 EAG susceptibility gene and the H2 were also identified, and the presence of an H2-linked susceptibility gene (Gasa4) confirmed by analysis of H2 congenic mice.

A familial component to the tendency to develop autoimmune gastritis has long been recognized. Although linkage to certain HLA alleles and an association with the endocrine autoimmune diseases thyroiditis and type 1 diabetes have been reported, little further progress has been achieved in clinical studies. In contrast, the mouse model of gastritis induced in the BALB/c strain by thymectomy in the third day of life has identified four linkage regions; two on distal chromosome 4 (Gasa1 and Gasa2), one on chromosome 6 (Gasa3) and one in the H2 (Gasa4). Three of these four genes colocalize with NOD mouse diabetes susceptibility genes--the strongest concordance identified to date between any two autoimmune diseases--reflecting the association between autoimmune diabetes and type 1 gastritis in humans.

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