Antigen density on target cells determines the immunosuppressive potential of rat IgG2b monoclonal antibodies

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Abstract
The current studies were designed to determine the relevance of T cell antigen density, besides antibody isotype, with regard to the success of antibody serotherapy. We compared the immunosuppressive effects of two rat IgG2b monoclonal anti‐Thy‐1 antibodies, RmT1 and 30‐H12, with distinct binding sites in a graft‐vs.‐host disease (GVHD) model of fully H‐2 and I‐A regionmismatched bone marrow transplantation, making use of the difference in Thy‐1.2 antigen density between homozygous (BALB/c) and heterozygous (BALB/c×AKR/J)F1 GVHD‐promoting donor cells. Antibodies RmT1 (directed against a monomorphic determinant on mouse Thy‐1) and 30‐H12 (reactive with the Thy‐1.2 allele‐specific determinant) did not differ in their anti‐GVHD activity with regard to Thy‐1.2 homozygous grafts. However, in the region of a critical number of binding sites a small difference in the amounts of the two antibodies bound (about 8×103 IgG molecules/cell) obviously accounts for a great difference in anti‐GVHD activity. This is shown in a two haplotype host‐graft disparity between C57BL/6 recipients treated with either RmT1 or 30‐H12 before challenging them with (BALB/c×AKR/J)F1 grafts, where the Thy‐1.2 antigen concentration is approximately 50% compared to the density on BALB/c lymphocytes. Here, mAb 30‐H12 loses its remarkable in vivo immunosuppressive quality, whereas RmT1 treatment protects mice against lethal GVHD. Binding sites were quantitated using a computerized approach for the analysis of data from ligand binding experiments of the respective mAb, RmT1 and 30‐H12, coated to LN cells of BALB/c and F1 hybrid origin. Furthermore, the in vivo immunosuppressive activity of rat IgG2b antibodies directed against Thy‐1 was found to correlate with their ability to generate stable antibody‐Clq complexes on the cell surface of immunocompetent T cells.