Characterization and functions of natural killer cells

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23rd FOR UM I N I M M U N O L O G Y

CHARACTE~TION

AND FUNCTIONS OF NATURAL KHJLER CELLS

J.R. Ovtaldo, B.J. Mathieson and R.H. Wiltrout

Laboratory of Experimental Immunology, Biological Response Modifiers Program, Frederick Cancer Research Facility, Bldg. 560, Room 31-93A Frederick, MD 21701 Introduction.

Natural killer (NK) cells have spontaneous cytotoxic activity which can be augmented or suppressed by a wide range of stimuli [1, 2], and which does not depend on prior sensitization by antigens [3]. NK cells also lyse targets without restriction related to the major histocompatibility complex. Large granular lymphocytes (LGL) mediate both NK and antibody-dependent cellmediated cytotoxicity (ADCC) in mice, rats and humans [3]. NK cells have also been suggested to mediate immunoregulatory effects, since they have been recently demonstrated to secrete a variety of biologically active cytokines upon stimulation with biological response modifiers [4]. Ontogeny of NK cells.

The haematopoietic lineage of the NK cell has remained controversial, despite a number of attempts to isolate NK progenitor cells and to evaluate their relationship to T cells and other leukocyte populations. The problems persist largely because NK cells share some characteristics with both T cells and myelomonocytic cells. The hypothesis that NK cells were derived from the T-cell lineage has been supported by the evidence that ~ cells share a number of phenotypic and functional characteristics with T cells. Both human [5] and rat [6] NK cells express CD8, although CD8 appears restricted to T cells in mice. Human T cells and NK cells also express CD2 and are able to form rosettes with sheep red cells [5].

In mice, a subset of NK cells expresses Thy-1 [7, 8] which was initially believed to be a T-cell-specific marker. Likewise, NK cells express Qa-4/5 and Ly-5/T200, markers initially reported to be T-cell aatigens. Moreover, NK cells have been shown to produce IL-2 [4, 9], a lymphokine primarily produced by T cells. In contrast, several other lines of evidence suggest that NK cells bear some relationship to myelomonocytic cells or other leukocytes. Beige (bg/bg) mice and Chediak.-Higashi patients display prominent defects in myeloid cells, NK cells and cytolytic T cells (CTL) because of a defect in the ability to effectively form lysosomal granules [10, 11]. In vitro experiments with bone marrow precursors have been used to support the idea that NK cells were related to promonocytes [12]. In the .human and mouse, NK cells express some myelomonocytic markers (e.g., C D l l / F c R , Mac-l). In addition, NK cells have been shown to secrete cytokines generally associated with monocytes, e.g. ILl, and TNF~ [4, 9]. More recently, evaluation of the severe combined immunodeficiency syndrome (SCIDS) in both human subjects [13] and in mice [14] has indicated that the combined deficiency for both T and B lymphocyte differentiation may not block the appearance of differentiated NK cells, although their activity may be suboptimal. These observations suggested that the NK-cell lineage diverges in development prior to the differentiation of the T-lymphocyte lineage. We have devised a mouse congenic transfer system to study the relationship

L YMPHOKL~IE-ACTIVA ~r~!) KILLER CELLS between these lineages. Transfer of immature thymocytes, isolated as dull Ly-1/CD5 cells which are negative t'or both CD4 and CD8 (dLy-I cells) [15], has resulted in functionally active donor-derived NK ceils that can be isolated from the liver of recipient mice. A portion of these donor-derived LGL express a newly reported NK cell surface marker, LGL-1 [16]. There are two important considerations that relate to this observation. First the dLy-1 ceils do not have the ability to form 12-day splenic colonies [15], indicating that they are committed cells no longer multipotent for haematopoietic activity. Secondly and more importantly, because they do not form 8-day splenic colonies or colonies in vitro, they also do not have the capacity to differentiate into myelomonocytic cells. Because the donor-derived cells do not uniformly express the LGL-1 marker, we axe currently attempting to determine whether the remaining cells are precursors for NK cells or a subset of NK cells that fail to express the LGL-1 marker. The proposed model (fig. 1) postulates a common T / N K lymphoid precursor cell (T/NKPC) which arises from a pleuripotent haematopoietic stem cell (PHSC). Some of these T/NK-PC differentiate in the periphery into m a t u r e NK cells. However, most of these T/N-K-PC enter the thymus, where they generally differentiate into the T-lymphocyte lineage. Within the immature dLyl + thymocytes, the capacity to differentiate into NK cells in the proper environment is retained. This hypothesis is supported by our results which demonstrate the co-transfer of both NK and T progenitors from this population. Experiments from others [17, 18] would suggest that a second dual-lineage pcogenitor exists for B and myeloid (MBPC) differentiation. The T/NK-PC cell may normally be rapidly c o m m i t t ~ to thymic/T-cell development upon reaching the thymus. However, this progenitor is unable to differentiate into other populations such as myelomonocytic CFU or B ceils ([15] and unpublished observations). Thus, our recent data suggest that NK cells are derived from a committed T/NK-PC lymphocyte lineage.

