lymphokine-activated killer cells, natural killer cells and cytokines

Lymphokine-activated killer cells, and cytokines

Bite Perussia

natural killer cells

Thomas Jefferson University, Philadelphia, Pennsylvania, USA

In the past year, natural killer cells have been the subject of much active investigation. The analysis of the effect of cytokines on the generation, proliferation and function of natural killer cells, and the definition of the

lymphokines that they produce, have been particularly important areas of

research in view of their possible application in adaptive immunotherapy,

combined with biological response modifiers.

Current Opinion in Immunology 1991, 3:4955

Introduction

Mature natural killer (NK) cells are a discrete subset of lymphocytes distinct from B and T cells and from cells of the myelomonocytic lineage [ 1,2]. NK cells lack the antigen-recognition molecules, T-cell receptor (TCR) and surface immunoglobulin, and express a chamcteris- tic combination of differentiation antigens on their mem- brane. The best studied of these is the low-aihnity receptor for the Fc portion of IgG (FcyRIIIA, CDl6) that serves as recognition structure for antibody-sensitized cells and immune complexes, and through which antibody-dependent cytotoxicity is mediated [3,&*]. Several antigens (FqRIIIA, CD2 and interleukin-2 receptor (IL-2R)-j3 chain in particular) represent important functional molecules through which ligand binding can trigger activation and through which NK cells interact with the humoral and cellular compartments of the adaptive and non-adaptive systems of defence. The majority of the cells strictly deiined as lymphokine-activated killer (LAK) cells have been shown to be, both at the progenitor and at the effector- cell stage, NK cells that, upon activation by interleukin (IL)-2, become able to kill several target cells, in particular freshly explanted tumor cells [ 51. Subsets of T lymphocytes, particularly CD3+/CDllb+/CDS+ cells I&], share with LAK cells the ability to kill NK cell-resistant target cells. These T-cell types, as well as the mechanism(s) by which NK and T cells mediate recognition of target cells and cytotoxicity have been the subject of recent re- views [7-Y] and will not be addressed here.

Many reports in the past year have pointed to a primary role for cytokines and specilic ligands in aifecting NK cell biological functions, and have helped demonstrate that NK cells mediate a broader range of functions than

simply spontaneous cytotoxicity in vitro. Much evidence has also accumulated that NK cells have a regulatory effect on several cell types. The effects are not only mediated through direct cellular interactions, but also in part via secretion of soluble factors that NK cells produce upon stimulation by lymphokines or upon interaction with specific ligands. The stimuli and mechanisms of activation participating in, and resulting from, such interactions are the central theme of this review. The review is limited to studies performed on NK cells either resting or activated by lymphokines and deiined as those TCR-/CD3-/CDl6+ and/or CD56+ lymphocytes that are cytotoxic to a variety of target cells in a major histocompatibility complex (MHC)-non-restricted fashion, and which mostly have large granular lymphocyte (LGL) morphology.

Regulation of NK cell functions by cytokines

Proliferation Several cytokines, including IL-2, interferon (IFN)-u-p and the recently described NKcell-stimulatory factor [lo**], modulate NK-cell function and induce their proliferation. Studies in vitro and in vivo in both the human and the murine system have shown that mature NK cells proliferate in response to IL-2, which they bind through the functional IL-2Rp (~75) chain with intermediate aifin- ity [11**,12**]. After the demonstration that IL-2 induces most human NK cells in vitro, but only a proportion of the T cells, to enter cell cycle [ 131, compelling evidence that the primary role of IL-~ is in vivo to induce NK-cell proliferation came from the studies of Biron et al. [ 14**] in the murine system. These authors demonstrated, that

Abbreviations CLMF--cytotoxic lymphocyte maturation factor; CSF-colony-stimulating factor;

CM-CSF-granulocyte-macrophage colony-stimulating factor; FCyRlRA-low-affinity receptor for the Fc portion of IgG; I-interferon; IL-interleukin; IL-2R-interleukin-2 receptor; LAK-lymphokine-activated killer;

LCL--large granular lymphocyte; LTRMC-long-term bone marrow culture; MHC-major histocompatibility complex; NK-natural killer; NK!W-natural killer cell-stimulatory factor; PRL-peripheral blood lymphocytes;

SCIkevere combined immunodeficiency; TCR-T-cell receptor; TNF-tumor necrosis factor.

