Enzyme Immunoassay with Cell Suspensions: Studies on Reactions between Serum Antibodies and Cell-Surface Antigens

The following reactions of serum antibodies with cell surface antigens were studied by means of an enzyme immunoassay with cell suspensions: (1) Rh antiserum with human red blood cells; (2) H-2 antisera with murine red blood cells; (3) H-2 antisera with murine thymocytes and hepatocytes; (4) Thy-1 antiserum with murine thymocytes; (5) polyvalent and monovalent HLA alloantisera with human leukocytes and human mononuclear cells, and (6) antisera of murine origin with human and marmoset lymphoid cell lines. In all instances, with the exception of monovalent HLA antisera, the assay proved to be a sensitive and highly specific procedure.</jats:p

The conformation of Ld induced by beta 2-microglobulin is fixed during de novo synthesis and irreversible by exchange or dissociation.

Abstract Data are presented that support the hypothesis that beta 2m controls the folding of the ligand binding site of newly synthesized class I molecules. This conclusion was indicated by comparisons of two antigenic forms of the Ld molecule separated by sequential immunoprecipitation. Whereas, mAb 30-5-7+ Ld molecules were found to exist either as free H chains or associated with beta 2m, 30-5-7- Ld molecules showed no beta 2m association. Chemical comparisons showed 30-5-7- Ld molecules to be highly sensitive to proteolysis relative to 30-5-7+ Ld molecules. Experiments employing a construct with the Ld gene juxtaposed to the inducible metallothionein promoter indicated that the ratio of the antigenic forms of Ld was determined by the relative synthesis of beta 2m vs class I proteins. Pulse-chase experiments demonstrated that the two antigenic forms of Ld do not share a precursor-product relationship, but do display disparate rates of intracellular transport. beta 2m dissociation or exchange at the cell surface was found not to affect the ratio of the two antigenic forms of Ld. In contrast to these findings with Ld, the Dd and Ddml molecules were not detected in alternative conformations, thus mapping this property to the N-terminus of the class I molecule. These findings support the notion that beta 2m induces conformation on the alpha 1/alpha 2 domains of Ld molecules during de novo synthesis and once beta 2m-conformed, the class I structure is fixed and irreversible.</jats:p

Mutation at amino acid position 133 of H-2Dd prevents beta 2m association and immune recognition but not surface expression.

Abstract The histocompatibility loss mutation H-2dm6 was derived from a mouse treated with the chemical mutagen ethylnitrosourea, and previous mapping studies implicated Dd as the affected locus. Southern blot analyses of DNA from H-2dm6 cells did not detect major deletions in the Ddm6 gene, suggesting that H-2dm6 was different from the previously characterized D region mutants H-2dm1 and H-2dm2. RNA blot analysis identified Ddm6 transcripts of appropriate size and a Ddm6 protein was immunoprecipitated from biosynthetically labeled H-2dm6 cells. Interestingly, the Ddm6 protein showed no beta 2m association and was only precipitated by a mAb to the alpha 3 domain. Furthermore, oligosaccharide maturation and low levels of surface expression of Ddm6 molecules were detected. However, the surface Ddm6 was nonfunctional as a target Ag in in vitro cytotoxicity assays, consistent with its original in vivo detection as a loss mutation. Sequence analyses of Ddm6 cDNA identified a single nucleotide base difference from wild-type, resulting in the substitution of a Trp to Arg at position 133. The significance of this substitution is discussed in the context of other class I expression variants.</jats:p

Full hematopoietic engraftment after allogeneic bone marrow transplantation without cytoreduction in a child with severe combined immunodeficiency

Abstract Bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in T-lymphocyte engraftment and correction of immune dysfunction but not in full hematopoietic engraftment. A case of a 17-month-old girl with adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic engraftment developed after BMT from her HLA-identical sister is reported. No myeloablative or immunosuppressive therapy or graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and chronic GVHD developed, her B- and T-cell functions became reconstituted, and she is well almost 11 years after BMT. After BMT, repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.</jats:p