445

Recognition of target cells by NK cells. A model has been proposed dividing the process of NK and CTL cytolytic acfi-dty into two clearly defined steps [3]. Tl~e first step is recognition and binding of the effector cell to the target cell. In the second step, post-binding events lead to lysis of the target cell. This involves m o v e m e n t of azurophilic granules and other cytoplasmic organeiles toward the binding site and granule release which is associated with the secretion of cytolytic factors that bind to the target cell and cause lysis. In the T-cell model, specific CTL employ components of the T-cell receptor (TCR) with rearrangements of their ~- and It-chains in strict association with MHC components. The fine specificity that CTL exhibit has recently been shown to be due to the antibody-like rearrangements of the VDJ portion of the TCR chains. NK cells possess different mechanisms o f recognition, since CD3- LGL lack gene rearrangeTaent and expression of the ~, ~ and 7 chains of the TCR

[19, 201. Numerous molecules including CD2, CD16, and LFAI, have been suggested to play a role ~a NK-mediated recognition of targets, but none of these molecules are exclusively associated with NK cells. Recently several alternative molecules have been proposed as potential NK recognition, receptors. Laminin [21] is expressed at low levels on fresh NK cells, and its expression is increased after IL2 activation. However, its appears to function mostly as a secondary, adhesion-~ctivation molecule. Recently, two proposed NK receptor molecules have been described. The first is a 40-Kd protein [22] that was identified by monoclonai antib~ies made against fish NK cells and the ~econd is an 80-Kd structure [23] unique to human NK cells that was identified by an antiidiotypic antisera made against a monoclonal antibody which recognizes a structure on NK-susceptible target cells. Both molecules have been shown to block binding of NK cells and subsequent killing by CD3- LGL. The 80-Kd molecule has been demonstrated

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23 rd F O R U M I N I M M U N O L O G Y

THYMUS .~-~

~

~TPC)

PC / :3~_.j

I CD, ,* ?Th

,~Tslc

\--.~../ I CD~ + .

/'i'INK~ --.

Aec/

~P".C}

/CD3"~

~_._~ Fio. 1. - - Proposed model for T/NK development. The proposed model highlights the develoI?ment of mature lymphocytes from T/NK precursor cells. Both T/NK and myelomonocytic/Bcell precursor cell (MBPC) arise from a pluripotent haematopoietic stem cell (PHSC). The T/NK-PC cell can then either further differentiate into a CD3- NK cell in the periphery or enter the thymus, where it can rapidly differentiate into a committed T-lymphocyte precursor cell (CTPC) and then proceed along the T-lymphocyte lineage.

to mediate lysis following cross-linking with heteroconjugated antibodies. Alternatively, it is possible that NK cells do not utilise a single, unique receptor, but that various receptors like CD2, CDI6 and C D I I participate in target cel~ ~ecognition and lysis. At present, there is some evidence that these molecules are involved in lysis but none seem to be discrete recognition receptors. The identification o f the ~ cell associated receptor(s) remains a critical issue, since the finding o f a unique receptor would provide an important means of distinguishing cells with NK activity from cells with other types of lytic activity.