@ Current Biology Ltd ISSN 0952-7915 49

50 Innate immunity

high dose IL-2 treatment induces the appearance of blast- size NK cells and proliferation of the NK1.l + cell popu- lation, which is preferentially driven to the S- and G2/M- phases of the cell cycle in the absence of detectable expression of IL-2Ra (~50, CD25) mRNA.

These data also support the notion that IL-2 interacts di rectly with the IL-2Rp chain constitutlvely expressed in NK cells, as previously indicated by the observation that anti-IL-2Rj!l antibodies inhibit IL-2induced proliferation and other functions of NK cells in vitro [ 12**]. The role of the IL-2Rj3 chain on NK cells in transducing signals leading to cell proliferation has been further documented in studies using anti-II2RP antibodies directed at epitopes of the molecule not involved in IL-2 binding in the absence of IL-2 production [ 15.1. However, evidence has also been presented that NK cells are unusually expanded in mice transgenic for both the IL-2 and IL-2Ra and not IL-~ genes alone [ I6**]. Such data support the possibility that IL-2/IL-2Ra, and not just the constitutive IL-2Rp, play an important role in the proliferation of NK cells in vivo. This may be reconciled with previous data of Biron et al. [l4**] that suggests that distinct subsets of NK cells are induced to proliferate, or have a proliferative advantage, under the two di@erent experimental conditions.

Data obtained in vivo from athymic nude mice [ 17**] clearly indicate that IL-2, although capable of inducing NK-cell proliferation, is not the major mediator under particular conditions, such as during acute proliferative responses to IFN-a-0 in vim. The IFNa-P-induced bias togenesis and proliferation of NK cells is not sensitive to cyclosporin A which is capable of inhibiting the poly I : C- induced IL-~ gene expression in athymic mice.

NK cells depend for their maturation on an intact bone marrow, and transplantable NK cell precursors have been identilied in the mouse system [ 181. The factors influ- encing or determining differentiation of these precursors have not yet been definitively identified. The use of long- term bone marrow culture systems has allowed several investigators to start dissecting the relevance of different cytokines in the generation of mature NK cells from bone marrow populations depleted of phenotypically mature cells. Sameva et ul. [ 191, using 5-fluorouracil and anti- asialo GM1 or NK-specific 3.2.3 antibodies, have shown that mature NK cells can be generated from rat bone marrow cells depleted of mature proliferating cells. Fur- ther studies indicate that m-cell generation in long-term bone marrow cultures requires not only IL-2 but also a factor (or co-factor) present in the conditioned medium and possibly produced by the bone marrow stromal cells [20**].

The possible participation of cytokines other than IL-2 in inducing proliferation of mature cells and possibly dif ferentiation of immature NK cells from peripheral blood, had been previously suggested on the basis of data from in vitro culture systems. It was reported that NK-cell proliferation is supported when B-lymphoblastoid cell lines are present as feeders [ 211. Although IL-2, endogenously produced in these cultures, is necessary for NK-cell expansion, IL-2 alone is not a sufficient stimulus for this effect. Also, although the B-cell lines by themselves do

not induce cell proliferation, they enhance induction by IL-2 [ 131. During studies on the preferential proliferation of NK cells in this co-culture system, a stimulating factor has now been isolated from the Epstein-Barr virus -transformed RPMI-8866 B-cell line. This factor has been provisionally named natural killer cell stimulatoiy factor (NKSF) [lo**] and is a heterodimeric cytokine of 7OkD, composed of two chains of 40 and 35 kD. Recently, the genes encoding the two chains contributing to the heterodimeric functional NKSF molecule have been cloned (Wolf et al, personal communication). The sequences of the proteins predicted from the cDNA sequence of each chain contain regions that are identical to those of the amino-termlni peptides reported for another B-cell- derived cytokine, described by Stem et al. [22**] as cy totoxic lymphocyte maturation factor (CLMF), that can induce LAK cell activity. It is likely that NKSF and CLMF represent homologous, if not identical, proteins. NKSF, although not specific for NK cells, directly induces their proliferation and modulates their proliferative response to IL-2 (Perussia et al, unpublished data). Although it cannot account alone for all the effects induced in NK cells by B-lymphoblastoid cell lines, it may represent one of several factors, in addition to IL-2 and IFN, involved in NK-cell maturation/proliferation.

