SEPARATION OF NORMAL AND IMMUNE LYMPHOID CELLS BY ANTIGEN-COATED COLUMNS : ANTIGEN-BINDING CHARACTERISTICS OF MEMBRANE ANTIBODIES AS ANALYZED BY HAPTEN-PROTEIN ANTIGENS

Specific fractionation of immunologically competent cells derived from normal or immune animals was achieved by filtration through antigen-coated bead columns. This selective retention of the relevant reactive cells could be shown to produce a cell population, which after passage through the column would behave like a suspension rendered immunologically tolerant by "classical" means. The immunologically specific elimination of potential antibody-forming capacity of the filtered cells could be shown to affect the IgM and the IgG system to a similar extent. Analysis of the binding characteristics of the membrane antibodies responsible for this selective retention indicate striking similarities to those of the humoral antibodies produced against the antigen. Thus, the surface receptor could distinguish isolated haptenic groups on a "foreign" carrier background and the receptor of the hapten-reactive cells in the present system (high-rate antibody-forming cells against NIP) failed to combine with carrier specific determinants in analogy with the binding behavior of the serum anti-hapten antibodies. The binding of anti-hapten reactive cells to bead-attached haptens could be specifically inhibited by the presence of free hapten in the columnar fluid during cellular filtration. The results strongly suggest that the potential humoral antibody-forming cell has preformed receptors on its outer surface with binding characteristics, indicating similarity, if not identity, to those of the eventual product of the cell, the humoral antibody

cell surface glycoproteins of murine cytotoxic T lymphocytes. I. T 145, a new cell surface glycoprotein selectively expressed on Ly 1-2(+) cytotoxic T lymphocytes

T lymphocytes at various stages of maturation and differentiation have been isolated by cellular fractionation procedures and characterized by cell surface markers and functional assays, The cell surface glycoproteins of the various T-cell preparations have been selectively radiolabeled by the galactose oxidase-tritiated sodium borohydride technique and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Details are presented on the appearance of a new cell surface glycoprotein (T 145), present on immunocompetent T lymphocytes after activation by either major histocompatibility complex alloantigens or by concanavalin A. The intensity of T 145 expression on T lymphoblasts is shown to be directly correlated in time and extent to the levels of cytotoxicity generated in a variety of T-cell activations. Specific enrichment procedures of purified populations of mixed leukocyte culture blasts have shown Ly 1(+)2(-) blasts to be T 145(-) and Ly 1(-)2(+) blasts to be strongly T 145(+). Similar enrichment procedures on normal peripheral T cells have failed to reveal any significant expression of T 145 on a highly enriched population of Ly 1(-)2(+) T cells, Further studies on the stability of T 145 expression after induction have shown it to be a more permanent-type differentiation structure whose expression is clearly not linked to the blast stage of activation. T 145 would thus appear to represent a membrane glycoprotein whose exclusive expression on T lymphoblasts is further restricted to a defined group of cells endowed with cytolytic activity and bearing the Ly phenotype Ly 1(-)2(+)

Identification of distinct lymphocyte subsets responding to subcellular fractions of Mycobacterium bovis bacille calmette-Guerin (BCG)

In order to investigate the ability of Mycobacterium bovis BCG vaccination to induce immune responses toward different classes of mycobacterial antigens and the cell populations involved in such responses, proliferation of distinct human lymphocyte subsets from BCG-vaccinated donors in response to different subcellular fractions of BCG was analysed and compared with that of not sensitized subjects. Proliferation of different cell subsets was evaluated by flow cytometric determination of bromodeoxyuridine incorporated into DNA of dividing cells and simultaneous identification of cell surface markers. Although a certain degree of variability was observed among different donors, after 6 days of in vitro stimulation BCG-vaccinated subjects displayed, as a mean, a stronger blastogenic response to all the classes of antigens compared with non-sensitized ones. PPD, culture filtrates and membrane antigens induced a predominant proliferation of CD4(+) T cells. In contrast, preparations enriched in cytosolic antigens elicited strong proliferation of gamma delta(+) T cells which, as a mean, represented 55% of the proliferating cells. Although to a lesser extent, proliferation of gamma delta(+) T cells was also elicited by preparations enriched in membrane and cell wall antigens. In response to the latter preparation proliferation of CD4(+) T cells and CD16(+)/CD3(-) (natural killer (NK)) cells was observed, as well. In particular, cell wall antigens were found to induce significantly higher levels of proliferation of NK cells compared with all the other classes of antigens