Heterogeneity of NK cells. NK activity has been largely associated with a morphological cell type, the C D 3 - LGL, which has characteristic cytoplasmic azurophilic granules. Although some minor differences exist between species, considerable evidence indicates that this CD3- LGL [24-30] is responsible for most endogenous NK activity in humans, mice and rats. LGL from these species do not adhere easily to either nylon or plastic; they possess Fc~-receptors but lack complement receptors and surface immunoglobulin. The morphological characteristics o f LGL have

L Y M P H O K I N E - A C T I V A TED K I L L E R CE LLS been a very useful means of identification. However, the distinctive cytoplasmic granules are not a lineagespecific character'stic, since they have been observed in some T-cell populations. Specifically, virus-specific T-cell blasts isolated directly from infected mice also contain cytoplasmic granules [31], as do CTL that have lost their antigenic specificity in vitro in the presence of high doses of IL-2 [32, 33]. The results from a variety of laboratories indicate that cells exhibiting NK function can express surface phenotype markers associated with other cell types [9]. In humans, some CD3- LGL express myelomonocytic (CDI 1) and T-related markers (CD2. CDS, OKT10). Most human NK activity (>i 95 %) is mediated by CD3-: Leul9[NKH1] +,CD16 + LGL [2~ 24-28]. CD3-,Leul9[NKH1] + ,CDI6cells are a small subset (~< 5 070)of LGL that is similar to the major LGL subset but does not express the CDI6 marker. The C D 3 - , L e u I 9 [ N K H I ] - , C D I 6 + LGL subset represents yet another very small subset of LGL. In addition, a CD3 +, LeuI9[NKI-II] + T-cell subset: can also mediate some anti-KS62 activity ( = 5 %), a p p a r e n t l y via the CD3-associated T-cell receptor. Heterogeneity also exists among CD3- LGL with regard to produ~ion of immunoregulatory cytokines such as IFN (u,y), IL-I, IL-2, IL-4 and CSF. Various subsets of CD3- LGL also secrete different quantities of cytotoxic/cytostatic factors (cytolysin, TNF). Thus, the potential of various CD3LGL subsets to mediate anti-tumour effects directly or indirectly through soluble factors must be considered further. However, it is apparent that the heterogeneity of T cells or monocyte/macrophages with regard to factor production is no less diverse than that observed for CD3- LGL. Role of NK cells in the in vivo antitumour effects o f LAK and IL2.

The use of <
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tion has led to a great deal of confusion regarding the nature of the cell populations being addressed. Similar problems exist in the characterization of cells which mediate lymphokine-activated killer (LAK) activity. Functionally, NK and LAK activity are quite similar. Most investigators have concluded from the numerous studies examining the progenitors and effectors of the LAK phenomenon) that no unique, single progenitor nor effector mediates the activity. Both CD3 + and CD3- lymphocytes from normal peripheral blood can mediate LAK activity after exposure to IL2. Although there is some variability in the phenotype of the LAK effector cell isolated from whole PBL based on the target cell used, the primary effector cell has the CD3- CD16 + LeuI9[NKHI] +, surface phenotype, coincident with activated NK cells. However, a CD3 + lymphocyte contributes a small but significant amount to rIL-2-generated cytotoxicity [25, 28]. Overall, these results suggest that LAK activity is not generated from a unique precursor nor mediated by a unique effector, but rather that several different lymphocyte subsets can serve as precursors and cytotoxic effector ceils. Clearly, the C D 3 - lymphocyte subset contributes the n a j o r portion of the IL2-induced activity from human PBL [24, 28], and rodent spleen [29, 30]. However, if LAK is generated from other sources such as thymus or lymph node, the conditions are modified (e.g. inclusion of anti-CD3, PHA, etc.), then the contribution of other subsets, particularly the CD3 + subset, may vary. In contrast to what is known about the phenotypic characterization of LAK precursors and effectors, little is known regarding the mechanism by which adoptively transferred LAK cells mediate antitumour effects. There is little convincing evidence to date that cytolytic LAK effector cells distribute to sites of tumour growth in vivo [34, 35]. In fact, enriched LAK effector cells have been reported to have limited distribution capacity following intravenous transfer into tumour-bearing rats [34] and preferential accumulation of LAK effector cells in tumour sites has

