What are killer cells and what do they do?

What Are Killer Cells and What Do They Do?

P. G. Hogan, A. Basten SUMMA R Y. Cytotoxic effector function of the immune system is mediated predominantly by killer cells and with the exception of complement-mediated lysis aU forms of immunedirected ceU death are attributable to these cells. The heterogeneous collection of cells with cytotoxic properties ranges from the phylogenetically primitive granulocyte and natural killer (NK) ceU lineages to the more complex and versatile macrophage and antigen-specific cytotoxic T lymphocyte. Most killer cells are capable of the dual functions of cytotoxicity and immunoregulatioo. Granulocytes, macrophages and NK cells constitute an effective ‘first line’ cellular defence against invasive microbial pathogens and emerging malignancies. Through direct cytotoxicity or by the secretion of cytokines, NK cells also prevent over-proliferationof precursor cell populations, thereby exerting a more discriminating control over antigen-speciUc T and B cell responses. The ultimate refinement of the killer ceU is the cytotoxic T cell which is directed by an antigen receptor compaiable in specificity and diversity to the immunoglobulin molecule. The mechanisms underlying the initial interaction between killer cell and target and the subsequent lytic event are imperfectly understood. Although many cytokines with lytic properties have been identified and in some cases cloned, their relative importance and intricate interactions with other components of the immune system are still largely unknown. In addition to the prevention of infection and malignancy, killer cells of all lineages are important in the pathogenesis of human disease. Of particular interest is the role of macrophages, NK cells and cytotoxic T cells in autoimmunity. Although there is little evidence that these cells account for defective immunoregulation, there is a strong suspicion that killer cells are important mediators of tissue injury and organ dysfunction in these diseases.

Killer cells together with specific antibody and delayed hypersensitivity responses comprise the effector arm of the immune system, the function of which is to protect the body against infection from without and tumour cell growth from within. All forms of immunemediated cytolysis except for complement-mediated lysis are mediated by killer cells. The human immune system is armed with an array of killer cells which vary markedly in the complexity and specificity of their cytotoxic activity (Table 1). Macrophages, granulocytes and natural killer (NK) cells are primitive P. G. Hogan, A. Baatea, Clinical Immunology Research Centre, The University of Sydney, Sydney, NSW 2006, Australia.

effector cells which phylogenetically form part of the constitutive immune system. While their phagocytic and bactericidal properties are paramount, granulocytes and macrophages also function as immunedirected killers by the process of antibody-dependent cellular cytotoxicity. Macrophages are also capable of lysing turnout cells spontaneously. NK cells lyse tumour cells and some nonmalignant cells by means of a complex cytotoxic mechanism which operates on first exposure and is independent of both extrinsic antigen stimulation and the major histocompatibility complex (MHC). A closely related mononuclear cell cohort known simply as killer (K) cells mediate antibody-dependent cellular cytotoxicity. By contrast,

BLOOD REVIEWS TaMe 1 Categories of human killer cells and their cytotoxic

properties Cell type

Cytotoxicity

MHC restriction

Reference

Neutrophils Eosinophils Monocyte/macrophage Monocyte/macrophage Natural killer (NK) cells Killer (K) cell Cytotoxic T cell (CTL) Lymphokine-activated killer (LAK) cell Lymphokine-activated killer (LAK) cell

ADCC’ ADCC MTCO ADCC NK activity ADCC CML@

No No No No No No Yes

1 2 3

LAK activity

No

ADCC

No

4

1 5 6

* Antibody-dependent cell-mediated cytotoxicity 0 Macrophage-mediated tumour cytotoxicity @ T cell-mediated lympholysis

the cytotoxic T lymphocyte typic receptor responsible

is equipped with a clonofor activation of the cell

and programming it for lysis after specific recognition of target cell surface antigen in association with self MHC structures. Lymphokine-activated killer (LAK) cells are a heterogeneous collection of cells capable of mediating potent NK-like activity against tumour cells which are resistant to resting NK cells. This review will focus on the cytotoxic and regulatory functions of NK and LAK cells which may be relevant to the prevention, pathogenesis or treatment of human disease. Important distinctions between NK cells and cytotoxic T lymphocytes are highlighted and the cytotoxic functions of macrophages and granulocytes will be covered in the discussion of antibody-dependent cellular cytotoxicity.

