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MHC restricted and non-restricted killer lymphocytes
MHC Restricted and Non-restricted Killer Lymphocytes
R. C. Rees SUMMA R Y. Cytotoxic lymphocytes are either MHC-restricted (cytotoxic T-cells) or aonrestricted (natural killer NKcells), although cells of the monocyte/macrophage lineage are also cytotoxic, aud lymphocytes or pkagocytic cells expressing Fc-receptors for immunoglobuUa can function as antibodydependent killer cells (referred to as antibodydepeudent cellular cytotoxicity: ADCC). Antigeuqeci6c T-lympkocytes recognise their target antigen in tbe context of MHC class I components, focusing tkeir attack only agaiti those cells expressing the relevant antigen specilIcity on their cell surface. A more primitive and alternative mechanism exists whereby NKcells, classilled as large grauular lymphocytes (LGL), are able to kill in a non-spec%c manner, not requiring prior semitisatiou to antigen. Both antigeu-speciUc T-cells and LGL mediate their cytotoxicity through the release of cytotoxic molecules at tbe target-effeetor cell interface. LGL also have a regulatory role in tbe immune system through the release of cytokines, and can be . . . dstmgdd from T-lympkocytes by the expression of distinct pkenotypic markers (CI)I6+, CD!%+) and they lack CD3 antigen expression or rearranged a/p or y/S T-cell receptor geae products. Cytot?xic activity is positively regulated by interleukiu-2 (IL-2) and interferon (IFN), wkilst prostaglaudins and transforming growth factor-l) (TGFjl) dimiuisb activation and effector patkways. Cytotoxicity mediated by NK- aud T-cell populations are principally involved in the defence against microbiaI infections and neoplasia; tbe abrogation of cytotoxicity either by direct interaction of ‘suppressor factors’ with effector cells, or imhectly by reducing cytokine production can inevitably lead to the prolireration of tke disease.
The rapid advance in our understanding of the immune system and its regulation has allowed us to define the roles played by lymphoid cells in establishing antigen-specific immunity and natural resistance, and represent important defences against microbial infection. Many cell types are involved in the induction and effector phases of the response to invading organisms and ‘non-self’ antigens expressed on cells. Cytotoxic attack directed against target cells expressing foreign antigens can occur through the generation of cytotoxic T-cells, which recognise a specific seR. C. Rees, Section of Tumour Biology and Immunology, Department of Experimental and Clinical Microbiology, The University of Sheffield Medical School, Beech Hill Road, Sheffield SlO 2RX, UK. Blood Reviews (1990) 4, 204-210 0 1990 Longman GroupUK Ltd
quence of amino acids and are defined as being antigen-specific. Natural killing occurs against ‘abnormal’ cell types, for example cells infected with viral agents or transformed (cancer) cells; both LGL and some T-cells are able to mediate natural killing, a function which does not require prior exposure and sensitisation to antigen. Iv2 The induction of antigenspecific immunity and the regulation of cytotoxicity is governed by the release of cytokines from T-cells, NK-cells and other cells of the immune system. Although the complexity of this signalling system has not been fully established, many of the interleukins (l-9), IFN a and y, tumour necrosis factor (TNF) and lymphotoxin are known to govern lymphocyte killing potential.3 In addition to lymphocytotoxicity, 0268-960X/90/000&0204
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cells of the monocyte and macrophage series can mediate killing through a phagocytic-endocytotic mechanism or by a non-phagocytic process similar to that of lymphocytes. B-lymphocytes can solicit the aid of LGL and monocyte/macrophages through an antibody-dependent killing process, involving the production of antigen-specific IgG, which forms a bridge between the target and effector cell and determines the specificity of the cytotoxic attack. This association allows the effector cell to deliver a lethal cytotoxic attack on its target, a process referred to as ADCC. We will consider here principally those effector cells of the lymphocyte lineage which mediate antigenspecific and antigen-nonspecific cytotoxicity, their regulation and activation pathways, and how these cells govern other immune functions and contribute to host immunity. Characteristics of the Effector Cells T-lymphocytes express CD3 antigen at their cell surface in association with the T-cell receptor (TCR); this represents the pathway for signal transduction and cell activation in response to foreign protein. The majority of T-lymphocytes also possess CD2 (the erythrocyte receptor), and are divided into two major subpopulations based on the expression of CD4 (Thelper cells) and CD8 (cytototoxic effector cells) antigens. This latter population is principally involved in mediating cytotoxic responses and in generating antigen-specific lymphocyte clones; the specificity of which is determined by the rearrangement of a,$ or y/S genes and the expression of their hypervariable products on the TCR.4 The heterodimers which form at the surface of T-cells recognise specific antigen-peptide sequences expressed on the target in the context of major histocompatibility complex (MHC) antigens. The initial recognition of the peptide sequence by T-cells occurs through a process of antigen presentation (by for example follicular dendritic cells), a process which initiates the clonal expansion of antigen-specific cytotoxic T-cell clones. The CD3/ TCR complex of antigen ‘primed’ cytotoxic T-cells is able to recognise antigen when expressed on the target in association with MHC class I gene products. Following recognition, the CD3/TCR complex transduces an activation signal which in turn causes the cell the deliver a lethal cytotoxic response. Certain stages of cellular activation and proliferation are controlled by cytokines which interact with appropriate cell membrane receptors. Cytotoxic Tcells express at their cell surface high affinity receptors for various cytokines, including IL-2 and IL-4, which upon activation induce cell proliferation and expansion of antigen-specific T-cell clones. The receptor for IL-2 is particularly well characterised and has been defined as having two major components: the a- and P-chain (also known as P55 or Tat and P70); together they constitute a high affinity receptor for IL-2, which upon association with IL-2 stimulates cell prolifer-
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ation and the expression of genes for other cytokines, namely IL-la and B, TNFa and IFNy. These in turn have major regulatory effects on lymphocyte function.6 It is important to real& that under normal physiological conditions, cytokines released from lymphocytes mediate their effects over short distances and have a limited half-life, so ensuring widespread activation of bystander cells is avoided. The major cell type involved in non-MHC restricted killing is the NK cell. These are morphologically large granular lymphocytes which have a kidney shaped nucleus and azurophilic granules in their cytoplasm. Unlike T-cells they do not rearrange a@ or y/6 genes and do not synthesise functional TCR mRNA, although they may express limited transcripts of the 8 and 6 portions of the TCR; the TCR complex is therefore not involved in NK-cell recognition or cytotoxic activity. Not all LGL are cytotoxic, and as will be discussed later, events other than target recognition and target cell binding are required for cytotoxicity to occur. NK-cells express at their surface Leu 19 (NKH-1 antigen or CD56) and CD16 antigens and are CD3 -. Populations of T-lymphocytes (TCR+) may also coexpress