Christopher S. Henney

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.

Lymphokine‐Driven “Differentiation” of Cytotoxic T‐Cell Clones into Cells with NK‐Like Specificity: Correlations with Display of Membrane Macromolecules

We have, for some time, had a good overall appreciation of how cytotoxic Tcells (CTL) act (Brunner & Cerottini 1974, Berke 1980, Green & Henney 1981). Under appropriate in vitro conditions, membrane-membrane interactions occur which involve the engagement of antigen-specific recognition units on the killer cell by homologous antigens on the target cell-surface. Shortly thereafter, membrane permeability changes can be measured in the target cell and these herald the eventual destruction of the cell, most probably by colloid-osmotic lysis. The killer cell survives the lytic encounter unscathed and can readily engage new targets. Despite this appreciation ofthe lytic event, little is known of the phenomenon in molecular terms. The nature ofthe antigen recognition unit and the lytic event itself remain largely uncharacterized (recently reviewed in Clark & Golstein, 1982). Since studies on the mechanism of cytotoxic T-cells were last reviewed in these pages, however, a major technical advance has had a substantial impact on cellular immunology and has allowed new strategies to be formulated for analysis of CTL. This finding, the identification and characterization of aT-cell growth factor (TCGF, also termed Interleukin 2; IL-2), has allowed considerable insight into the manner in which the expansion of T-cell clones is regulated (see Immunological Reviews, volume 63, 1982). In a more practical setting,

T-Cell-Mediated Cytolysis: An Overview of Some Current Issues

It is the purpose of this brief review to summarize some recent developments in our understanding of the mechanism of T-cell-mediated cytolysis. As is customary in this series, the account will be a rather personal one, and the “developments” to be discussed will center principally on observations made in this laboratory during the course of the last two years.

DISPLAY OF THE NEUTRAL GLYCOLIPID GANGLIO-N-TETRAOSYLCERAMIDE (ASIALO GM1) ON CELLS OF THE NATURAL KILLER AND T LINEAGES

Analysis was made of the display of the neutral glycolipid asialo GM1 on cells involved in the differentiation and expression of natural killer (NK) and T cell-mediated cytotoxicity in vitro. Removal of asialo GM1-bearing cells from CBA spleens, by treatment with a specific rabbit antibody in the presence of complement, led not only to the abrogation of NK cell activity but also to the lack of responsiveness of such populations to polyinosinic: polycytidylic acid (poly I:C) and to interferon, indicating that both NK cells and interferon-responsive cells of the NK cell lineage bear asialo GM1. Cytotoxic T lymphocytes (CTLs) induced by mixed lymphocyte culture in vitro were unaffected by treatment with antiasialo GM1 serum in the presence of complement, but normal spleen cells subjected to this treatment failed to mount CTL responses to alloantigen, even in the presence of an exogenous source of Interleukin-2 (IL-2). Furthermore, spleen cell populations depleted of asialo GM1-bearing cells showed a decreased ability to produce IL-2 in response to mitogenic stimulation.

Effect of anti-lymphotoxin on cell-mediated cytotoxicity. Evidence for two pathways, one involving lymphotoxin and the other requiring intimate contact between the plasma membranes of killer and target cells.

Abstract A rabbit anti-lymphotoxin serum produced against partially purified, antigeninduced, guinea pig lymphotoxin, was used to study the role of lymphotoxin in lymphocyte-mediated cytotoxicity in vitro . The anti-lymphotoxin serum inhibited cytolysis resulting from incubation of ovalbumin-immune guinea pig spleen cells with either mouse (P815 mastocytoma) or guinea pig (line 10 hepatoma) target cells in the presence of soluble ovalbumin. The antiserum also inhibited the cytolysis of ovalbumin-coupled target cells by ovalbumin-immune guinea pig spleen cells. In contrast, the anti-lymphotoxin serum did not inhibit: (a) the lysis of line 10 (strain 2) hepatoma cells by spleen cells from alloimmunized Hartley or strain 13 animals; (b) the lysis of line 10 hepatoma cells by spleen cells from tumor-bearing syngeneic animals; or (c) the lysis of P815-mastocytoma cells by spleen cells from P815-immune guinea pigs. These results support the hypothesis that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro , one which involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another which probably requires intimate contact between the plasma membranes of the target and killer cells.

Specific cytolytic activity of lymphocytes, effect of antibodies against complement components c2, c3, and c5.

Abstract The role of complement in the specific cytolytic activity of immune lymphoid cells has been directly evaluated. Rabbit antisera monospecific for guinea pig C 2 , C 3 , and C 5 were added to a mixture of “sensitized” guinea pig lymphocytes and the mastocytoma “target” cells (P815 of the DBA/2 strain) with which the guinea pigs had previously been immunized. The target cells were labeled internally with 51 Cr and target cell destruction assessed by 51 Cr release. Addition of an excess of anti C 2 , C 3 , and C 5 to the reaction medium had no demonstrable effect on lymphocyte-mediated cytolysis, but at identical concentrations effectively inhibited lysis of the same target cells by antibody in the presence of complement. These results provide no evidence that complement participation is a prerequisite of the immune lysis of target cells by lymphocytes.