The Natural Killer Cell Lineage: Cytotoxic and Regulatory Functions DeJnition

Natural killer (NK) cells constitute a unique lineage of large granular lymphocytes which arise from precursors in the bone marrow.’ Approximately 10 to 15% of peripheral blood lymphocytes are large granular lymphocytes and of these 70% have the potential to act as NK cells. *,’ The remainder of the large granular lymphocyte pool lack NK cell properties and consist predominantly of T cells. All large granular lymphocytes have an indented, eccentric nucleus, plentiful cytoplasm containing an active secretory apparatus and prominent azurophilic granules with the enzymatic profile of lysosomes.” Despite superficial similarities to the monocyte, NK cells are peroxidase-negative, nonadherent and nonphagocytic. In the human, small numbers of NK cells are detectable in the bone marrow, spleen and lung but are surprisingly rare in the extensive mucosal surface of the intestine.’ 1’12S13NK cells were known to constitute the major ‘null cell’ in the peripheral blood but phenotypic studies now reveal that NK cells express a

51

heterogeneous array of surface antigens.‘“i7 Some are shared with the T cell (CD2, CD3, CD8) or myeloid (OKMl) lineages while others are more specific for NK cells (Leu-7 and Leu-19). Most NK cells carry the sheep erythrocyte receptor (CD2) and Fc receptors for IgG which bear an antigenic determinant designated as the Leu-1 1 (CD16) antigen. Analysis of cell phenotype using flow cytometry indicates that peripheral blood NK cell activity is mediated by multiple subpopulations of cells (Table 2). The most potent NK cells are the CD3- (non T) Leu-11 +Leu-7- minority subset which accounts for 30% of all NK activity. Paradoxically, a significant population of large granular lymphocytes mediating NK activity are CD3+Leu-7+Leu-19+ but Leu-lland appear to utilize the T cell receptor (Ti)/CD3 complex to mediate their cytotoxic activity in a nonMHC-restricted manner.” Southern blot analysis of DNA from CD3- NK cells reveals germ-line configuration of the a, p and y chain Ti genes whereas CD3+ cells with NK activity possess the c1and l3 rearrangements characteristic of mature T cells.17,18 A possible precursor of the CD3+ cell with non-MHC-restricted cytotoxicity has been isolated from peripheral blood and appears to express an early form of the Ti coded for by y genes. ’ 9 On the basis of these findings, CD3 + cells with NK activity should be classified as cytotoxic T lymphocytes rather than NK cells. Tumour-cell Killing

Before the advent of flow cytometry, NK cells were defined operationally by their ability to lyse tumour cells in a non-MHC-restricted manner.2o,21 Targets susceptible to NK activity in vitro include cell lines derived from leukaemias, B and T cell lymphomas and some solid tumours whereas freshly isolated cells from resected solid tumours or leukaemias are generally resistant to resting NK cells. However, following incubation with alpha interferon (IFN-a) or interleukin 2 (IL-2), resting NK cells become activated and within 1 to 4 h can lyse both freshly isolated tumour cells and leukaemic blasts.22*23 Convincing evidence of an antitumour effect by NK cells in vivo has come Table 2

Surface phenotypes of mononuclear cells with natural killer (NK) or lymphokine-activated killer (LAK) cell activity Surface phenotype*

NK activity

LAK activity

+++ ++ + +

-

_ _

+++ +++ +++ ++

NK cells

CD33Leu-ll+Leu-19+Leu7CD3-Leu-l l ‘Leu-19*Leu-7+ CD33Leu-ll-Leu-19+ CD3’Leu-II-Leu-19+Leu-7+0 LAK cells

CD3-Leu-ll+Leu-19+Leu-7CD33Leu-l f +Leu-19+Leu-7+ CD33Leu-ll-Leu-19+ CD3+Leu-ll-Leu-19+ * 0

Presence indicated by +; absence by This cell should be classified as a CTL because it carries a functional Ti/CD3 complex and the CDS antigen

52

WHAT ARE KILLER CELLS AND WHAT DO THEY DO?