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been noted [34, 35]. This distribution pattern appears similar ~:othat previously reported for peript~eral blood NK cells [36]. These results further support a similarity between NK cells and LAK cells and suggest that at least some o f the antitumour effects mediated by NK cells may be indirect, probably through the production and release o f immunoregulatory cytokines. Since the LAK effector cells, which are largely generated from NK cells, do not selectively localize in tumours, it is impossible to determine at this point whether they are the critical antitumour effectors in vivo. It will be necessary to perform two types o f studies to determine the in vivo role for NK cells in the LAK + IL2 therapeutic response. First, various phenotypically distinct subsets o f cultured LAK cells must be tested for therapeutic efficacy in vivo. If all of the antitumour effects in vivo are found in the cytotoxic LAK effector population, then the results will : n p l y that NK cells are important. Second, it seem likely that some host component, possibly NK cells or macrophages, may be stimulated by the transfer of LAK cells and the administration o f IL2. Therefore more studies need to be performed whereby LAK + IL2 is used to treat tumours in mice which are generally immunodeficient, or have been selectively depleted

of various lymphoid subsets. Such experiments will determine to what degree NK cells contribute to the effects of LAK + IL2 therapy, both at the level of the donor cell transferred and at the level of a possible role for the recipient's immune response. IL2 is a potent stimulator o f NK activity and has also been reported to mediate regression o f tumours in mice and humans in the absence of LAK transfer. Recently, we have reported that the investigational drug flavone aceuc acid (FAA) synergizes with IL2 for the treatment of murine renal cancer [37]. F _ ~ has been shown to potently augment NK act!:vity in both lymphoid and non-lymphoid sites, suggesting a possible role for NK cells in the therapeutic effects of F A A and IL2. Further studies have shown that the therapeutic effect o f FAA and IL2 are significantly reduced by the treatment o f tumour-bearing mice with antiasialo-GM1 serum [38], which potently suppresses NK activity. These results suggest that host asialo-GM1 + lymphocytes may be a critical component o f the therapeutic effects o f F A A and IL2 against murine renal cancer. Further studies involving depletion of NK cells by more selective antibodies will be required in order to definitively determine the contribution of NK cells to the therapeutic effects of FAA and IL2 in vivo.

References.

[1] ORTALDO,J.R., Regulation of natural killer activity. Cancer and Metastasis Reviews, in

press. [2] TRmCHEPa,G. & P~USSlA, B., Biology of Disease. Human natural killer cells: biologic and pathologic aspects. Lab° Invest., 1984, 50, 489-513. [3] H ~ R n ~ , R.B., P~WOLDS, C.W. & ORT~LDO,J.R., Mechanism of cytotoxicity by T natural killer (NK) cells. Ann. Rev. Immunol., 1986, 4, 651-80. [4] ORTALDO,J.R., Cytokine production by CD3- large granular lymphocytes. In: Functions of the Natural Immune system, eds. C.W. Reynolds and R.H. W'dtrout, Plenum Press, NY, 1988 (in press). [5] ORT~DO,J.R., Sruau~ow,S.O., T~ol~n~N,T. & I ~ . ~ , R.B., Determination of surface antigens on highly purified human NK cells by flow cytometry with monoclonal antibodies. J. ImmunoL, 1978, 121, 304-15. [6] Rm~ou>s, C.W., S ~ o w , S.O., ORTALOO,J.R. & ~ , R.B., Natural killer activity in the rat. - - II. Analysis of surface antigens on LGL by flow cytometry. J. lmmunol., 1982, 127, 2204-11. [7] Koo, G.C., JACOBSON,J.B., ~ m G , G.J. & ~ I N O , U., Antigenic prof'fle of murine natural killer cells. J. ImmunoL, 1980, 125, 1003-6.