Several cytokines, able or not to induce NK-cell proliferation directly, have been reported to modulate the induction by IL-2. IFN-a-p, which on its own induces blastogenesis of immature but not mature NK cells in vivo, inhibits NK-cell proliferation induced by IL-2 in vitro [WI. Analagously, IL-4 inhibits IL-2induced pro- filferation of mature CDl6+ NK cells [24,25**], which it does not induce to proliferate. The same lymphokine induces minimal, but detectable, proliferation of the human CDl6-NK cells, possibly containing NK-cell precursors, and synergizes with IL-2 in this effect [25*-l. The mechanism(s) by which such effects are mediated has not been elucidated, but it is possible that the same cytokine me- diates different effects on cells at diiferent stages of activation. Other cytokines, such as IL-3, have been shown to synergize with low doses of IL-2 to induce the appearance of cells with the NKcell phenotype [26*]. In this case, the effect is indirect and results from IL-3-dependent enhanced expression of IL-2R on immature NK cells.

Cytotoxicity

NK cells mediate their cytotoxic functions spontaneously, but several cytokines modulate this activity by acting on targets : effector cell binding-recognition steps and possibly by modulating expression of molecules (pore form ing protein and serine esterases) through which, at least in part, the qtotoxic mechanisms may be mediated. The observation that expression of some of the same molecules is downregulated in cytotoxic T lymphocytes by cytokines (e.g. IL-4) that inhibit cytotoxicity [ 27’1 sup- ports this possibility.

IFN-a--P, IL-2 and NKSF are the most potent known enhancers of spontaneous cytotoxicity and their direct effect on m-cell populations, purified to homogeneity

Lymphokine-activated killer cells, natural killer cells and cytokines Perussia 51

and monitored for absence of accessory cells, has been demonstrated using antibodies that neutralize their activity. The mechanism(s) by which these three cytokines af- feet cytotoxicity may, in part, be distinct, as suggested by several observations: granulogenesis occurs in NK cells stimulated with IL-2 [ 281 or NKSF (Perussia et al, unpublished observation), but not with IFNa-p; the inactiva tion of NK cells induced upon recognition of target cells sensitized or not with specific IgG antibodies [29,30*] is only partially reversed by simultaneous stimulation with IFN-fJ or NKSF (Perussia et al, unpublished data) [29] but is completely reversed with IL-2 [30*]. Our unpublished observations indicate that the different effects of these three cytokines on cytotoxicity are accompanied by different effects on expression of pore-feting protein and serine esterase ~RNA.

Other cytokines variably modulate cytotoxic functions through indirect mechanisms that are still poorly understood. IL-4 inhibits cytotoxicity of both CD16+ and CDlG- NK cells [25**] but it induces cytotoxicity in peripheral blood lymphocytes (PBL) preincubated with IL-2 in viva or in vitro [31-l. The inhibitory effect may depend, in part, on induction of increased intracellular cAMP levels that are accompanied by decreased serine esterase and tumor necrosis factor (TNF) production [ 32.1. This suggests the possibility that mechanisms controlling transduction pathways vary, depending on the activation state of the effector cells. IL-5 alone does not induce killer activity but enhances the IL-2-induced IAK activity against a variety of tumor target cells and acts at late stages of IAKcell induction in the presence of IL-2 [ 33*]. Whether the action of IL-5 is directly or indirectly mediated via release of other cytokines remains to be established. Indi rect stimulation of MHC-non-restricted cytotoxic activity of the CD3- PBL, via induced production of IL-2, has been reported in the case of IL-~ [34*].

Production of cytokines by NK cells

Most of the evidence reported to date for the production of cytokines by NK cells has been obtained in vitro. It has proved difficult and remains premature to extrapolate these results to the in vivo situation. With this limitation, however, the experimental evidence available clearly in- dicates that NK cells are capable of producing cytokines when appropriately stimulated and pathological or physi- ological conditions can be envisaged in which such stimulation can occur.

