Leu-2 Cells Research Articles

35] Transformation of nuclear respiratory deficient mutants of yeast

This chapter focuses on the transformation of the nuclear respiratory deficient mutants of yeast. The chapter describes the methods for (1) transformation of pet mutants with a plasmid pool and the isolation of plasmid(s) carrying the wild-type genes complementing the mutations; (2) transfer of the selected plasmid into Escherichia coli to amplify and purify the wild-type DNA insert for restriction analyses and sequencing; and (3) transformation of the pet mutant with a plasmid preparation from E. coli to confirm the presence of the wild-type gene copy in the insert of the isolated plasmid. The vector CV13 plasmid (YEp 13) has three components: pBR322, Eco RI fragment, and a Pst I. The Pst I fragment of nuclear yeast DNA contains the LEU2 gene that confers a means of positive selection for leu2 cells acquiring plasmid during transformation. To complete the identification of the CV13 plasmid isolated in E. coli as the carrier of the wild-type gene complementing a yeast nuclear mutation, a small-scale E. coli DNA preparation is used to transform the original pet mutant.

Dissection of immunoregulatory subpopulations of T lymphocytes within the helper and suppressor sublineages in man.

The helper/inducer (Leu-3) and suppressor/cytotoxic (Leu-2) sublineages of human peripheral blood T cells can both the subdivided into functionally distinct subsets with anti-Leu-8, a new monoclonal antibody that identifies 75 +/- 10% of the Leu-3 cells and 60 +/- 10% of the Leu-2 cells. Using the autologous MLR as a T cell-dependent stimulus for immunoglobulin synthesis, we have shown that the major helper effect for antibody formation lies within the numerically minor Leu-3,8- subset. In addition, neither Leu-2,8- cells alone suppress Leu-3-induced immunoglobulin synthesis, but in combination these subsets are markedly inhibitory. These results indicate that at least two phenotypically distinct cell types of suppressor lineage interact to produce suppression of an immune response in man.

Immunoglobulin secretion in the human autologous mixed leukocyte reaction. Definition of a suppressor-amplifier circuit using monoclonal antibodies.

The induction of immunoglobulin (Ig) synthesis in the autologous MLR has an absolute requirement for helper/inducer (Leu-3) T cells, whereas an excess of suppressor/cytotoxic (leu-2) cells suppresses the response. The current study was an effort to assess the immunoregulatory potential to T cells activated in the autologous mixed-leukocyte response (MLR). T cells were cultured with autologous non-T cells for 8-9 d, after which the activated T cells were fractionated into subsets with monoclonal antibodies to T cell markers and HLA-DR antigen. Each population was co-cultured in fresh autologous MLR, and on the 8th day of culture, Ig-secreting cells were measured in a reverse hemolytic plaque assay. The results show that activated Leu-2, DR+ T cells, but neither Leu-2, DR- nor Leu-3 T cells, were at least 50 times more potent as suppressors of IgM and IgG synthesis than fresh Leu-2 cells alone. The activation of this Leu-2, DR+ subpopulation required Leu-3 cells in the primary culture. Furthermore, in the absence of Leu-2 cells in the second culture, little or no suppression was observed, suggesting that the Leu-2, DR+ cells act to amplify or induce suppressor effects of fresh Leu-2 cells. This indicates that at least two distinct subpopulations of Leu-2 cells are required for maximal suppression of an immune response, and that immunoregulatory circuits analogous to those described in the mouse exist in man.

Activation of human T lymphocyte subsets: helper and suppressor/cytotoxic T cells recognize and respond to distinct histocompatibility antigens.

This study was directed at determining the major histocompatibility complex (MHC) antigens recognized by helper (Leu-3) and suppressor-cytotoxic (Leu-2) T lymphocyte subsets in man. These 2 subsets were isolated from peripheral blood with monoclonal antibodies and challenged in vitro with various stimuli. Only Leu-3 cells proliferated in response to autologous nonrosetting cells and soluble antigens, suggesting that helper but not suppressor-cytotoxic T cells recognize autologous HLA-DR antigen. Furthermore, only Leu-3 T cells responded to allogeneic DR antigen; this was shown in reactions between 2 siblings who were HLA-identical except for a single disparity at HLA-DR caused by a crossover event. Leu-3 cells activated in primary allogeneic mixed leukocyte reactions (MLR) responded equally in secondary allogeneic MLR to the priming cells and to cells that were identical at HLA-DR but discordant at HLA-A, B, and C to the priming cell. The antigens responsible for stimulating Leu-2 cells in allogeneic MLR were not identified, although the results are compatible with a role for HLA-A and B antigens and exclude a dominant role for HLA-DR. These data indicate that the helper and suppressor-cytotoxic T cell subsets in man respond differentially to MHC antigens in a manner analogous to the murine Lyt-2- 3- and Lyt-2+ 3+ populations.

Induction of immunoglobulin-secreting cells in the allogeneic mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets in man.

The existence of functionally distinct T lymphocyte subsets in man, initially demonstrated with heteroantisera, has been confirmed with monoclonal reagents. Two major subsets have been defined: Leu-2 cells, which effect cell mediated lymphocytotoxicity (CML), and Leu-3 cells, which amplify CML and other T cell functions. This study is an effort to determine the effects of these subsets on the immunoglobulin secretion induced in the human mixed leukocyte reaction (MLR). T cells were separated from non-T cells by rosetting with sheep erythrocytes and were fractionated into Leu-2 and Leu-3 subsets by solid phase immunoabsorption with monoclonal antibodies. T cell subsets were cultured with autologous non-T cells and irradiated allogeneic stimulator non-T cells. Secretion of IgM and IgG was measured by a reverse hemolytic plaque assay. MLR-induced antibody secretion was specifically dependent on the Leu-3 T lymphocyte subset. The Leu-2 subset was incapable of generating large numbers of IgM- or IgG-secreting cells, and, in fact, suppressed the Leu-3-induced response. Exposure of Leu-3 cells to a dose of irradiation sufficient to prevent their proliferation in MLR did not reduce induced immunoglobulin secretion. Leu-2-mediated suppression, however, was sensitive to low dose irradiation. Thus Ig secretion in human MLR is regulated by a balance of helper activity from the Leu-3 subset and suppressor activity from the Leu-2 subset.