NKH-1 antigen but are not cytotoxic for NK-sensitive targets. In addition, both CD3+ and CD3- lymphocyte clones can express the CD16 marker (the receptor for the Fc portion of IgG FcRIII) which can itself act as an activation pathway for the induction of cytotoxicity. It has also been recently shown that certain a/8 or y/S T-cell subpopulations mediate ‘NK-like’ killing without the requirement for MHC-antigen recognition. Whether NK-cells and non-MHC restricted cytotoxic T-lymphocytes arise from a common progenitor cell type has not been resolved, and we await definitive evidence to resolve this issue. Cytotoxic Mechanism Cytotoxic effector cells mediate target cell lysis through a series of well defined stages: (1) Target cell recognition; (2) Cell adhesion; (3) Effector cell triggering; (4) Effector cell activation; (5) Secretion of cytotoxic factors; (6) Factor binding to the target cell; (7) Target cell changes; (8) Cell death, and (9) Effector cell recycling. T-lymphocytes recognise peptide sequences associated with MHC class I antigen via their CD3/TCR complex, whereas unique surface molecules are associated with target recognition by NKcells. Adhesion of lymphocytes to the target occurs through a series of well character&d adhesion proteins present on the effector cell surface, for example fibronectin, laminin and leukocyte function-associated (LFA) molecules are involved in T-lymphocyte adhesion to target cells, and on NK-cells the CD56 molecule (NKH-l/Leu 19 antigen) appears to be involved in target cell binding.“*” The expression of LFA-1 on T-cells maybe of considerable importance as an attachment molecule, binding to intercellular adhesion molecule-l (ICAM-l), a common mem-
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KILLER
brane component expressed by many cell types. Attachment of lymphocytes to target cells is a prerequisite for killing, although transduction of the activation signal occurs through other membrane components, for example by CD3/TCR triggering in the case of antigen-specific cytotoxic T-cells. Alternative activation pathways for lymphocytes can occur through triggering CD2 or CD3 antigens (e.g. using monoclonal antibodies against CD2 or CD3); this activates protein kinase C and causes either a persistant (through CD2) or transient (through CD3) production of LFA- 1.lo Recently a unique 48 kDa molecule, which shares some homology with the 8 2-chain of the matrix laminin and kappa light chain, has been described on NK-cells and IL-2 activated NK-cells, and appears to be involved in IL-2 dependent cell proliferation and esterase secretion from NK-cells. Following the binding of NK-cells to targets, transmembrane signalling activates certain intracellular processes, mediated through an increase in phosphatidyl-inositol (PI) metabolism, calcium influx and eventually the rearrangement and release of intracellular granules.’ This leads to the secretion of granules from cytotoxic T-cells or NK-cells which are responsible for delivering the ‘lethal hit’ to the target. Although the process of killing by NK-cells and cytotoxic Tlymphocytes (CTL) is in many respects similar, granules only appear in CTL upon activation, whereas lytic LGL already contain these granules. One of the major biochemical components of lymphocyte granules is a pore-forming protein, perform, a 70 kDa molecule with partial sequence homology with complement C9. l2 Insertion of perform into the lipid bilayer of target cells results in polymerisation of the membrane constituents and the formation of a tubular transmembrane pore. Other molecules thought to contribute to the lytic mechanism include 6 serine esterases and high molecular weight proteoglycans which are probably physically associated with both perforin and the serine esterases; cytolysin, chemotactic factors for leukocytes, a heat stable macrophage activating factor, and NK cytotoxic factor (NKCF) have also been isolated from NK-cells, and contribute to the lytic events occurring at the membrane interface.13*i4 Cytolysin (a 70 kDa molecule) has been shown to be a potent cytotoxic factor released from NK-cells, and TNFu may play a role in the killing mediated by subsets of naturally cytotoxic cells.’ 5 It is thought that either fusion of the membrane of the cytotoxic granules with the target cell membrane or endocytosis of the granules occurs, so allowing the delivery of cytotoxic substances to the target cell.“j Additional evidence suggests that CTL can kill their target cell by a process of apoptosis which is accompanied by DNA fragmentation within the target cell prior to rupture of the cell membrane.” Exposure of target cells to IFN induces partial resistance to cytotoxic attack, due to the failure of the effector granules to become reorientated after target cell binding; binding to IFN-treated targets is therefore in-
LYMPHOCYTES
effective in triggering lytic events. A distinction which has been made previously between NK and cytotoxic T-cell killing relates to the role of MHC antigen expression on target cells. Thus, whereas T-cells require the expression of MHC class I for efficient T-cell receptor interaction to occur, an inverse correlation exists between target cell susceptibility to lysis by NKcells and the expression of MHC class I, although this may not be true for all effector-target systems. Regulation and Activation of Cytotoxicity It has been 8rmly established that the expansion of cytotoxic T-cell clones is governed by growth factors produced by other lymphocyte subsets including Thelper cells and LGL. The main influences on T-cell growth occur following their interaction with the Tcell growth factors IL-2 and IL-4; IL-2 for example can act as a growth stimulant for the expansion of clones following interaction with antigen presenting cells. The P70 subunit of the high affinity IL-2 receptor is constitutively expressed on T-cells, and upon association with its ligand will cause P55 (tat antigen) subunit expression; these together constitute a heterodimer (IL-2 receptor) with a high binding affinity for IL-2; the P70 subunit transduces the activation signal across the membrane whilst the combination of P55 and P70 allows for continual IL-2 stimulation of the ~ell.~ IFNa, /3and y and IL-2 represent the main positive regulatory signals for NK-cells. LGL and non-MHC restricted cytotoxic T-cells are activated by these cytokines to increase their proliferation and lytic potential, and although the two events are not necessarily interdependent. In addition, CD3- LGL produce IL-l and colony stimulating factors (CSF), which not only influence the production, tumover, migration (chemotaxis) and functional activity of NK-cells but also affect other cells of the immune system.3 Activating agents, such as IFN and IL-2, may alter the pattern of conjugate formation, rate of lysis, recycling or modulate the effect of cytotoxic molecules on the target cell. IFNa for example will increase the rate of recycling of NK-cells, and so affect the kinetics of cytotoxicity. The time course of activation may vary for different cytokines, for example IFNu activates NK-cells maximally within l-2 h, whereas IFNy induces maximum activation of NKcells only after a prolonged (48 h) incubation period, and this is probably as a result of the production of additional cytokines which influence NK cytotoxicity.‘* It has recently been shown that IL-6 significantly augments the cytotoxic activity of human NKcells.” Previously known as B-cell stimulatory factor 2, this 26 kDa protein activates human CD3- NKcells but not CD3 + non-MHC restricted cytotoxic Tcells by increasing the cytotoxic potential of the cell rather than increasing the number of effecters capable of binding to target cells. The most likely explanation for this effect is that IL-6 promotes the production of