from murine studies which clearly demonstrate protection against both spontaneously arising and experimentally-induced neoplasms.24~25 Due to the nonspecific suppressive effects of large tumour burdens, a definite role for NK cells in human malignancy has been more difficult to prove. Nevertheless, NK cell numbers and/or the activity of individual cells are depressed in a variety of human tumours, notably lymphomas more often than solid tumours.26*27 Furthermore, the increased rate of malignancy in primary and secondary immunodeficiency has been attributed to reduced NK activity, although this defect rarely occurs on its own and is usually accompanied by numerous other immune abnormalities which could equally well predispose to neoplasia.28*2g On balance NK cells are more likely to be important as a surveillance mechanism early in tumour development rather than a significant influence after the tumour has presented clinically. The capacity of IFN and IL-2 to stimulate NK cells in vitro has provided a basis for the use of these cytokines in clinical trials. Repeated intravenous administration of purified or recombinant human IFNa causes a significant increase in NK activity in tumour-bearing recipients. The enhanced NK activity is limited to NK-sensitive targets and after a peak at approximately day 7, usually declines to pre-therapy levels.30 Although only one of the many immunomodulatory effects of IFN, NK cell activation in vivo may contribute to the dramatic response of Hairy Cell Leukaemia to IFN therapy, particularly since pretreatment NK cell function is frequently reduced.31 Preliminary clinical trials with megaunit doses of IL-2 have failed to lead to a consistent increase in NK activity in the recipients.32 Nevertheless, IL-2 therapy does appear to result in complete or partial remissions in a small proportion of patients with disseminated cance.r.33 NK cell-mediated lysis of tumours proceeds through a sequence of programmed steps which include: (i) NK-tumour cell binding, (ii) NK cell activation and delivery of the ‘lethal hit’, (iii) tumour cell lysis and (iv) NK cell recycling for further lysis.34 As illustrated in Figure 1A, each step is guided by a different set of receptors which collectively confer specificity upon the interaction. The target cell structures recognized by NK cells have proved difficult to isolate and characterize. T cells with NK activity bind to a 120 kD heterodimer which can be detected on a wide variety of proliferating lymphoid cell~.~~ The proposed target for non-T NK cells is an unrelated glycoprotein which may be located close to the transferrin receptor on the cell surface.36 The strength of the NK-target cell interaction is further enhanced by nonspecific binding of the target to specialized leucocyte function-associated molecules on the NK cell surface. The leucocyte function-associated family of glycoproteins perform a similar accessory function for cytotoxic T lymphocytes3’ Delivery of the lethal hit involves microtubule-directed migration of cyto-

plasmic granules to the vicinity of membrane contact followed by exocytosis of granule contents either directly into or close to the target cell membrane38 (Fig. 1A). The cytolysins are stored in a sulphated proteoglycan matrix in NK cell granules and include perforin (60-70 kD), NK cell cytotoxic factor (2G30 kD) and serine proteases and esterases.3g*40 The relative importance of these molecules in causing target cell lysis is unresolved. In a process analogous to the action of the ninth component of complement, protease-activated perform molecules polymerize into hollow 16 mm diameter tubular structures which insert into the tumour cell membrane allowing influx of water and solutes with resultant osmotic lysis. The NK cell cytotoxic glycoprotein appears to interact with specific receptors on NK-sensitive targets but its overall contribution to target cell lysis is uncertain. In addition to cytotoxic factors, NK cells secrete a 44 kD cytostatic factor called leukoregulin which inhibits tumour cell proliferation without causing lysis. The non-identity of NK cell-derived cytolysins with lymphotoxin and tumour necrosis factor has been clearly established. Virus-infected Cells

The lytic machinery of NK cells is also a ‘front line’ defence against microbial insults to the host. In murine models of cytomegalovirus infection NK cells boosted by high levels of IFN-u provide early protection against virus dissemination several days before specific cytotoxic T lymphocytes deveiop.41 Virusinfected host cells rapidly acquire susceptibility to NK cells and are therefore destroyed before intact virions can be replicated. IFN provides additional benefit by increasing the resistance of uninfected cells to NK activity. 42 The expanding list of viral pathogens susceptible to NK cells includes herpes simplex virus, varicella zoster, measles, mumps, influenza, human T cell lymphotrophic virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-l) as well as cytomegalovirus.43*44 Regulatory Role

An unexpected level of complexity was added to the NK cell repertoire when their regulatory potential was discovered. In addition to cytolysins human NK cells secrete potent immunoregulatory cytokines in physiologically significant amounts.45A8 Exposure to microbes, tumours or cytokines stimulates NK cells to release IL-l, IL-2, IFNs-a and y, B cell growth factor, colony stimulating factors and tumour necrosis factor. The growth and differentiation of lymphoid and haematopoietic precursor populations in human thymus and bone marrow are also subject to downregulation by the lytic and secretory. activity of NK cells.4g.50 Furthermore, the functions of mature B and T cells can be influenced in vitro by NK cells