The treatment of induced immune deficiency with interleukin-2

In vivo generation of alloreactive cytotoxic T lymphocytes (CTL) was found to be inhibited by treatment of mice with either cyclophosphamide or the glucocorticoid, hydrocortisone acetate. The effects of these immunosuppressive agents could be overcome, however, by the in vivo administration of IL-2 from both murine and human sources. Both human IL-2 derived by recombinant DNA techniques as well as the natural protein from mouse and man all reverse the unresponsive state. A single injection of IL-2 was sufficient to reverse the effect of cyclophosphamide treatment, while additional injections with as little as 8 micrograms of protein ablated the steroid-induced suppression. Furthermore, the responder cells generated in vivo following IL-2 therapy were shown to be antigen specific in terms of their lytic capacity. Thus, IL-2 therapy appears to restore the in vivo responsiveness of immunosuppressed recipients to allogeneic tumor cell challenge. These data demonstrate the importance of IL-2 as an immunoregulatory molecule in vivo and suggest its future use as a potent therapeutic.

Interleukin-2 and the Regulation of Natural Killer Activity in Cultured Cell Populations

The recent immunology literature is replete with examples of effector T-cell populations, the proliferation and function of which are potentiated by culture medium containing Interleukin-2 (IL-2), formerly T-cell growth for factor (TCGF). Indeed, it is often taken as axiomatic that all T cells can respond to IL-2, in conjunction with other signals, and, conversely, that all cells that respond to IL-2 are T cells. An apparent exception to this general rule was provided by the observation that IL-2 could enhance natural killer (NK) activity and that medium containing this lymphokine could support the continuous growth of cell lines with NK-like activity. This chapter reviews the now extensive literature pertaining to these phenomena and describes some newer data that demonstrate some interesting and previously undocumented interrelationships between cytotoxic T cells (CTL) and NK cells.

The Relationship between Lymphokines and Cell-Mediated Cytotoxicity

Publisher Summary The demonstration of cytotoxic activity in the supernatant of lymphocytes, cultured with either antigen or mitogen, potentially ranks as one of the most important findings in the short history of lymphokine biology. The significance of the observation lies in the possibility that the toxic factor, termed lymphotoxins, serves an important physiological role in various types of tissue destruction mediated by the cells of the immune system. This chapter discusses some of the issues inherent in developing the case that lymphotoxin is involved in cell-mediated cytotoxicity. It focuses on those cytotoxic reactions in which the effector cell is a thymus-derived lymphocyteor T-cell. T-cell mediated lysis is a phenomenon of unquestioned physiological significance both in allograft rejection and in the regulation of growth of certain neoplasms. Cytolysis mediated by T-cells is independent of either antibody or the complement system. Lysis mediated by T-cells exhibits exquisite immunological specificity. Cytolysis requires an intimate contact between the effector cell and its homologous target. When killer and target cell are separated, either by a semipermeable membrane or by suspension in a viscous medium, such as dextran or agarose, cytolysis does not occur. Lymphocyte production requires a viable protein-synthesizing cell but is independent of DNA synthesis.

The cytolytic action of thymus-derived lymphocytes with reference to the destruction of connective tissue.

Until the last decade, it was axiomatic that discussions of immunologically specific cell destruction referred to a process mediated by serum complement in the presence of antibody. Recently, however, we have become aware of complement-independent cytolytic pathways that demonstrate immunologic specificity. In these cases, viable cells, often lymphoid in origin, mediate the lytic process, and although antibody against the target cell is occasionally required to demonstrate cytolysis, other lytic pathways exist in which neither antibody nor complement play a role (for review, see Reference 1). Particular attention has focused on antibody-independent lytic pathways that are mediated by immune, thymus-derived (T) lymphocytes.*. This interest was summoned by demonstrations that T-cell-mediated cytolysis plays a predominant role in allograft rejection and often contributes significantly to limiting neoplastic cell growth. Although these conclusions are to date based on observations made in the mouse, it is widely considered that they are also applicable to mart. We now recognize that cytolytically active T cells are a characteristic component of the mammalian cell-mediated immune response and that such cells provide an effective host defense mechanism for controlling the proliferation of cells that bear %onself”-antigens. Though cytolytically active T cells might well be considered primarily of “defensive” significance, recent studies indicate that they can also cause the destruction of host tissue in a manner that is clearly not beneficial. Thus, Cole et al. have shown that the pathologic features that result from lymphocytic choriomeningitis (LCM) virus infection are caused by the host’s immune response to LCM virus.4 Virus-bearing meningeal cells are destroyed in vivo by cytolytically active T cells produced in response to the viral antigen challenge. It is this destruction that accounts for the pathologic condition associated with LCM infection. These observations give cause for considering a more general issue: DO cytolytically active T cells contribute to tissue destruction in other diseases in which there is reason to suppose an autoimmune etiology? More specifically,