NK cells, cocultured with bone marrow cells, inhibit myeloid colony growth and the cell-free supematant Ilu- ids from short-term cocultures of NK cells with NK-susceptible, but not NUnsensitive, target cells or with precursor cell-enriched bone marrow cell preparations, contain an activity that suppresses colony formation [35]. This activity can be inhibited by anti-TNF antibodies and synergizes with IFN-y to suppress hematopoietic colony formation [36]. These data suggested that NK cells are activated to produce cytokines upon binding of recogniz- able cell types and may regulate myeloid cell diEerentia- tion, at least in part, through production of lymphokines.

Additional reports have recently confirmed these Iind- ings [37*] and extended them to indicate that interaction of tumor target cells [38*], or of B lymphoblastoid cells [39-l, with NK cells results in IPN-y production. It can be hypothesized that one or more molecules engaged in binding-recognition of the target cells transduce the signals intracellularly, leading to cytotoxicity and lymphokine production. The biochemical pathways responsible for this are incompletely understood. Binding of target cells results in Ca2+ mobilization in NK cells [38*] (Perussia et al, unpublished data); the magnitude of these effects does not correlate with that of the specilic lysis of these same cells.

Direct demonstration that NK cells produce TNF and IFN-y, comes from studies performed using homoge- neous NKcell preparations that have beeri stimulated with immune complexes, or with IL-~, in the absence of target or contaminant cells that may produce cy tokines spontaneously or upon interaction with the effector cells [40,41**]. In this system it has been demonstrated that NK cells produce IFN-y, TNF, granulocyte- macrophage colony-stimulating factor (GM-CSF), colony- stimulating factor (CSF) and IL-3. To date, the only char- acterized receptors that have been demonstrated to be able to transduce signals leading to lymphokine production in NK cells are IL-2RP and FcyRIIIA (CDl6). Other molecules, such as CD2, are unable to transduce these signals although, like CD16 and unlike IL- 2 [42-j, they do induce NK-cell activation. This may be determined by their ability to induce increased intracellular Ca2+ levels and their ability to trigger cytotoxicity in redirected antibody-dependent cytotoxicity systems. These data suggest that either increased intracellular Ca2+, although a prerequisite for lymphokine secretion/induction under specific circum- stances, is not always sufficient to result in IFN-y production, or Ca2+ mobilized from different cellular pools has different effects on lymphokine production. It is also possible that molecules associated with FcyRIIIA, or af fected by the interaction of FcyRIIIA with its ligands, but not with other receptors, are those directly involved in signals responsible for specific effector functions. The most likely candidates would be the FcyRIIIA associated r-chain or Fc&RI y-chain [4,43**].

Besides IL-2, only NKSF has been unambiguously shown to induce production of cytokines from NK cells. NKSF acts both directly and in synergy with IL-2, and other spe cific or mitogenic stimuli, to induce production of IFNy [44] and other cytokines from both NK and T cells. IL-2 and the FcyR ligands act with very fast kinetics, inducing transcription of the mRNA for lymphokines within 10 min and accumulation of their mRNA within 1 h of stimulation in the absence of de nova protein synthesis [40]. Both FcyR ligands and target cells (Perussia et al, unpublished data) synergize with IL-2 to induce mRNA accumulation and secretion of the lymphokines. Induction of these lym phokines by immune complexes, but not by IL-2 and NKSF, occurs through activation of molecular pathways that result in both Ca2+ mobilization and phosphoinositide turnover; induction is completely inhibited in the absence of extracellular Ca2+ [42-j. Synergy between

52 Innate immunity

NKSF and IL-2 to induce IFN-y production depends, at least in part, on post-transcriptional events such as mRNA stability, which are induced by these two stimuli in combination. As my colleagues and I previously described for NK cells stimulated with IL-2 and FcyRIIIA ligands, NKSF also induces IFNy mRNA transcription, but no significant synergy between IL-2 and NKSF is detectable at this level. Although the majority of the lymphokines are produced following stimulation with immune complexes and IL-2, IL-3 is not induced at detectable levels by either stimuli, acting alone or in combination [41**], indicating that this gene is diIferentially regulated in NK cells. Altogether these observations indicate that immune complexes, target cells and lymphokines induce signals that are trans- duced in a distinct way to result in cytokine production. When more than one receptor is occupied, the final out- come of the stimulation depends, in part, on how (in a synergistic or inhibitory way) these intracellular pathways interact.

