Cell Surface Immunoglobulin

Abstract

There is accumulating evidence that the capacity of lymphocytes to bind antigen specifically (1-6) is due to Ig on the cell surface: anti-Ig causes the transformation of lymphocytes (7-12) and other changes in membrane properties (13,14); anti-L chain and anti-Ig inhibit both binding of specific antigen and stimulation of cellmediated immune reactions by antigen (15-18); anti-Ig of allotype or class specificity binds to lymphocytes (19-23); "rosette" formation around lymphocytes occurs when hybrid antibody to both Ig and specific antigen is added along with red cells coated with antigen (24, 25); lymphocytes treated with anti-Ig form rosettes with Ig-coated red cells (26,27); and specific adherence of lymphocytes to antigen-coated columns is blocked by pretreatment of cells with anti-Ig serum (28). There is conflicting evidence, however, as to whether the major class of cell surface immunoglobulin on normal splenic lymphocytes is IgM (12,20) or IgG (7, 21), as well as the distribution of Ig on cells (20,21).In evaluating the above results, I t should be emphasized that the methodologies used rely on the capacity of anti-Ig antibody to make effective contact with antigenic determinants of the receptor. Such contact depends upon the presence of determinants on portions of the receptor available to anti-Ig antibody in the medium and retention of antigenicity when the receptor is bound to the plasma membrane. These problems may account for the several contradictory reports mentioned above and the finding that individual antisera directed against the same immunoglobulin can give conflicting results, presumably depending on differences in the specificities of the antisera (20,29).For these reasons we developed an approach in which surface Ig could be studied by standard immuno-and biochemical techniques after its removal from the celt surface (30).

Cell Surface Immunoglobulin

“…The enzyme was removed by one to two washes with the same buffer. In several experiments, iodinated cells were fractionated into small and large lymphocytes on albumin gradients as previously described (3,6). In experiments in which radioiodinated cells were cultured, cells were washed only once before iodination and once afterwards.…”

“…Preparation of Iodinated Spleen Cells.--Splenic l)wnphocytes were prepared and iodinated as previously described (3). Cells were labeled at a concentration of 5 M 107-1 M l0 s cells/ml with 50/zg (0.1 ml) of lactoperoxidase (Calbiochem, Los Angeles, Calif.), 1-2 mCi 125I (1.0 ml), and 0.03% H202 (0.025 ml).…”

Induction of Immunity and Tolerance in Vitro by Hapten Protein Conjugates

“…Poisson analysis shows that there would be only 4% of DNP HgG molecules with more than one DNP group. Multivalent DNP HgG with 12 moles DNP/mole of HgG (DNP12HgG) was used as a DNP compound of approximately the same diameter as the receptor on B cells, a monomeric IgM molecule (13). Nonimmunogenic DNP3POL (1) was employed as a multivalent and polymeric D N P compound.…”

Cell Surface Immunoglobulin

“…The value for the control immunoprecipitate was subtracted from the experimental value to determine the amount of radioactive Ig that was specifically immunoprecipitated. Immunoprecipitates were dissolved in 2% SDS, reduced and alkylated and electrophoresed on SDS-5% acrylamide gels (1). Radioiodinated 7-and L-chains were electrophoresed on companion gels as molecular weight markers.…”

“…This finding was unexpected because the H chains synthesized b y and on the surface of splenocytes from similar animals are almost exclusively/z-chain (1). T o investigate this finding further, thoracic duct l y m p h o c y t e s and splenocytes of y o u n g B a l b / c mice were labeled with [3H]tyrosine or were surface radioiodinated with 125I.…”