Diane Y. Mochizuki

Molecular Characterization of Interleukin 2

The isolation of specific T cell growth factors termed interleukin 2 (IL 2) is changing the approach to understanding T cell function. This class of growth factors has allowed te establishment of a number of human and murine T cell lines. We summarize the biochemical properties of human and murine IL 2. Studies have been initiated to isolate mRNA encoding for IL 2. Such RNA can be translated in rabbit reticulocyte lysates yielding IL. 2. This RNA may be useful for the development of probes to isolate lymphokine genes.

Interleukin 2: A Class of T Cell Growth Factors

ABBREVIATIONS: IL 1 IL 2 Con A CTL HT FCS DEAE lEF LAF LPS MTLC pi PHA SRBC TCGF TSF TRF Interleukin 1 Interleukin 2 Concanavalin A cytotoxic T lymphocytes helper T lymphocytes fetal calf serum diethyl-amino-ethyl isoelectric focusing lymphocyte activating factor E. coli lipopolysaccharide mixed tumor-lymphocyte culture isoelectric point phytohemagglutinin sheep erythrocytes T cell growth factor thymocyte stimulating factor T cell-replacing factor

Expression, purification and characterization of recombinant marine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.

T-cell growth factors: interleukin 2

One of the key aspects of immunoregulation is the ability of T-cell subpopulations to help or suppress the expression of antigen-sensitive lymphocytes. The cloning of lymphoid cells - usually neoplastic cells - has long been a basic approach to the analysis of cellular function within the immune system but in the investigation of how T-cells act, T-cell lymphoma lines have been of limited value because the antigen specificity of their effector functions could not be detected. Now, however, lymphokines have been isolated which permit the growth of continuous lines of human and mouse T-cells with known specificity for antigen(2-8). In this review, James Watson and his colleagues discuss these lymphokines and some of the outstanding questions about their nature and function.

Biochemical separation of interleukin 2.

Interleukin 2 (IL-2) is a class of T cell growth factors produced by T cells of a number of species. The unique growth-promoting properties of these molecules allow the development of antigen-specific effector T cell lines which can be used to analyze the molecular basis of lymphocyte interactions. A murine T cell tumor line has been identified as a source of IL-2. The purification and biochemical properties of murine IL-2 are described, and compared with rat and human Il-2.

Comparative mitogenic effects of herpes simplex virus and mycoplasma on murine lymphocytes.

Previous work indicated that herpes simplex virus type 1 (HSV-1) is a mitogen for mouse spleen cultures, as monitored by uptake of 3H-thymidine. We observed variable responses of mouse spleen cultures to different viral preparations. The variable responses, which did not follow normal dose-response relationships, were not due to HSV-1 strain differences, altered response kinetics or the presence or absence of defective viral particles, but to mycoplasma contamination of viral stocks. Mycoplasma-free (MF) HSV-1 stocks were prepared by transfection of MF NHF cells with HSV-1 DNA. MF HSV-1 infection of spleen cultures resulted in a five- to sixfold stimulation of DNA synthesis and stimulation of a polyclonal antibody response. Heat treatment (56 degrees C for 1 h) and antibiotics were used to distinguish mycoplasma and HSV-1-induced spleen culture mitogenic responses. The mycoplasma-induced mitogenic activity was found to be heat labile and sensitive to gentamicin and chloramphenicol. In contrast, the HSV-1 induced response was not affected by gentamicin or chloramphenicol and heat treatment resulted in only a 50% loss of activity.

BIOCHEMISTRY OF LYMPHOCYTE COMMUNICATION

ABSTRACT Humoral factors secreted by T lymphocytes and macrophages appear to play a role in cell communication leading to the triggering of immune responses. A factor has been purified from the culture supernatants of Concanavalin A-activated murine spleen cells with helper T cell-replacing (TRF) activity in three assay systems: (i) stimulation of antibody responses to erythrocyte antigens in T cell-depleted cultures, (ii) amplification of production of cytotoxic T cells in thymocyte cultures, and (iii) stimulation of mitogenic responses to Con A in thymocyte cultures where the cell density is too low to support responses to Con A alone. The biologic activity has been purified by salt precipitation, gel filtration, ion exchange chromatography, and isoelectric focusing (IEF). TRF activity is found in protein-containing molecules with a Stokes radius corresponding to a globular protein of 30–35,000 daltons molecular weight, and a pI ranging from 4–5. Quantitative assays reveal this material is active at concentrations of less than 10 −9 M in each lymphocyte response system used. The production of TRF requires the presence of T cells, and limiting dilution analyses reveal one in 20,000 spleen cells are capable of producing TRF. When produced in conditions of limiting dilution, the TRF show a segregation of biologic activities as revealed by an ability to stimulate immune responses to one erythrocyte but not another. These results indicate that TRF produced in Con A-treated spleen cultures may have antigenic specificity. In an attempt to develop cloned cell lines that produce TRF, antigen-specific helper T cells have been established in continuous culture. These cells require TRF for proliferation. This finding raises the question of whether there exists one or two factors in the TRF preparations, a T cell growth factor (TCGF) and a helper T cell replacing factor (TRF). The identification of the molecules required for each of these biological assays may lead to an understanding of the nature of specific and nonspecific helper T cell-replacing factors.