Effects of NK-cell-produced cytokines

Through the array of cytokines they produce, NK cells may mediate homeostatic functions and regulate activity of several cell types. Such an effect would only be detectable on those cells with which NK cells come into di rect contact, because the concentration of lymphokines at the site of interaction between the two cell types would be relatively high. This is easily envisaged in peripheral blood, in particular in conditions in which NK cells may be stimulated by lyrnphokines produced in response to pathogenic stimuli, or in the bone marrow if NK cells are recruited or altered in their trafhcking; however, NK cells may mediate their effect at a distance, on cells sensitive to very low doses of the lymphokines produced.

NK cells may regulate the function of cells of the myeloid and monocytic lineage, both positively and negatively, by acting at each stage of myelomonocytic differentiation. Through production of GM-CSF and IL-3, they can exert burst-promoting activity. On the other hand, NK cells can also inhibit myeloid colony formation via TNE and IFN-y production. An interesting possibility is that they affect proliferation of the CD34+ hematopoietic progenitor cells on which TNE potentiates the proliferative effect of GM-CSF and IL-3 [45*]. TNF, GM-CSF, IEN-y and IL-3 induce activation of mature myeloid and monocytic cells and eosinophils by activating their functions, by inducing or enhancing expression of surface receptors mediating specific functions (e.g. FcyR, HIA-DR-antigen), or by ren- dering them susceptible to the effect of other activatory cytokines (e.g. IL-2). Effects on lymphoid cells can be mediated through release of IFN-y. Using a coculture system of B and NK cells, Michael et UC [39**] have shown that interaction with B cells stimulates NK cells to produce IFN-y and this results in inhibition of polyclonally induced B-cell prolif eration. IFN-y has been shown to enhance immunoglobulin secretion from resting B cells. Numerous examples exist in the literature that NK cells also participate in the defence against bacteria and par-

asites [2]. A very interesting observation strongly suggests that a cause-and-effect relationship exists between protection against the intracellular pathogenic bacterium Lkteria monocytogenes and TNF production by mono- cytes in T-cell-deficient severe combined immunodefi- cient SCID mice infected with this pathogen [ 46.1. SCID mice do not respond to T-cell stimuli (e.g. concanavalin A) with IFN-y production, but they produce this lymphokine in response to injection of heat-killed L mono cytogenes Depletion of asialo GMl-positive cells results in inhibition of macrophage activation and IEN-y production. Also, like neutralization of the TNE produced soon after challenge, it results in exacerbation of the infection. Administration of recombinant TNF to both T-cell-intact and T-cell-deficient mice, protects against challenge with lethal doses of the bacteria [47*]. Resistance to other pathogens (e.g. Mjzoph.vnupuZmonis) has been corre- lated by Iai et al. [48-l to IFN-y produced by activated NK cells.

It is likely that NK cells interact with pathogens through a receptor(s) and mechanism(s) not yet understood. This results in an induction of lymphokine production that is similar to that observed upon interaction with ligands and lymphokines, the receptors for which have been char- acterized. The cytokines produced may exert direct effects on the pathogens and potentiate indirectly the anti- pathogenic activity of other cell types directly responsible for defence.

Conclusions

In mediating their functions, NK cells are regulated by a complex network of humoral and cellular interactions. The interplay between NK cells and other cells of natural and specific immunity occurs, in part, through release of cytokines. The fast kinetics with which NK and IAK cells are induced to produce soluble mediators upon specific stimulation render these cells important effector cell types of non-adaptive immunity, when specific antigen-dependent effector mechanisms have not yet been elicited. On the other hand, the expression and maintenance of their functions at later times is regulated by lymphokines, particularly IL-2, produced by antigen-stimulated cells. Cytotoxic activity, while serving initially to identify NK and LAK cells, may or may not represent their most physiologically significant function. With the relatively recent accumulation of knowledge and reagents, there is the exciting possibility of analyzing both the functions of these cells in vz’vo and the cytokine- dependent expression and regulation of genes important to their biology.

Acknowledgements

This work was supported in part by National Institutes of Health grants, CA 37155 and CA 45284, and by a Scholarship from the Leukemia Society of America. I wish to thank the members of my laboratory, the colleques who contributed to produce part of the experimental work raiewed here, and Mrs M. Kaplan for secretarial assistance.

