A Quantitative Study at the Single Cell Level of Immunoglobulin Antigenic Determinants Present on the Surface of Murine B and T Lymphocytes

Abstract

An enzyme-immunoassay for the quantitation at the single cell level of immunoglobulin antigenic determinants present on the membrane of murine lymphocytes (MIg) has been developed. This immunoassay makes use of specific Fab:anti-Ig, anti-κ, anti-µ, and anti-γ conjugated to Escherichia coli β-galactosidase of high specific activity in conjunction with the micromanipulation of lymphocytes and the measurement of enzyme activity with the fluorogenic substrate 4-methylumbelliferyl-β-D-galactopyranoside with a microassay system. With this technique MIg was detected and could be measured in substantial amounts on the surface of almost all splenic and lymph node lymphocytes examined. Depending on the specific Fab employed, 0 to more than 79,000 MIg per lymphocyte were measured, but a larger proportion of cells with higher amounts of MIg were found in spleen than in lymph nodes. However, when the same Fab′s were used to localize MIg by a conventional immunohistochemical staining, only a fraction of cells, corresponding to published values, could be scored as positive.The fact that MIg was associated with the vast majority of lymphoid cells implied that T cells carried such antigens. In order to investigate this further, experiments were performed on splenic B and T cell fractions obtained either with an anti-Ig immunoadsorbent or after treatment with anti-θ serum plus complement. The B cell fraction possessed greater amounts of MIg (1500 to 79,000) than the T cell fraction (0 to 39,000). However, on the average, B cells carried only 2 to 2.5 times more µ, κ, and γ antigenic determinants than T cells. Thus, B cells carried a mean of 27,300 to 47,800 Ig, 17,400 to 30,400 κ, 13,000 to 23,000 µ, and 4200 to 7300 γ antigenic determinants on their surface. T cells carried 4600 to 8000 Ig, 7200 to 12,600 κ, 5600 to 9800 µ, and 2400 to 4200 γ determinants. Immunoabsorbent column-fractionated T cells from which MIg was removed by anti-mouse Ig stripping were able to regenerate MIg after 24 hr of culture. The quantities reexpressed were equal to those found on T cells before anti-Ig treatment. These results strongly suggest that T cells synthesize and express MIg.Two nonmutually exclusive hypotheses are advanced that may account for the observations made: 1) Irrespective of the molecular nature of the T cell structure detected in this study, it is proposed that the difficulty in detecting MIg by standard immunohistochemical staining methods may reflect differences in the membrane composition of B and T cells. Local concentrations of MIg on B cells could be higher than on T cells, or a microredistribution of MIg may be achieved more easily on B than on T cells. 2) The T cell MIg may be conceived of as a molecule structurally different from the MIg on B cells.