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IL-2 from either T-cells or LGL, since the enhancing properties of IL-6 can be abrogated by the presence of neutralising antibody to IL-2. Other cytokines, for example IL-l and TNFcz may act synergistically with IL-2 to promote LGL cytotoxic activity,*’ and IL-4 and TNFtz appear to be intimately involved in the regulation of non-MHC restricted T-cell activation.*’ Recently it has been shown that the addition of exogenous IL-2 to blood lymphocyte cultures for periods of 4 or more days results in the activation of both CD3+ and CD3- lymphocyte subsets which are non-MHC restricted killer cells, now referred to as lymphokine activated killer (LAK)cells.6 This is not the subject of the present review, although it illustrates the potential of IL-2 for inducing both lymphocyte proliferation and cytotoxic effector cells; this is an approach which has been used recently in the treatment of patients with malignant disease where antigen-specific and non-MHC restricted effector cells expanded in vitro with IL-2 have been adoptively transferred back into the same individual; 20-30% of patients with melanomas or renal cell carcinoma show a positive clinical response (complete or partial tumour regression) to this form of therapy.22v23 Negative regulation of cytotoxic lymphocyte activity may occur as the result of exposure of effector cells to prostaglandins of the E series or to TGFB.24,25 TGFB is a known mediator of inflammation, repair, and angiogenesis, and negatively regulates the growth of epithelial cells, and T- and B-lymphocytes.*’ Released in a latent form from platelets and in less quantity from other cell types, proteolytic or acid cleavage of the molecule yields a 25 kDa homodimer which is a potent suppressor of lymphocyte activation. TGFB inhibits lymphocyte proliferation, reduces IL-2 induced cytotoxicity and has assumed a prominent position as a regulator of immunity. It is likely that we will see an expansion in the members of the TGFB family with continued research, and an increase in its involvement in various disease states. Immueoregdation by Cytokines Released from ‘Killer’ Cells It is now clear that the molecular signals released from lymphocytes in the form of cytokines govern the induction and maintenance of specific and innate immunity. T-cells for example, secrete a variety of cytokines, which include IL- 1 to 6, IFN ct and -y, TNFcr, and colony stimulating factors.26*27 IL-2 influences both B-and T-cell function, but only recently have the effects of other cytokines on immune function/regulation been fully appreciated. IL-4 induces B-cell growth, 28 but will also supp ort T-cell proliferation and influence mast cell and haematopoietic cell activity. TNFcl has now been shown to enhance the activation of T-cells via CDZdependent and independent pathways while IL-4 appears to function via the CD3 pathway only and can inhibit IL-2 activation of T-cells.*’ LGL, in addition to being cytotoxiz, pro-
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duce a variety of cytokines including IL-I (u and B), IL-2, B-cell growth factor, colony stimulating factors and IFNol and y. This implicates NK-cells in a variety of immunological responses, including the proliferation and differentiation of B-cells; however, besides providing a positive signal for B-cell development, a subset of CD16+/CD3NK-cells suppresses antibody production. *’ LGL release of IFNa and -y occurs during the process of target cell binding, although this property may be restricted to those LGL possessing DR antigen and Fc receptor.2 Both T-lymphocytes and LGL produce IL-2 but differences in its regulation and secretion have become apparent: T-cell IL-2 production can be increased by IL-l, whereas the secretion of IL-2 by NK-cells is not influenced by the addition of exogenous IL-l. Furthermore, the analysis of NK clones has revealed that not all cells mediating NK activity are able to secrete cytokines, supporting the view that subpopulations of LGL have defined immunoregulatory functions. Using a molecular approach to the examination of activation of human peripheral blood lymphocytes by IL-2, Kovacs et al 3o showed that the genes for IL101and IL- 1b were expressed very early following IL-2 activation (within 2 h of IL-2 treatment), whereas the genes for IFNy, TNFct and lymphotoxin were expressed at later time points. Clearly, the available evidence implicates NK- and T-lymphocytes not only in cytotoxic interactions but also as principle influences in the regulation of a variety of immune responses. Cytotoxic Effector Cells and Diseases Cytotoxic T- and NK-cells perform an essential role in the defence against microbial and neoplastic disease. The specificity of CTL for varial antigens (expressed in the context of self MHC antigens) allows the ‘lethal hit’ to be focused towards infected, but not uninfected cells, and they appear to be necessary in mediating recovery from virus infection. Factors released from T-cells may activate macrophages and NK-cells to kill in a MHC unrestricted manner, and LGL respond chemotactically and proliferate in response to interferon generated by virusinfected cells in vivo. The conception that NK-cells are important as a host defence mechanism against viral infection is supported by the experimental observation that mice depleted of LGL (by treatment with asialo GM 1 or alloantigen NK 1.I antisera) are rendered more susceptible to a variety of viruses including murine cytomegalovirus, vaccinia virus, Coxsackie B virus, encephalomyocarditis virus and influenza virus; resistance to infection in NK-depleted mice can be re-established by adoptive transfer of NK-cells.’ In vitro, virus-infected target cells can be shown to be more susceptible to cytotoxic attack than uninfected targets at a time before late viral proteins and infectious virus are produced. Both CD3+ and CD3 - non-MHC restricted killer cells lyse virus-
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infected cells, although CD3+ cells mediate a greater degree of selection for targets. The pathogenesis of viral disease is further influenced by the production of IFN by lymphocytes and infected cells, which render normal (non-infected) cells resistant to virus infection and cytotoxic attack by NK-cells. Dendritic cells may in some cases produce IFN and consequently activate NK-cells at the site of infection, and in some instances, as in herpes simplex infection, NK-cells may contribute to the host defence as the effector cell responsible for antibody-dependent killing (ADCC).31 The action of NK-cells in bacterial infections is less clear, although it can be inferred from the evidence available that they mediate antibacterial effects. Mammalian cells infected with intracellular bacteria are more susceptible to NK-cell mediated lysis, as are human monocytes infected with Legionella pneumophilia. NK-cells may contribute to the destruction of bacteria in antibody-dependent killing (ADCC), directly affect bacteria by reducing their plating efficiency, or produce cytokines which activate other antimicrobial defence mechanisms, for example by releasing NK macrophage activating factors.