Lymphokineactivated killer cells, natural killer cells and cytokines Perussia 53

References and recommended reading

Papers of special interest, published within the annual period of review, have been highlighted as: . of interest . . of outstanding interest

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A very important paper which shows that NK blasts are recovered from the spleen of IL-2 injected mice and directly demonstrates that IL-2 can induce NK-ceU proliferation and expansion in oiw.

2. TTUNCHIEIU G: Biology of Natural KiUer CeUs. Adv fmmunol 1989, 47:187-376.

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Interesting paper indicating that anti-IL-2Rfi antibodies not blocking the IL-2 binding sites, transduce signals of activation on LGIz, and on NK cell lines.

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4. RAVETCH JV, KINET J~P: Fc Receptors. Annu Rev Immunol . . 1991, in press. A most complete raiew of the structure and function of Fc receptors.

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not T CeUs in Human Interleukin Z/InterIeukin 2 Receptor (Tat) Transgenic Mice. J Exp Med 1990, 170:1103-1115.

An important paper showing NKceU expansion in IL-2/lL-2Ra gene transgenic mice and clearly pointing to a role for IL-2Ra in NKceU activation.

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Interesting paper showing that within CD3+ T iymphocytes the CD8+/CDllb+ subset is the only one able to respond to IL-2 stimulation by generating cells with w< activity.

Demonstration of the role of IFN-a-p in inducing proliferation of NK cells independently from IL-2 and through a cyclosporin A-independent pathway in viva.

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This paper provides strong evidence that NK-ceU activity can be generated from bone marrow cell preparations depleted of mature NK cells.

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First report of a heterodimeric cytokine that is distinct from IL-2 and affects NKceU and T-cell functions independently of IL-2.

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A careful study describing a long-term bone marrow culture system in which generation of NK cells from NK-cell precursors is shown to re- quire a factor in addition to IL-2 and which is possibly produced by the bone marrow stromal microenvironment.

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. . TSUDO M, SoNDEL P: Increased Expression of the lnterleukin 2 (IL-2) Receptor b Chain @70) on CD56+ Natu- ral Killer CeIIs After In Vfvo IL-2 Therapy: p70 Expression Does Not ALone Predict the Level of Intermediate A&&y IL-2 Binding. J Exp Med 1990, 172:1101-1114.

First evidence that increased expression of IL-2Rp (~70) on the surface of NK cells in ztivodoes not necessariiy result in an increased functional capability of the cells.

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A most complete study demonstrating that anti-IL-2RP (~70) antibod) completely blocks the IL-2-induced activation of NK cells.

A report of the biochemical characterization and amino acid sequence of the two subunits of a heterodimeric cytokine (CLMF) that induces proliferation of activated T cells and LAK-ceU generation,

13. LONDON L, PERUSSIA B, TIUNCHIEIU G: Induction of ProIifera- tion In Vftfv of Resting Human Natural KiIIer CeIIs: IL-2 Induces into Cell Cycle Most Peripheral Blood NK CeUs, But Only a Minor Subset of Low Density T Cells. J Immunol 1986, 137:38453854.

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. B: The Proliferation and Life-Span of Rat Large Granular Lymphocytes: Effects of Cytokines. Eur J Immunol 1989, 19189%1902.

Interesting report showing effects of IFN-a-p and IL-2 on NK-cell pro- liferdtion in the mouse.

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54 Innate immunity

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Human Natural KiIIer CeIIs: AnaIysis of the Conditions that InIluence the Release and Detection of Colony-Stimulating Activity. Blood 1989, 74:156-164.

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This paper reports evidence that IL-3 can induce generation of NKceU activity from murine bone marrow cells depleted of matllre NK cells by 5-RuofouraciI treatment.

Documents evidence that a colony-stimulating activity, not completety abolished by anti-GM-CSF antibodies, is produced by NK cells.

38. CHONG AS-F, SCUDERI P, GRIMES W, HERSH EM: Tumor Targets . Stimulate IL-2 Activated KiIIer CeIIs to Produce Interferon-y

and Tumor Necrosis Factor. J Immunol1989, 142:21332139. Reports aidence that IFN-y and TNF are induced in NK cells upon stimulation with tllmor cells.

