Increased numbers of rabbit blood lymphocytes with allotypic determinants demonstrated by double-coating indirect rosette formation.

Using single-coating indirect rosette formation (SIRF), a modification of the mixed antiglobulin reaction, the percentage of rabbit peripheral blood lymphocytes with demonstrable a1 immunoglobulin allotypic determinants is far smaller than the percentage with b4 or b5 allotypic determinants. The number of lymphocytes with a1 determinants, which are located on the Fd portion of the immunoglobulin molecule, is consistently increased (two to eight-fold) using double-coating indirect rosette formation (DIRF). In contrast, the percentage of lymphocytes with b4 or b5 determinants, which are located on the immunoglobulin light chains, remains the same or is only slightly increased, so that the lymphocytes from four a1a1 b5b5 rabbits, when tested separately for a1 and b5 by DIRF, have similar percentages of a1 and b5 containing cells. SIRF lymphocytes are formed by coating lymphocytes with antiallotypic antibody which will bind to erythrocytes coated with subagglutinating doses of anti-RBC antibody which carries the same allotypic specificity as the lymphocyte donor. DIRF lymphocytes are formed by interposing two antiallotypic molecules between the lymphocyte and the indicator red cell. For example, b4 lymphocytes coated with b5 anti-b4 molecules are double coated by the addition of anti-b5 made in a b6 rabbit and rosettes formed by the addition of sheep red cells coated with b5 anti-RBC. The consistent increase in the numbers of lymphocytes with a1 allotypic determinants by DIRF as compared to b group (b4 or b5) determinants suggests that the Fd portion of lymphocyte surface immunoglobulins is less exposed than light chains or the Fc piece.