‘*32 Natural immunity can also contribute to host resistance against protozoa1 and mycotic infections, although relatively little detailed information is available.1*33v34 A role for NK-cells in the control of haematopoiesis has been suggested from observations demonstrating human NK cytotoxicity towards bone marrow cells; in addition cytokines produced by NK-cells are likely to influence bone marrow cell differentiation and maturation. In autoimmune disease lymphocyte activity plays a decisive role. Production of cytokines (IFNa or -y, or TNFu) by lymphocytes could result in the inappropriate expression of class II MHC antigens, leading to the presentation of ‘self’ antigens to the immune system (antigen presentation occurs when class II MHC and antigen are appropriately presented to T-lymphocytes). This could lead to a breakdown of immune tolerance, and the development of antigen specific cytotoxic T-cells reactive against ‘self’ antigens expressed on normal cells. Immune Surveillance and Tumour Immunity Immune surveillance against cancer cells could be mediated either by the natural defence systems, (i.e. NK-cells or macrophages) or by T-cells able to recognise ‘foreign’ tumour-associated transplantation antigens (TATA). Experimental tumours induced by viruses or carcinogens express antigenic determinants which stimulate T-cell responses; however, the presence of similar components on human turnours, with the exception of malignant melanoma and renal cell carcinoma, has not been conclusively demonstrated. Reduced immunoreactivity towards tumours may occur either because they fail to express an appropriate ‘foreign’ antigen (tumour rejection antigen) or MHC class I antigen,35-37 or because tumours subvert the induction of antigen-specific immune responses or
effector function through the production of ‘suppressor’ factors. Antigen-specific T-cells can therefore only function as a defence system against newly arising cancers expressing relevant immunogenic components. There is also a substantial body of evidence favouring the view that cells mediating nonMHC restricted killing are more likely to operate as an immune surveillance mechanism against human neoplasia. For example, the incidence of malignant lymphoma has been shown to be increased in NKdeficient mice carrying the beige (bg+/bg+) mutation. In several studies the NK status of mice inversely correlates with the growth rate of transplantable tumours,38*3g and an increase in tumour takes is apparent where animals have been depleted of their NK-cells by treatment with asialo-GM 1 antiserum.40 A higher frequency of spontaneous lymphoproliferative disorders has also been shown in patients with Chediak Higashi syndrome, where NK-cell activity is considerably lower than in normal individuals.41 Tlymphocyte and NK-cell responses are depressed in patients bearing advanced malignant disease, which in part, may be due to immune suppression mediated by tumour cell products. 42-45 In some instances a correlation between the level of NK activity and the survival of patients with solid malignancies has been shown,46 indirectly inferring a role for natural immunity in restricting tumour growth. In vivo activation of cytotoxic T- and NK-cells using IL-2 or other cytokines and the induction of LAK-cells maybe an acceptable way of augmenting/restoring antitumour host immunity. Collectively, there is evidence to support the view that NK-cells play a role in immune surveillance against cancer. In a number of studies it has been shown that NKcells play a role in limiting the haematogenous spread of tumour cells to secondary organs. Metastasis is a complex multistep process, and for cells to form secondary tumours it is necessary for them to possess the properties necessary for invasion, thus allowing them to extravasate the primary site and exit the blood via endothelial cell and basement membrane barriers into secondary organs, and avoid host defence mechanisms present in the blood system and tissues. NK-cells appear to play a central role in destroying tumour cells in the blood circulation, and experimentally at least the ability of tumour cells to survive when injected into the bloodstream of mice can be related to the NK status of the host.47*48 This view is further supported by studies showing that cloned tumour cells show high, low and intermediate metastatic ability, and that the spontaneous metastatic potential of these clones is inversely related to their susceptibility to natural killing.36 Far less is known of how antigen-specific and non-MHC restricted killer cells affect human tumour growth and metastasis, and the available experimental evidence provides only a simplistic view of the complex cellular interactions which occur. Our knowledge of the genetic control of turnour metastases and immune resistance is limited
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and further evidence establishing the immunobiology of human tumours is necessary if we are to understand the precise role of natural and antigen-specik responses in controlling tumour growth and metastasis.
21.
22.
Acknowledgements I would like to acknowledge the Yorkshire Cancer Research Campaign for their support of these studies and Mrs C. Mullan for typing this manuscript.
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natural cytotoxicity of spleen effector cells from patients with carcinoma. Journal of Cancer Research and Clinical Gncology 106:202-209 43. Steinhauer E, Doyle A, Reed J, Kadish A 1982 Defective natural cytotoxicity in patients with cancer: normal number of effector cells but decreased recycling capacity in patients with advanced disease. Journal of Immunology 129: 2255-2259 44 Jermy A, Lilleman J S, Jennings R, Rees R C 1987 Spontaneous natural killer cell activity in childhood acute lymphoblastic leukaemia. European Journal of Cancer and Clinical Oncology 23: 1365-1570 45. Healy F, Rees R C, Hancock B W 1985 An assessment of
cell mediated cytotoxicity on patients with malignant lymphoma. European Journal of Cancer and Clinical Oncology 21: 115-184 46. Shantz S P, Brown B W, Lira E, Taylor D L, Beddingfleld N 1987 Evidence for the role of natural immunity in the control of metastatic spread of head and neck cancer. Cancer Immunology Immunotherapy 25: 141-148 41. Hanna N, Fidler I J 1980 The role of natural killer cells in the destruction of circulating tumour emboli. Journal of the National Cancer Institute 65: 801-809 48. Kasai M, Yoneda T, Habu S et al.1981 In vivo effect of anti-asialo GM1 antibody on natural killer activity. Nature 291:334-335
R. C. Rees SUMMA R Y. Cytotoxic lymphocytes are either MHC-restricted (cytotoxic T-cells) or aonrestricted (natural killer NKcells), although cells of the monocyte/macrophage lineage are also cytotoxic, aud lymphocytes or pkagocytic cells expressing Fc-receptors for immunoglobuUa can function as antibodydependent killer cells (referred to as antibodydepeudent cellular cytotoxicity: ADCC). Antigeuqeci6c T-lympkocytes recognise their target antigen in tbe context of MHC class I components, focusing tkeir attack only agaiti those cells expressing the relevant antigen specilIcity on their cell surface. A more primitive and alternative mechanism exists whereby NKcells, classilled as large grauular lymphocytes (LGL), are able to kill in a non-spec%c manner, not requiring prior semitisatiou to antigen. Both antigeu-speciUc T-cells and LGL mediate their cytotoxicity through the release of cytotoxic molecules at tbe target-effeetor cell interface. LGL also have a regulatory role in tbe immune system through the release of cytokines, and can be . . . dstmgdd from T-lympkocytes by the expression of distinct pkenotypic markers (CI)I6+, CD!%+) and they lack CD3 antigen expression or rearranged a/p or y/S T-cell receptor geae products. Cytot?xic activity is positively regulated by interleukiu-2 (IL-2) and interferon (IFN), wkilst prostaglaudins and transforming growth factor-l) (TGFjl) dimiuisb activation and effector patkways. Cytotoxicity mediated by NK- aud T-cell populations are principally involved in the defence against microbiaI infections and neoplasia; tbe abrogation of cytotoxicity either by direct interaction of ‘suppressor factors’ with effector cells, or imhectly by reducing cytokine production can inevitably lead to the prolireration of tke disease.