BLOOD REVIEWS

A. NK ACTIVITY

B. CML

esterases

53

C. ADCC

Lymphotoxin esterases

(i)

Binding

(ii)

Activation

via NKTS

(iii)

The ‘lethal

hit’

Fig. 1 (A) Natural killer (NK) cell activity. NK cells bind to a target cell structure (NKTS) and are activated for delivery of the lethal hit by the receptor-mediated steps shown. NK cell-derived cytotoxic factor (NKCF) is released from granules and causes lysis by interaction with a receptor on the target cell. Perforin and serine proteases and estarases with nonspecific cytotoxic activity are also released in close proximity to the target cell. (8) Ceil-mediated lympholysis (CML). Specific recognition of target cell antigen (TA) associated with a class I MHC molecule by the T cell receptor (Ti) results in activation of the CD8+ cytotoxic T lymphocyte (CTL) through the associated CD3 activation complex. Interaction between the CD8 and target MHC molecules and identity of class I MHC structures on CTL and target are also necessary for lysis to proceed. The principal cytotoxic molecules of CTL are perforin, lymphotoxin and serine proteases and estarasas. A similar mechanism can occur with CD4+ CTL except that class II molecules are involved in the Ti-TA interaction. (C) Antibody-dependent cellular cytotoxicity (ADCC). Contact of a target cell coated with specific antibody and any cytotoxic cell bearing Fc receptors results in lysis of that target call. Cells capable of mediating ADCC include NK/K cells, macrophages/monocytes, eosinophils, neutrophils and Tr cells. Depending on the cell type involved, various cytolysins such as lymphotoxin, perforin, tumour necrosis factor, free oxygen radicals, cytolytic protease and major basic protein may ba released from the Fc receptor-positive cell.

acting as nonspecific suppressors or antigen-presenting cells, depending on the experimental system.51*52 General Disease

In view of the wide range of NK cell activities, their role in nonmalignant disease is more difficult to evaluate. Impairment of spontaneous and IFN-inducible NK activity in peripheral blood is a consistent feature of systemic lupus erythematosus and similar findings have been reported in other autoimmune diseases such as inflammatory bowel disease.53*54 This observation does not form an integral part of the accepted theories of the immunopathogenesis of autoimmune diseases like lupus. Indeed the reduction in NK cell function in this situation is generally considered to be secondary to the effects of the disease or its treatment or to recruitment of cells from blood to disease-affected tissue. Furthermore, even when mononuclear cells have been isolated from diseaseaffected tissue (e.g. intestinal mucosa in inflammatory

bowel disease) very little NK activity has been observed.54 Thus NK cells are unlikely to play an important primary role in autoimmunity, although these observations do not discount a significant role for other cytotoxic cells such as cytotoxic T lymphocytes, macrophages or granulocytes. Transplantation

A more convincing role for NK cells has been demonstrated in bone marrow transplantation. After transplantation, NK cells are one of the first lymphoid populations to regenerate, attaining normal levels of activity in 4 to 6 weeks. 55 In the early post-transplant period, NK cells are in an activated state as evidenced by cytotoxic activity against normally resistant targets and increased secretion of cytokines. An antimicrobial function for these cells was suggested by the correlation between delayed recovery of NK activity and an increased frequency of opportunistic infections. 56 In addition to this benefit, there is evidence to

54

WHAT ARE KILLER CELLS AND WHAT DO THEY DO?