27. I~J C-C, JOAG SV, KWON BS, YOUNG JD: Induction of Perforin . and Serine Esterases in a Murine Cytotoxic T Lymphocyte

Clone. J Immund 1930, 144:119&1201. Most complete study of the ability of cytokines to induce PFP and SE in murine CTL.

39. MICHAEL A, HACKETT JJ, BENNETT M, KUMAR V, YUAN D: Reg- . . uIation of B Lymphocytes by Natural KiUer CeIIs: Role of

IFN-y. J Immunol 1989, 142:109~1101. Reports that IFN-1 is produced by NK cells upon interaction with B cells and demonstrates that this results in decreased immunoglobulin production, suggesting a mechanism by which NK cells regulate B lym phocyte functions.

28. ZAP&ONE D, PRASTHOFER EF, MAL&VA% F, PISTO~A V, IOBUGLIO AF, GROSSI C: UItrastructuraI AnaIysis of Human Natural KUIer CeU Activation. Bkxd 1987, 69:17251736.

29. PERUSSJA B, TRI~CHIERI G: Inactivation of Natural Wer CeU Cytotoxic Activity After Interaction with Target CeIIs. J Immund 1981, 126:754758.

40. ANEGON 1, CUTURI MC, TRINCHLERI G, PERUSSIA B: Interaction of Fey Receptor (CD16) with Ligands Induces Transcrip- tion of IL-2 Receptor (CD25) and Lymphokine Genes and Expression of their Products in Human Natural KiIIer CeIIs. J .%p Med 1988, 167452472.

30. XLAO J. BRAHMI 2: Target Cell-Directed Inactivation and IL-2 . Dependent Reactivation of LAK CeIIs. Cell Immunol 1989,

122:29>306.

41. CUTURI MC, ANEGON I, SHERMAN F, LOUDON R, CL4RK SC, . . PERUSSV~ B, TRINCHIERI G: Production of Hematopoietic

Colony-Stimulating Factors by Human Natural KiIIer CeIIs. J Ekp Med 1989, 169~569-583.

This paper reports that IL-2 aUows recovery of cytotoxic activity in NK ceUs after their target cell-induced inactivation.

The first evidence that NK cells produce M-CSF, GM-CSF, and IL-3. De- fines the cytokines atfecting haematopoiesis produced by NK cells upon maximal and specific stimulation.

31. HIGUCHI CM, THOMPSN JA, LLNDGREN CG, GILUS S, WIDMER . MB, KERN DE, FEFER A Induction of Lymphokine-Activated

KiIIer Activity by Interleukin 4 in Human Lymphocytes Pre- activated by lnterleukin 2 In Vivo or In ViWo. Cancer Res 1989, 496487-6492.

Characterization of differential effects of IL-4 on resting and activated PBL and of its interaction with IL-2.

42. CASSATEUA MA, &EGON 1, CUTUIU MC, GMKEY P, TRINCHIEIU . . G, PERUSS~A B: FcyR(CD16) Interaction with Ligand Induces

Ca*+ Mobilization and Phosphoinositide Turnover in Hu- man Natural KiIIer CeIIs. Role of Ca*+ in FcyR(CDl6)- Induced Transcription and Expression of Lymphokine Genes. J Exp Med 1989, 169:54%567.

First direct demonstration that cytokine production by NK cells is dif- ferentIy regulated by FqRIIL4-Iigand binding and IL-2.

32. BRAY J-Y, BRANEIJ.EC D, ROBINET E, DUGAS B, GAY F, CHOUAIB . S: InvoIvement of Cyclic Adenosine Monophosphate in the

Interleukin 4 Inhibitory Effect on Interleukin-2.Induced Lymphokine-Activated KiIIer Generation. J Clin Invest l%Q, 85:190%1913.

Demonstration that IL-4 inhibits the IL-2 induced expression of TNF mRNA tbrcqh, at least in part, increased CAMP levels.

33. AOKI T, KIKUCHI H, MNATAKE S-I, ODA Y, IW&%KI K, YAKASAKI . T, KINA.SHI T, HONJIO T: Interleukin 5 Enhances InterIeukin

2-Mediated Lymphokine-Activated Wer Activity. J Exp Med 1989, 1703583588.