The rapid advance in our understanding of the immune system and its regulation has allowed us to define the roles played by lymphoid cells in establishing antigen-specific immunity and natural resistance, and represent important defences against microbial infection. Many cell types are involved in the induction and effector phases of the response to invading organisms and ‘non-self’ antigens expressed on cells. Cytotoxic attack directed against target cells expressing foreign antigens can occur through the generation of cytotoxic T-cells, which recognise a specific seR. C. Rees, Section of Tumour Biology and Immunology, Department of Experimental and Clinical Microbiology, The University of Sheffield Medical School, Beech Hill Road, Sheffield SlO 2RX, UK. Blood Reviews (1990) 4, 204-210 0 1990 Longman GroupUK Ltd
quence of amino acids and are defined as being antigen-specific. Natural killing occurs against ‘abnormal’ cell types, for example cells infected with viral agents or transformed (cancer) cells; both LGL and some T-cells are able to mediate natural killing, a function which does not require prior exposure and sensitisation to antigen. Iv2 The induction of antigenspecific immunity and the regulation of cytotoxicity is governed by the release of cytokines from T-cells, NK-cells and other cells of the immune system. Although the complexity of this signalling system has not been fully established, many of the interleukins (l-9), IFN a and y, tumour necrosis factor (TNF) and lymphotoxin are known to govern lymphocyte killing potential.3 In addition to lymphocytotoxicity, 0268-960X/90/000&0204
$10.00
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cells of the monocyte and macrophage series can mediate killing through a phagocytic-endocytotic mechanism or by a non-phagocytic process similar to that of lymphocytes. B-lymphocytes can solicit the aid of LGL and monocyte/macrophages through an antibody-dependent killing process, involving the production of antigen-specific IgG, which forms a bridge between the target and effector cell and determines the specificity of the cytotoxic attack. This association allows the effector cell to deliver a lethal cytotoxic attack on its target, a process referred to as ADCC. We will consider here principally those effector cells of the lymphocyte lineage which mediate antigenspecific and antigen-nonspecific cytotoxicity, their regulation and activation pathways, and how these cells govern other immune functions and contribute to host immunity. Characteristics of the Effector Cells T-lymphocytes express CD3 antigen at their cell surface in association with the T-cell receptor (TCR); this represents the pathway for signal transduction and cell activation in response to foreign protein. The majority of T-lymphocytes also possess CD2 (the erythrocyte receptor), and are divided into two major subpopulations based on the expression of CD4 (Thelper cells) and CD8 (cytototoxic effector cells) antigens. This latter population is principally involved in mediating cytotoxic responses and in generating antigen-specific lymphocyte clones; the specificity of which is determined by the rearrangement of a,$ or y/S genes and the expression of their hypervariable products on the TCR.4 The heterodimers which form at the surface of T-cells recognise specific antigen-peptide sequences expressed on the target in the context of major histocompatibility complex (MHC) antigens. The initial recognition of the peptide sequence by T-cells occurs through a process of antigen presentation (by for example follicular dendritic cells), a process which initiates the clonal expansion of antigen-specific cytotoxic T-cell clones. The CD3/ TCR complex of antigen ‘primed’ cytotoxic T-cells is able to recognise antigen when expressed on the target in association with MHC class I gene products. Following recognition, the CD3/TCR complex transduces an activation signal which in turn causes the cell the deliver a lethal cytotoxic response. Certain stages of cellular activation and proliferation are controlled by cytokines which interact with appropriate cell membrane receptors. Cytotoxic Tcells express at their cell surface high affinity receptors for various cytokines, including IL-2 and IL-4, which upon activation induce cell proliferation and expansion of antigen-specific T-cell clones. The receptor for IL-2 is particularly well characterised and has been defined as having two major components: the a- and P-chain (also known as P55 or Tat and P70); together they constitute a high affinity receptor for IL-2, which upon association with IL-2 stimulates cell prolifer-
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ation and the expression of genes for other cytokines, namely IL-la and B, TNFa and IFNy. These in turn have major regulatory effects on lymphocyte function.6 It is important to real& that under normal physiological conditions, cytokines released from lymphocytes mediate their effects over short distances and have a limited half-life, so ensuring widespread activation of bystander cells is avoided. The major cell type involved in non-MHC restricted killing is the NK cell. These are morphologically large granular lymphocytes which have a kidney shaped nucleus and azurophilic granules in their cytoplasm. Unlike T-cells they do not rearrange a@ or y/6 genes and do not synthesise functional TCR mRNA, although they may express limited transcripts of the 8 and 6 portions of the TCR; the TCR complex is therefore not involved in NK-cell recognition or cytotoxic activity. Not all LGL are cytotoxic, and as will be discussed later, events other than target recognition and target cell binding are required for cytotoxicity to occur. NK-cells express at their surface Leu 19 (NKH-1 antigen or CD56) and CD16 antigens and are CD3 -. Populations of T-lymphocytes (TCR+) may also coexpress NKH-1 antigen but are not cytotoxic for NK-sensitive targets. In addition, both CD3+ and CD3- lymphocyte clones can express the CD16 marker (the receptor for the Fc portion of IgG FcRIII) which can itself act as an activation pathway for the induction of cytotoxicity. It has also been recently shown that certain a/8 or y/S T-cell subpopulations mediate ‘NK-like’ killing without the requirement for MHC-antigen recognition. Whether NK-cells and non-MHC restricted cytotoxic T-lymphocytes arise from a common progenitor cell type has not been resolved, and we await definitive evidence to resolve this issue. Cytotoxic Mechanism Cytotoxic effector cells mediate target cell lysis through a series of well defined stages: (1) Target cell recognition; (2) Cell adhesion; (3) Effector cell triggering; (4) Effector cell activation; (5) Secretion of cytotoxic factors; (6) Factor binding to the target cell; (7) Target cell changes; (8) Cell death, and (9) Effector cell recycling. T-lymphocytes recognise peptide sequences associated with MHC class I antigen via their CD3/TCR complex, whereas unique surface molecules are associated with target recognition by NKcells. Adhesion of lymphocytes to the target occurs through a series of well character&d adhesion proteins present on the effector cell surface, for example fibronectin, laminin and leukocyte function-associated (LFA) molecules are involved in T-lymphocyte adhesion to target cells, and on NK-cells the CD56 molecule (NKH-l/Leu 19 antigen) appears to be involved in target cell binding.“*” The expression of LFA-1 on T-cells maybe of considerable importance as an attachment molecule, binding to intercellular adhesion molecule-l (ICAM-l), a common mem-