suggest that early-appearing NK cells assist B cell redevelopment because of their secretion of B cell growth and differentiation factors.” Possible adverse effects of NK cells include graft rejection and graft versus host disease. In myrine experiments, NK cells were convincingly shown to be responsible for rejection of bone marrow grafts between MHC-identical animals.58 In humans, the probability of GVHD has been correlated directly with abnormally high pre- or post-transplant NK activity in the recipient.56*5g Although convincing evidence supports the prime role of T cells in GVHD, the participation of CD3 + NK cells acting either as cytotoxic cells or as antigen presenting cells for T lymphocytes cannot be excluded. NK Cell Malignancy To date the only malignancy of NK cells which has been identified is included in the spectrum of disorders designated Ty-lymphoproliferative disease. The hallmark of Ty-lymphoproliferative disease is the finding of excess numbers of large granular lymphocytes in the peripheral blood, liver, spleen and bone marrow. It is a rare, indolent lymphoma characterized by hepatosplenomegaly, lymphocytosis, anaemia, neutropaenia and recurrent bacterial infections. Early attempts to classify the disease using rosetting techniques identified the large granular lymphocytes as T cells bearing Fc receptors for immunoglobulin G (Ty cells). More recent analysis of larger series has revealed the heterogeneous nature of this disorder despite the apparent uniformity of large granular lymphocytes. 60*61*62Most cases (SO-90%) of Tylymphoproliferative disease are clonal proliferations of CD3+CD8+ T cells which were detected by uniform rearrangements of the Ti 8 gene. In the remaining cases, non-T NK cells expressing Leu-7 and Leu11 but lacking CD3 are the predominant cell type. In a variable proportion of cases, large granular lymphocytes of either T cell or NK cell derivation mediate NK activity and/or antibody-dependent cellular cytotoxicity. Evidence from in vitro experiments suggesting that the abnormal large granular lymphocyte can inhibit the formation of haematopoietic colonies provides a plausible explanation for the neutropaenia and anaemia complicating Ty-lymphoproliferative disease.

Cytotoxic T Lymphocytes In contrast to NK cells, the antigen receptor (Ti) of cytotoxic T lymphocytes recognizes specific target cell antigens ,in association with self MHC class I or less commonly class II antigens, resulting in T cell-mediated lympholysis (Fig. 1B). As a necessary part of this interaction, CD8 molecules on the cytotoxic T lymphocyte are thought to bind reversibly to target MHC molecules. Alloantigens on foreign cells are also potent stimulators of cytotoxic T lymphocytes but the induction mechanisms involved are uncertain. Although cytotoxic T lymphocytes are predominantly CD8+, some CD4+ lymphocyte clones have been

shown to possess specific cytotoxic properties.63 As a result of the Ti-antigen interaction, cytotoxic T lymphocytes are programmed for lysis through the Tiassociated CD3 activation structure. In addition to stabilizing the cytotoxic T lymphocyte-target bond, one of the glycoproteins (CD2) on the cytotoxic T lymphocyte may also act as an additional pathway for T cell activation.37 The principal cytolysins involved in cell-mediated lympholysis are perforin, lymphotoxin (formerly known as TNF-B) and a collection of serine proteases and esterases. Human lymphotoxin is a 19 kD glycoprotein which is released by either CD4+ or CD8+ lymphocytes after antigen-specific stimulation.‘j4 It has cytotoxic and cytostatic effects on tumour cells which may be enhanced by IFN-y. As with NK cells the precise biochemical mechanisms underlying cell-mediated lympholysis are controversial. Contrary to the conventional view of cytolysininduced passive target cell destruction, it is argued that cell-mediated lympholysis may be a more active process of programmed target cell destruction triggered by an inducer secreted by the cytotoxic T lymphocyte. 65 In addition to the perforin-mediated membrane lesions, a feature peculiar to cell-mediated lympholysis is the early degradation of target cell DNA into small fragments.64 This effect may be induced by lymphotoxin alone or may be due to diffusion of endonucleases from the cytoplasm of the target cell. Manipulations such as lectin binding which also bring cytotoxic T lymphocytes and target cells into close contact can result in nonspecific lysis. However, lectin-induced cellular cytotoxicity is essentially a laboratory artifact and probably has no in vivo relevance, except possibly in phylogenetically primitive species where lectins are thought to be a major defence mechanism. In the normal host, cytotoxic T lymphocytes play a versatile role in the elimination of virus infected cells and transformed cells expressing tumour-associated antigens. In the case of infectious mononucleosis, specific cytotoxic T lymphocytes recognizing Epstein Barr virus-infected B cells are primarily responsible for controlling the acute infection and for maintaining the virus in a latent state.66 In the setting of autoimmunity, cytotoxic T lymphocytes reactive against self antigens may be the main perpetrators of organ injury in diseases such as Hashimoto’s thyroiditis and juvenile onset diabetes mellitus. Many of the features of acute GVHD have been attributed to the ability of cytotoxic T lymphocytes to react to foreign MHC antigens. Although not proven, it is possible that cytotoxic T lymphocytes release significant amounts of lymphokines such as IL-2, IFN and colony stimulating factors, all of which can exert protean immunoregulatory effects.