L YMPHOKINE-ACTIVA TED KILLER CELLS

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[8] MATTES,M.J., SHARROW,S.O., HERBERMAN,R.B. & HOLDEN,H.T., Identification and separation of Thy-l-positive mouse spleen cells active in natural cytotoxL~ty and ADCC. J. Immunol., 1979, 123, 2851-60. [9] ORTALVO,J.R. & I'I~ge~AN, R.B., Heterogeneity of natural killer cells. In: Ann. Rev. Immunol. W.E. Paul, C.G. Fathman and H. Metzger, eds. Ann. Rev. Inc., Palo Alto, CA, pp. 359-94, 1984. [10] ROVER,J.C., HALIOTIS,T., KLBIN,Mo, KOREC,$., J~Tr, J.R., ORTALVO,J.R. & HEV.BERMAN,R.B., A new immunodeficiency disorder in humans involving NK cells. Nature (Lond.), 1980~ 284, 553-8. [11] BmoN, C.A., I~DERS~, K.F. & WELSH,R.M., Aberrant T cells in beige mutant mice. J. Immunol., 1987, 138, 2050-6. [12] LOHMAsN-MAT~tW.S,M.L., DOMZI~,W. & ROVES,J., Promonocytes have the functional characteristics of natural killer cells. J. Immunol., 1979, 123, 1883-6. [13] PETER,H.H., The origin of human NK cells. An ontogenic model derived from studies in patients with immunodeficiencies. Blur, 1983, 46, 239-45. [14] HAc~rr, J. Jr, BosuA, G.C., BosuA, M.J., BENN~rr, M. & Kt~aAR, V., Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocyte, Proc. natl. Acad. Sci. (USA), 1986, 83, 3427-31. [15] MArm~SON, B.J. & FowLr~S, B.J., Cell surface antigen expression on thymo~vtes: development and phenotypic differentiation of intrathymic subsets. Immunol. Rev., 1984, 82, 141-72. [16] MASON,L., G~.vn~A, S.L., ~ , T., ORT.ArgO, J.R. & MATron.SON,B.J., LGL-1 : a non. polymorphic antigen expressed on a major population of mouse natural killer cells. J. Immunol., 1988, 140, in press. [17] A B ~ s o ~ , S., MILLER,R.G. & PmLLWS,R.A., The identification in adult bone marrow of pluripotent and restricted stem cells for the myeloid and lymphoid systems. J. exp. Meal., 1977, 14,5, 1567-79. [18] HOLMES,K.L., PW.RC~,J.H., DAV~oON,W.F. & MORSE,H.C. !IL Murine hematopoietic cells with pre-B or pre-B/myelbid characteristics are generated by in vitro transformation with retroviruses containing fes, ras, abl and arc oncogenes. Z exp. Med., 1986, 164, 443-57. [19] L~mR, L.L., CWmLA,S., FEVEP.SPmL,N. & PHILLIPS,J.H., Human natural killer cells isolated from peripheral blood do not rearrange T-cell antigen receptor beta chain genes. J. exp. Med., 1986, 163, 209-214. [20] YouNg, H.A., ORTALVO,J.A., H~RB~V.UAS,B.H. & I~VUOLVS,C.W., Analysis of T cell receptors in highly purified rat and human large gr~mular lymphocytes (LGL): lack of functional 1.3 kb [~-chain mRNA. J. Immunol., 1986, 136, 2701-2704. [21] H1sERour,J.C., LAVaOURN,K.A. & VAn~, J., Laminin inhibits the recognition of tumor targets cells by murine natural killer and natural cytotoxic lymphocytes. Am. J. Pathol., 1985, 121, 148-55. [22] I4A~IS, D., EVANS,B., F R m D ~ , L. & KOlU~N,H., Identification of a putative human natural killer (N-K) cell antigen receptor. J. Leuk. Biol., 1987, 42, 368, [23] ORTALDO,J.R., KAmOR, R., S~AL, D., BOLnUIS,R.H. & BINO, T., Identification of a proposed NK receptor. In: Proceedings of the 5th International Workshop on Natural Killer Cells. 1988. E. Ades and C. Lopez, eds. [24] ITOH,K., TILV~S,A.B., KVMAG~,K. & BALCH,C.M., Leull + lymphocytes with natural killer (N'K) activity are precursors of recombinant interleukin 2 (rIL-2)-induced a¢-~ tivated killer (AK) cells. J. Immunol., 1985, 134, 802-7. [2~] PmLurS, J.H. & L^Nmn, L.L., Dissection of the lymphokine-activated killer phenomenon: relative contribution of peripheral blood natural killer cells and T lymphocytes to cytolysis. J. exp. Med., 1986, 136, 1579-85. [26] LAN~, L.L., L~, A.M., C~vn%C.I., LIKEN,M.R. & PmLLrPS, J.G., The relationship of CD16 (Leu-I 1) and Leu-19 (NKH-I) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J. ImmunoL, 1986, 136, 4480-6. [27] H~c~No, T., R~mn~sz, E.L., ~ , S., SCHLOSSMAS,S.F. & R~z, J., Phenotypic and functional heterogeneity of human cloned natural killer cell lines. Nature (Lond.), 1983, 301, 158-60. [28] OnTALDO,J.R., MASON,A. & OVeRTON,R., Lymphokine-activated killer (LAK) cells: analysis of progenitors and effectors. J. exp. Meal., 1986, 164, 1193-205. [29] SALUP,R.R., BAC~, T.A. & W~LTSOtrr, R.H., Successful treatment of advanced murine renal cell cancer by bicompartmental adoptive chemoimmunotherapy. J. Immunol., 1987, 138, 641-647.