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AND NON-RESTRICTED
KILLER
brane component expressed by many cell types. Attachment of lymphocytes to target cells is a prerequisite for killing, although transduction of the activation signal occurs through other membrane components, for example by CD3/TCR triggering in the case of antigen-specific cytotoxic T-cells. Alternative activation pathways for lymphocytes can occur through triggering CD2 or CD3 antigens (e.g. using monoclonal antibodies against CD2 or CD3); this activates protein kinase C and causes either a persistant (through CD2) or transient (through CD3) production of LFA- 1.lo Recently a unique 48 kDa molecule, which shares some homology with the 8 2-chain of the matrix laminin and kappa light chain, has been described on NK-cells and IL-2 activated NK-cells, and appears to be involved in IL-2 dependent cell proliferation and esterase secretion from NK-cells. Following the binding of NK-cells to targets, transmembrane signalling activates certain intracellular processes, mediated through an increase in phosphatidyl-inositol (PI) metabolism, calcium influx and eventually the rearrangement and release of intracellular granules.’ This leads to the secretion of granules from cytotoxic T-cells or NK-cells which are responsible for delivering the ‘lethal hit’ to the target. Although the process of killing by NK-cells and cytotoxic Tlymphocytes (CTL) is in many respects similar, granules only appear in CTL upon activation, whereas lytic LGL already contain these granules. One of the major biochemical components of lymphocyte granules is a pore-forming protein, perform, a 70 kDa molecule with partial sequence homology with complement C9. l2 Insertion of perform into the lipid bilayer of target cells results in polymerisation of the membrane constituents and the formation of a tubular transmembrane pore. Other molecules thought to contribute to the lytic mechanism include 6 serine esterases and high molecular weight proteoglycans which are probably physically associated with both perforin and the serine esterases; cytolysin, chemotactic factors for leukocytes, a heat stable macrophage activating factor, and NK cytotoxic factor (NKCF) have also been isolated from NK-cells, and contribute to the lytic events occurring at the membrane interface.13*i4 Cytolysin (a 70 kDa molecule) has been shown to be a potent cytotoxic factor released from NK-cells, and TNFu may play a role in the killing mediated by subsets of naturally cytotoxic cells.’ 5 It is thought that either fusion of the membrane of the cytotoxic granules with the target cell membrane or endocytosis of the granules occurs, so allowing the delivery of cytotoxic substances to the target cell.“j Additional evidence suggests that CTL can kill their target cell by a process of apoptosis which is accompanied by DNA fragmentation within the target cell prior to rupture of the cell membrane.” Exposure of target cells to IFN induces partial resistance to cytotoxic attack, due to the failure of the effector granules to become reorientated after target cell binding; binding to IFN-treated targets is therefore in-
LYMPHOCYTES
effective in triggering lytic events. A distinction which has been made previously between NK and cytotoxic T-cell killing relates to the role of MHC antigen expression on target cells. Thus, whereas T-cells require the expression of MHC class I for efficient T-cell receptor interaction to occur, an inverse correlation exists between target cell susceptibility to lysis by NKcells and the expression of MHC class I, although this may not be true for all effector-target systems. Regulation and Activation of Cytotoxicity It has been 8rmly established that the expansion of cytotoxic T-cell clones is governed by growth factors produced by other lymphocyte subsets including Thelper cells and LGL. The main influences on T-cell growth occur following their interaction with the Tcell growth factors IL-2 and IL-4; IL-2 for example can act as a growth stimulant for the expansion of clones following interaction with antigen presenting cells. The P70 subunit of the high affinity IL-2 receptor is constitutively expressed on T-cells, and upon association with its ligand will cause P55 (tat antigen) subunit expression; these together constitute a heterodimer (IL-2 receptor) with a high binding affinity for IL-2; the P70 subunit transduces the activation signal across the membrane whilst the combination of P55 and P70 allows for continual IL-2 stimulation of the ~ell.~ IFNa, /3and y and IL-2 represent the main positive regulatory signals for NK-cells. LGL and non-MHC restricted cytotoxic T-cells are activated by these cytokines to increase their proliferation and lytic potential, and although the two events are not necessarily interdependent. In addition, CD3- LGL produce IL-l and colony stimulating factors (CSF), which not only influence the production, tumover, migration (chemotaxis) and functional activity of NK-cells but also affect other cells of the immune system.3 Activating agents, such as IFN and IL-2, may alter the pattern of conjugate formation, rate of lysis, recycling or modulate the effect of cytotoxic molecules on the target cell. IFNa for example will increase the rate of recycling of NK-cells, and so affect the kinetics of cytotoxicity. The time course of activation may vary for different cytokines, for example IFNu activates NK-cells maximally within l-2 h, whereas IFNy induces maximum activation of NKcells only after a prolonged (48 h) incubation period, and this is probably as a result of the production of additional cytokines which influence NK cytotoxicity.‘* It has recently been shown that IL-6 significantly augments the cytotoxic activity of human NKcells.” Previously known as B-cell stimulatory factor 2, this 26 kDa protein activates human CD3- NKcells but not CD3 + non-MHC restricted cytotoxic Tcells by increasing the cytotoxic potential of the cell rather than increasing the number of effecters capable of binding to target cells. The most likely explanation for this effect is that IL-6 promotes the production of