Antibody-dependent Cellular Cytotoxicity Interaction between cell surface antigens and specific antibody may result in destruction of the cell by

BLOOD

complement-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis. At concentrations of cell-bound antibody too low to initiate complement activation or opsonization, ADCC remains an effective mechanism causing cell lysis. In ADCC, antibody directed specifically towards the cell surface acts as a bridge between the target and the Fc receptor of the cytotoxic effector cell (Fig. 1C). By poorly understood mechanisms which may act through the Fc receptor, the cytotoxic cell becomes activated and lyses the target. Cells capable of ADCC express Fc receptors for IgG and therefore include K cells, monocytes/macrophages, eosinophils, neutrophils and lymphokine-activated killer cells (Table 1). For unknown reasons each cell type has a restricted range of targets which it can destroy by ADCC. The ability to mediate ADCC is an important facet of host defence against microbes and also possibly malignant transformation. Bacteria, protozoan and helminth parasites and fungi appear to be susceptible to ADCC.67,68*2*69 In addition, many NK-resistant tumour cells can be lysed by ADCC once targeted by specific antibody. The term killer (K) cell refers to large granular lymphocytes which express the Fc receptor for IgG (the Leu-ll antigen) and are therefore capable of ADCC. The majority of the K cell population are phenotypically identical to CD3 - NK cells and are regarded by many investigators as a subset of NK cells which have the additional capacity to mediate ADCC. Visualization of NK cells mixed with both NK-susceptible and antibody-labelled target cells confirms that a single NK cell can bind to and lyse both targets simultaneously.70 Furthermore, many cloned NK cell lines are capable of both forms of cytotoxic activity. A small proportion of K cells are Leu-1 1 + T cells which were previously designated as Ty cells.71 These CD3+Leu-l l + lymphocytes lack the CD4, CD8 and Leu-19 antigens and are incapable of NK activity. In vitro studies show that K cells can lyse antibody-labelled tumour cell lines and the pathogens, salmonella, shigella and giardia.67*68 A special feature of intestinal lymphocytes isolated from mice is the expression of Fc receptors for IgA which facilitate ADCC of enteric pathogens recognized by specific secretory IgA. Both neutrophils and eosinophils mediate ADCC against tumour cells, but only eosinophils possess the ability to kill schistosomes by a similar mechanism.‘** Parasite-specific IgG can initiate ADCC by binding to the appropriate Fc receptor on eosinophils and following this interaction the contents of eosinophil granules are observed adherent to and eroding the parasite integument. The highly toxic proteins found in eosinophil granules include major basic protein, eosinophil cationic protein and eosinophil peroxidase. The peroxidase enzyme catalyzes the reaction between halide ions (chloride or iodide) and hydrogen peroxide to produce highly reactive hypohalite ions. A potential disadvantage of these powerful defences is

REVIEWS

55

diffusion of cytotoxins away from the target causing damage to uninvolved cells or tissues such as cardiac muscle and glial cells which appear to be peculiarly susceptible to injury by mediators of eosinophil origin.72 Examples of this occurrence include the cardiomyopathy associated with the hypereosinophilic syndrome and the Gordon phenomenon (encephalomyelitis) occasionally seen in patients with eosinophilia complicating Hodgkin’s disease. Activated macrophages and monocytes can cause the extracellular lysis of tumour cells either spontaneously by macrophage-mediated tumour cytotoxicity, or by ADCC in the presence of tumour-specific antibodies.3 MTC is controlled by receptors which are poorly understood and in vitro the process takes up to 2 days to complete. In the case of activated macrophages, ADCC of antibody-coated tumour cells can be complete within 6 h. Macrophages produce a variety of cytolysins which include tumour necrosis factor (also called TNF-a or cachectin) and a recently described cytolytic protease which kills neoplastic cells selectively. The effect of tumour necrosis factor on tumour cells is both cytotoxic and cytostatic and is enhanced by the presence of IFN-Y.~~ Free oxygen radicals are important in destroying the contents of phagocytic vacuoles, but it is undecided to what extent this mechanism participates in ADCC. Monocytes, neutrophils and K cells also mediate ADCC against human erythrocyte targets sensitized with antibodies specific for the blood group antigens. 73 Under conditions favouring opsonization, sensitized erythrocytes are ingested by monocytes and destroyed in phagolysosomes by oxygen-derived free radicals and enzymes. In autoimmune haemolysis, the density and class of antibody binding to erythrocytes dictates whether their elimination occurs by complement-mediated lysis and/or clearance by the hepatic and splenic mononuclear phagocytic system. The main mechanism of erythrocyte disposal is probably by opsonization, but ADCC mediated by macrophages and K cells may also be important.74 The human antibody isotypes IgGi and IgG3 are most efficient in mediating ADCC of erythrocytes by both K cells and monocytes. According to recent observations, ADCC may be the main mechanism responsible for clearing sensitized Rh+ foetal erythrocytes in Rhmothers who are given anti-Rh (D) antibody perinatally. 74 In the treatment of malignancy using tumourspecific monoclonal antibodies which may not be able to fix human complement, ADCC may contribute to tumour cell eradication. 75 Injury to parenchymal cells, circulating leucocytes and hormone receptors in organ specific and multi-organ autoimmune diseases may be mediated by autoantibody-directed killer cells. Lymphokine-activated