43. ANDERSON P, CALIGIURI M, O’BRIEN C, MANLF~ T, Rnz J, . . SCHLosSMAN SF: Fey Receptor Type III (CD16) is Included in

the 6 NK Receptor Complex Expressed by Human Natural KiIIer CeIIs. Prcc Nat1 Acad Sci USA 1990, 87~2274-2278.

Direct biochemical evidence of association between CD3c and FcyRIIl on NK cells. Two-dimensional gel electrophoresis of FqRIIl precipi- tates demonstrates the coexistence of c-chains in the complex, open- ing the possibility that this molecule participates in signal transduction mediated via FcyRIlI.

Description of the effects of IL-5 on cytotoxic activity of IL-2-activated killer cells.

34. LUGER TA, KRLJTMANN J, KIRNBAUER R, URBANSKI A, SCHWARZ T, . KLAPPACHER G, KICK 4 MICKSCHE M, MALEJCZYK J, SCHAUER E,

MAYL LT, SEHGAL PB: IFN-flZ/lL-6 Augments in the Activity of Human Natural KiiIer CeIIs. J Immunoll989, 143:120&1209.

Reports that the ability of IFN-02 to enhance NKceII flotoxicity is indirect and is blocked by anti-IL-2 antibodies.

44. CHAN SH, PERUSSIA B, GUPTA JW, KOBAYASHI M, POSPISIL M, YOUNG D, CLARK SC, PRINCHIEIU G: Induction of IFN-y Pro- duction by NK CeII-Stimulating Factor (NKSF); Characteri- zation of the Responder CeIIs and Synergy with other In- ducers. J Eq Med 1991, in press.

35. DEGLLWTONI G, MURPHY M, KOBAYA~HI M, FRANCIS M-K, PERUSS~A B, TRINCHIERI G: NaturaI KiIIer (NK) Cell-Derived Hematopoietic Colony-Inhibiting Activity and NK Cyto- toxic Factor. ReIationship with Tumor Necrosis Factor and Synergism with Immune Interferon. J E.ap Med 1985, 162:1512-1530.

45. CHAUX C, SAEIAND S, FAVRE C, DIJVERT V, MANNONI P: Tu- . mor Necrosis Factor-AIpha Strongly Potentiates lnterleukin-

3 and GranuIocyte-Macrophage Colony-StimuIating Factor- Induced ProIiferation of Human CD34+ Hematopoietic Progenitor CeIIs. BIood 1990, 75:2292-2298.

This paper demonstrates that TNF-a increa~s the frequency of IL-3- responding ceUs and the size of IL-3-dependent clones.

46. BANCROFT GJ, SHEEHAN KCF, SCHREIBER RD, UNANUE ER: Tu- . . mor Necrosis Factor is InvoIved in the T Cell-Independent

Pathway of Macrophage Activation in Scid Mice. J Immund 1989, 143:127-130.

36. DEGLIANTONI G, PERU%% B, MANGONI I+ TTUNCHIERI G: lnhibi- don of Bone Marrow Colony Formation by Human Natural

An important paper, demonstrating that in KID mice, in the absence of T-cell responses, IFNy is produced by NK cells in vivo upon injection of L mona_ytogenes Also shows that the T-cell independent produc-

Lymphokine-activated killer cells, natural killer cells and cytokines Perussia 55

tion of this cytokine is central for resistance of the host at early times post-infection.

47. HAVEU EA: Evidence that Tumor Necrosis Factor has an . Important Role in Antibacterial Resistance. J Immunol1989,

143:28942899.

Mediated by Activated Natural Killer Cells. J In&Y Dis 1990, 161:12691275.

Interesting report that provides evidence that NK cells, through IFNy production, play a primary role in resistance to M. pulmonis

Demonstration of the role of TNF in listeria-infected orpans as a T-cell- independent resistance mechanism in athymic nude mice.

B Perussia. Deoartment of Microbiolow and Immunology, Thomas Jef- 48. Lu WC, BENNETT M, PAKES SP, KUMAR V, STEUTERMANN D, ferson Uni~erky, 1020 Locust Street, Philadelphia, Pen&yhania 19iO7, . OWLJSU I, MIKHAEL k Resistance to Mycoplasma Puhnonis USA.