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IL-2 from either T-cells or LGL, since the enhancing properties of IL-6 can be abrogated by the presence of neutralising antibody to IL-2. Other cytokines, for example IL-l and TNFcz may act synergistically with IL-2 to promote LGL cytotoxic activity,*’ and IL-4 and TNFtz appear to be intimately involved in the regulation of non-MHC restricted T-cell activation.*’ Recently it has been shown that the addition of exogenous IL-2 to blood lymphocyte cultures for periods of 4 or more days results in the activation of both CD3+ and CD3- lymphocyte subsets which are non-MHC restricted killer cells, now referred to as lymphokine activated killer (LAK)cells.6 This is not the subject of the present review, although it illustrates the potential of IL-2 for inducing both lymphocyte proliferation and cytotoxic effector cells; this is an approach which has been used recently in the treatment of patients with malignant disease where antigen-specific and non-MHC restricted effector cells expanded in vitro with IL-2 have been adoptively transferred back into the same individual; 20-30% of patients with melanomas or renal cell carcinoma show a positive clinical response (complete or partial tumour regression) to this form of therapy.22v23 Negative regulation of cytotoxic lymphocyte activity may occur as the result of exposure of effector cells to prostaglandins of the E series or to TGFB.24,25 TGFB is a known mediator of inflammation, repair, and angiogenesis, and negatively regulates the growth of epithelial cells, and T- and B-lymphocytes.*’ Released in a latent form from platelets and in less quantity from other cell types, proteolytic or acid cleavage of the molecule yields a 25 kDa homodimer which is a potent suppressor of lymphocyte activation. TGFB inhibits lymphocyte proliferation, reduces IL-2 induced cytotoxicity and has assumed a prominent position as a regulator of immunity. It is likely that we will see an expansion in the members of the TGFB family with continued research, and an increase in its involvement in various disease states. Immueoregdation by Cytokines Released from ‘Killer’ Cells It is now clear that the molecular signals released from lymphocytes in the form of cytokines govern the induction and maintenance of specific and innate immunity. T-cells for example, secrete a variety of cytokines, which include IL- 1 to 6, IFN ct and -y, TNFcr, and colony stimulating factors.26*27 IL-2 influences both B-and T-cell function, but only recently have the effects of other cytokines on immune function/regulation been fully appreciated. IL-4 induces B-cell growth, 28 but will also supp ort T-cell proliferation and influence mast cell and haematopoietic cell activity. TNFcl has now been shown to enhance the activation of T-cells via CDZdependent and independent pathways while IL-4 appears to function via the CD3 pathway only and can inhibit IL-2 activation of T-cells.*’ LGL, in addition to being cytotoxiz, pro-
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duce a variety of cytokines including IL-I (u and B), IL-2, B-cell growth factor, colony stimulating factors and IFNol and y. This implicates NK-cells in a variety of immunological responses, including the proliferation and differentiation of B-cells; however, besides providing a positive signal for B-cell development, a subset of CD16+/CD3NK-cells suppresses antibody production. *’ LGL release of IFNa and -y occurs during the process of target cell binding, although this property may be restricted to those LGL possessing DR antigen and Fc receptor.2 Both T-lymphocytes and LGL produce IL-2 but differences in its regulation and secretion have become apparent: T-cell IL-2 production can be increased by IL-l, whereas the secretion of IL-2 by NK-cells is not influenced by the addition of exogenous IL-l. Furthermore, the analysis of NK clones has revealed that not all cells mediating NK activity are able to secrete cytokines, supporting the view that subpopulations of LGL have defined immunoregulatory functions. Using a molecular approach to the examination of activation of human peripheral blood lymphocytes by IL-2, Kovacs et al 3o showed that the genes for IL101and IL- 1b were expressed very early following IL-2 activation (within 2 h of IL-2 treatment), whereas the genes for IFNy, TNFct and lymphotoxin were expressed at later time points. Clearly, the available evidence implicates NK- and T-lymphocytes not only in cytotoxic interactions but also as principle influences in the regulation of a variety of immune responses. Cytotoxic Effector Cells and Diseases Cytotoxic T- and NK-cells perform an essential role in the defence against microbial and neoplastic disease. The specificity of CTL for varial antigens (expressed in the context of self MHC antigens) allows the ‘lethal hit’ to be focused towards infected, but not uninfected cells, and they appear to be necessary in mediating recovery from virus infection. Factors released from T-cells may activate macrophages and NK-cells to kill in a MHC unrestricted manner, and LGL respond chemotactically and proliferate in response to interferon generated by virusinfected cells in vivo. The conception that NK-cells are important as a host defence mechanism against viral infection is supported by the experimental observation that mice depleted of LGL (by treatment with asialo GM 1 or alloantigen NK 1.I antisera) are rendered more susceptible to a variety of viruses including murine cytomegalovirus, vaccinia virus, Coxsackie B virus, encephalomyocarditis virus and influenza virus; resistance to infection in NK-depleted mice can be re-established by adoptive transfer of NK-cells.’ In vitro, virus-infected target cells can be shown to be more susceptible to cytotoxic attack than uninfected targets at a time before late viral proteins and infectious virus are produced. Both CD3+ and CD3 - non-MHC restricted killer cells lyse virus-
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KILLER LYMPHOCYTES
infected cells, although CD3+ cells mediate a greater degree of selection for targets. The pathogenesis of viral disease is further influenced by the production of IFN by lymphocytes and infected cells, which render normal (non-infected) cells resistant to virus infection and cytotoxic attack by NK-cells. Dendritic cells may in some cases produce IFN and consequently activate NK-cells at the site of infection, and in some instances, as in herpes simplex infection, NK-cells may contribute to the host defence as the effector cell responsible for antibody-dependent killing (ADCC).31 The action of NK-cells in bacterial infections is less clear, although it can be inferred from the evidence available that they mediate antibacterial effects. Mammalian cells infected with intracellular bacteria are more susceptible to NK-cell mediated lysis, as are human monocytes infected with Legionella pneumophilia. NK-cells may contribute to the destruction of bacteria in antibody-dependent killing (ADCC), directly affect bacteria by reducing their plating efficiency, or produce cytokines which activate other antimicrobial defence mechanisms, for example by releasing NK macrophage activating factors.‘*32 Natural immunity can also contribute to host resistance against protozoa1 and mycotic infections, although relatively little detailed information is available.1*33v34 A role for NK-cells in the control of haematopoiesis has been suggested from observations demonstrating human NK cytotoxicity towards bone marrow cells; in addition cytokines produced by NK-cells are likely to influence bone marrow cell differentiation and maturation. In autoimmune disease lymphocyte activity plays a decisive role. Production of cytokines (IFNa or -y, or TNFu) by lymphocytes could result in the inappropriate expression of class II MHC antigens, leading to the presentation of ‘self’ antigens to the immune system (antigen presentation occurs when class II MHC and antigen are appropriately presented to T-lymphocytes). This could lead to a breakdown of immune tolerance, and the development of antigen specific cytotoxic T-cells reactive against ‘self’ antigens expressed on normal cells. Immune Surveillance and Tumour Immunity Immune surveillance against cancer cells could be mediated either by the natural defence systems, (i.e. NK-cells or macrophages) or by T-cells able to recognise ‘foreign’ tumour-associated transplantation antigens (TATA). Experimental tumours induced by viruses or carcinogens express antigenic determinants which stimulate T-cell responses; however, the presence of similar components on human turnours, with the exception of malignant melanoma and renal cell carcinoma, has not been conclusively demonstrated. Reduced immunoreactivity towards tumours may occur either because they fail to express an appropriate ‘foreign’ antigen (tumour rejection antigen) or MHC class I antigen,35-37 or because tumours subvert the induction of antigen-specific immune responses or