Killer (LAK) Cells

Lymphokine-activated killer (LAK) cells were first prepared by culturing unfractionated peripheral

56

WHAT ARE KILLER CELLS AND WHAT DO THEY DO?

blood lymphocytes in the presence of IL-2 or mitogenie lectins. 76 Lymphokine-activated killer cells prepared in this way display potent non-MHC restricted cytotoxicity against a wide range of target cells including NK-resistant tumour cell lines, freshly isolated tumour cells and leukaemic blasts. By contrast, normal parenchymal cells of the same type as the tumours were generally resistant to LAK cell activity. The LAK cell effect is responsible for the high levels of nonspecific cytotoxicity apparently mediated by alloantigen-activated cytotoxic T lymphocyte derived from mixed lymphocyte cultures. Dissection of the LAK cell phenomenon reveals a heterogeneous population of cytotoxic cell phenotypes, 6*77*78*7Q but all LAK cells were basically either T or NK cells which have been activated by exposure to high concentrations of IL-2 (Table 2). The NK-derived CD3- LAK cell is probably the more important, as it mediates higher levels of cytotoxicity and can lyse tumour cells which are resistant to CD3 + LAK cells. The LAK cell population generated from resting NK cells retains functional Fc receptors and thus also mediates ADCC. When cultured in the presence of IL-2, NK cells do not acquire the CD3 antigen. It is still unclear whether LAK cells bearing T cell antigens arise from the small pool of CD3+ NK cells or from the much larger population of cytotoxic T lymphocytes which mediate specific MHC-restricted cell-mediated lympholysis. Both cell types can probably act as precursors and furthermore, there is evidence to suggest that a single CD3 + cell is capable of mediating both LAK activity and specific cell-mediated lympholysis.80 Because the LAK cell was originally described as a culture-generated phenomenon, there was doubt about its spontaneous existence and relevance in the intact animal. However, cells with LAK activity have now been described in rheumatoid arthritis,‘l Tylymphoproliferative disease62 and after bone marrow transplantations5 The LAK cell phenomenon has revived interest in adoptive immunotherapy as a treatment for human cancer. Using leucopheresis and tissue culture methodology, adoptive immunotherapy is being investigated by infusing large numbers of culture-generated autologous LAK cells and IL-2 into patients with disseminated cancer.33 In both clinical and animal studies, the combination of LAK cells and IL2 was significantly more effective than either modality administered alone. The malignancies most susceptible to this protocol appear to be renal cell carcinoma and melanoma. The results of these trials have provoked controversy because the modest rate of complete (8%) and partial (14%) responses must be balanced against the great expense involved and the serious complication rate. Immunotherapy with LAK cells is an attractive treatment modality with a sound theoretical basis, but many problems have to be addressed before it gains widespread acceptance. Most serious complications are related to the intermittent injection of megaunit doses of IL-2 which

causes acute renal failure and fluid overload with consequent cardiopulmonary failure. Reduction of this side effect by continuous infusion of IL-2 has recently been achieved. 82 Responses to therapy in many patients are transient, possibly because of the short life span and uncertain tissue distribution of LAK cells after infusion. In the future it is likely that LAK cell immunotherapy will prove most useful in combination with other forms of treatment for malignancies which are resistant to present day conventional therapy.

Acknowledgement Dr P. Hogan is in receipt of a Clinical Fellowship from the Commonwealth Tertiary Education Commission’s Special Research Centre fund.

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