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[30] VuJ~ovlc, N.L., HERBERMAN,R.B., SALtW, R.R., OLSzowY, M.T., CRAMER. D.V., RaSVNOLDS,C.W. & HISERODT,J.C., Lymphokine-activated Miler cells in the rat. -II. Analysis of progenitor and effector cell phenotype and relationship to natural killer cells. Cancer Res., 1988, 48, 884-91. [31] BIRON,C.A., NATUK,R.J. & WELSH,R.M., Generation of large granular T lymFhocytes in vivo during viral infection. J. ImmunoL, 1986~ 136, 2280-6. [32] SHORTSt~,K.A., WmSON,A., SCOLLAY,R. & C~mN,W.F., Development of large granular lymphocytes with anomalous non-specific cytotoxicity in clones from Lyt-2 + T cells. Proc. natl. Acad. Sci., 1983, 80, 2728-35. [33] BRoom, C.G., UV.DAL,D.L. & HENm~Y,C.S., Lymphokine-driven <~differentiation, of cytotoxic T-cell clones into cells with NK-like specificity: correlation with display of membrane molecules, lmm. Rev., 1983, 72, 43-50 [$4] MA6HAZACm,A.A., HERBEnMma,R.B., VU,ANOVlC, N.L. & HISERODT, J.C., In vivo distribution and tissue localization of highly purified rat lymphokine-activated killer (LAK) cells. Cell ImmunoL, 1988 (in press). [35] HoRmmO,R.L., SALUP,R.R. & WiL~otrr, R.H., Tissue distribution and localization of IL2-activated killer cells after adoptive transfer in vivo. In <
EFFECTORS, REPERTOIPJE AND RECE~wrORS INVOLVED IN LYMPHOCYTE-MEDIATED MHC-UNRESTRICTED CYTOTOXICITY L.L. Lanier and ff.H. Phillips Becton Dickinson Monoclona! Center, Inc., Mountain View, CA ./,USA) We recently proposed that three types of human cytotoxic lymphocyte exist: (1)antigen-specific, MHCrestricted cytotoxic T lymphocytes (CTL), (2) MHC-unrestricted CTL, and (3) natural killer (NK) cells. The first type recognizes specific antigen using the ~/~-T-cell antigen receptor (TCR) heterodimer only in association with major histocompatibility complex

(MHC) class I or II antigens [1]. By contrast, NK cells and MHC-unrestricted CTL apparently do not require the presence of polymorphic MHC determinants on the target cell for recognition and lysis. Although apparent|y m e d i a ~ g the same functional activity, NK cells and MHC-unrestricted CTL are distinguished by the fact that CTL arise from the T cell lineage and rear-