effector function through the production of ‘suppressor’ factors. Antigen-specific T-cells can therefore only function as a defence system against newly arising cancers expressing relevant immunogenic components. There is also a substantial body of evidence favouring the view that cells mediating nonMHC restricted killing are more likely to operate as an immune surveillance mechanism against human neoplasia. For example, the incidence of malignant lymphoma has been shown to be increased in NKdeficient mice carrying the beige (bg+/bg+) mutation. In several studies the NK status of mice inversely correlates with the growth rate of transplantable tumours,38*3g and an increase in tumour takes is apparent where animals have been depleted of their NK-cells by treatment with asialo-GM 1 antiserum.40 A higher frequency of spontaneous lymphoproliferative disorders has also been shown in patients with Chediak Higashi syndrome, where NK-cell activity is considerably lower than in normal individuals.41 Tlymphocyte and NK-cell responses are depressed in patients bearing advanced malignant disease, which in part, may be due to immune suppression mediated by tumour cell products. 42-45 In some instances a correlation between the level of NK activity and the survival of patients with solid malignancies has been shown,46 indirectly inferring a role for natural immunity in restricting tumour growth. In vivo activation of cytotoxic T- and NK-cells using IL-2 or other cytokines and the induction of LAK-cells maybe an acceptable way of augmenting/restoring antitumour host immunity. Collectively, there is evidence to support the view that NK-cells play a role in immune surveillance against cancer. In a number of studies it has been shown that NKcells play a role in limiting the haematogenous spread of tumour cells to secondary organs. Metastasis is a complex multistep process, and for cells to form secondary tumours it is necessary for them to possess the properties necessary for invasion, thus allowing them to extravasate the primary site and exit the blood via endothelial cell and basement membrane barriers into secondary organs, and avoid host defence mechanisms present in the blood system and tissues. NK-cells appear to play a central role in destroying tumour cells in the blood circulation, and experimentally at least the ability of tumour cells to survive when injected into the bloodstream of mice can be related to the NK status of the host.47*48 This view is further supported by studies showing that cloned tumour cells show high, low and intermediate metastatic ability, and that the spontaneous metastatic potential of these clones is inversely related to their susceptibility to natural killing.36 Far less is known of how antigen-specific and non-MHC restricted killer cells affect human tumour growth and metastasis, and the available experimental evidence provides only a simplistic view of the complex cellular interactions which occur. Our knowledge of the genetic control of turnour metastases and immune resistance is limited
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and further evidence establishing the immunobiology of human tumours is necessary if we are to understand the precise role of natural and antigen-specik responses in controlling tumour growth and metastasis.
21.
22.
Acknowledgements I would like to acknowledge the Yorkshire Cancer Research Campaign for their support of these studies and Mrs C. Mullan for typing this manuscript.
References 1. Ades E W, Lepes C (eds) 1989 Natural Killer Cells and Host Defence. Karger, Base1 2. Lotzova E, Herberman R B (eds) 1986 Immunobiology of Natural Killer Cells. CRC Press, Florida 3. Balkwill F R, Burke F 1989 The cytokine network. Innumology Today 10: 299-304 4. David M M, Kappler J (eds) 1988 The T Cell Receptor. Alan R Liss, New York 5. Theofilopoulos A N, Kofler R 1989 Molecular aspects of autoimmunity. Immunology Today 10: 180-183 6. Rees R C (ed) 1990 The Biology and Clinical Application of Interleukin-2. Oxford University Press I. Lanier L L, Cwirla S, Federspiel N, Phillips J H 1986 Human natural killer cells isolated from peripheral blood do not re-arrange T cell antigen receptor g-chain genes. Journal of Experimental Medicine 163: 209-214 8. Anderson P, Caligiuri M, Ritz J, Schlossman S F 1989 CD3negative natural killer cells express TCR as part of a novel molecular complex. Nature 341: 159- 162 9. Lamer L L, Yu G, Phillips J H 1989 Co-association of CD3 with a receptor (CD16) for IgG on human natural killer cells. Nature 342: 803-805 10. von Kooyk Y, Kemenade W, Weder P, Kuijpers T W, Findor C G 1989 Enhancement of LFA-1 mediated cell adLesion by triggering through CD2 or CD3 on T lymphocytes. Nature 342~ 811-813 11. Nitto T, Yagita H, Sato K, Okumura K 1989 Involvement of CD56 (NKH-l/Le.u 19 antigen) as adhesion molecule in natural killer-target cell interaction. Journal of Experimental Medicine 170: 1757- 1761 12. Lichtenheld M G, Olsen K J, Lu P, et al 1988 Structure of human perforin. Nature 335: 448-451 13. Kuta A E. Revnolds C R. Her&art P A 1989 Mechanism of lysis by large gramdar lymphocyte granule cytolysin: generation of a stable cytolysin-RBC intermediate. Journal of Immunology 142: 4378-4384 14. Kobayashi M, Fitz L, Ryan M et al 1989 Identification and nurification of natural killer cell stimulatorv factor (NKSF). a cytokine with multiple biological effects on human ” lymphocytes. Journal of Experimental Medicine 170: 827-845 15. Lattime E C, Stoppacciano A, Khan A, Stutman 0 1988 Human natural cytotoxic activity mediated by tumour necrosis factor: regulation by interleukin-2. Journal of the National Cancer Institute 80: 1035-1038 16. Peters P J, Geuze H J, van der Donk H A, Borst J 1990 A new model for lethal hit delivery by cytotoxic T lymphocytes. Immunology Today 11: 28-32 17. Martz E, Howell D M 1989 CTL: virus control cells first and cytolytic cells second? DNA fragmentation, apoptosis and the prelytic halt hypothesis. Immunology Today 10: 79-86 18. Rawlinson L, Dalton B J, Rogers K, Rees R C 1989 The influence of interferon alpha and gamma, singly or in combination on human natural cell-mediated cytotoxicity. Bioscience Reports 9: 549-557 19. Luger T A, Krutmann J, Kimbauer R et al 1989 IFN-8 2/IL-6 augments the activity of human natural killer cells. Journal of Immunology 143: 1206-1209 20. Gwen-S&tub L B, Gutterman J U, Grimm E A 1988 Synergy of tumour necrosis factor and interleukin-2 in the activation of human cytotoxic lymphocytes: effect of tumour necrosis factor and interleukin-2 in the generation of human
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