Immunology 1982 46 619

Lectin-binding characteristics of human natural killer cells

B. M. VOSE, G. BLACKLEDGE*, D. CROWTHER* & J. GALLAHER* Department of Immunology, Paterson Laboratories and *Cancer Research Campaign Department of Clinical Oncology, Christie Hospital and Holt Radium Institute, Manchester

Acceptedfor publication 26 January 1982

Summary. Human natural killer (NK) cells separated used to separate the NK active population in E+ cells initially by density centrifugation oflymphocytes (E+) by fluorescence-activated cell sorting (FACS). These forming rosettes with sheep red blood cells (SRBC), data add a new dimension to the cell surface properties were further fractionated on gradients ofbovine serum of human NK cells and suggest the presence of albumin (BSA). Low density fractions contained LCA-reactive which are either enriched effector cells which displayed high cytotoxicity against in, or uniquely associated with, cells ofthe NK subset. the NK-sensitive erythroleukaemic cell line, K562. The experiments indicate that can serve as These low density cells, which expressed receptors for useful probes of lymphocyte function and provide the Fc and the monoclonal antibody OKM 1, showed basis for effective cell sorting. enhanced cytotoxicity when treated with lymphoblas- toid interferon (IFN-a). They also showed an in- creased response to phytomitogen in comparison with INTRODUCTION unseparated cells or those recovered from high density fractions. Two lymphocyte subsets one of high and Natural killers (NK) are non-adherent, non-phagocy- one oflow binding capacity were identified in the tic cells with receptors for the Fc portion of immuno- E+ populations by their reactivity with Lens culinaris globulin and are found in the peripheral blood and agglutinin (LCA). High LCA binding was observed spleen of all species examined (Herberman, 1980). only in low density fractions and was associated with a They have the capacity to lyse a wide range of targets marked enrichment ofNK activity. This property was ofmalignant and non-malignant origin (Herberman & Abbreviations: LGL, large granular lymphocytes; SRBC, Holden, 1979; Nunn, Herberman & Holden, 1977) sheep red blood cells; NK, natural killing; RPMI, Roswell and have been implicated in resistance to the out- Park Memorial Institute; 1640, medium; FCS foetal calf growth of certain tumour cells and to the transplan- serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PHA, phytohaemagglutinin; PPO, 2,5-diphenylox- tation ofhaemopoietic tissues (Herberman & Holden, azole; FITC, fluorescein isothiocyanate; FACS, fluorescent- 1979). Until recently the analysis of NK cells has activated cell sorter; F: P ratio fluorescein: molecules; relied solely upon their functional evaluation, i.e. the Con A ; LCA, lens culinaris; PNA, peanut killing of susceptible target cells in short term 5"Cr agglutinin; WGA, . release assays. However, in murine (Koo, Jacobson, Correspondence: Dr B M Vose, Department of Immu- nology, Paterson Laboratories, Christie Hospital and Holt Hammerling & Hammerling, 1980) and human sys- Radium Institute, Manchester M20 9BX. tems (Zarling & Kung, 1980; Ortaldo et al, 1981a) 0019-2805/82/0700-0619$02.00 the cell surface phenotype of NK cells has lately ©O 1982 Blackwell Scientific Publications been delineated by serological methods, including 619 620 B. M. Vose et al. monoclonal antibodies to the major leucocyte subset Corning culture flasks in RPMI +10% FCS. Non- antigens. This approach has been facilitated by the adherent cells were washed once, resuspended in FCS development of techniques by which NK cells can be and cell concentration adjusted to 5 x 106/ml. Cells enriched with respect to other lymphocytes on the were over 95% viable by trypan blue exclusion and basis ofdensity (Timonen & Saksela, 1980; Koo et al., > 97% were lymphocytes. 1980). Isolated NK cells have a characteristic size and morphology (high cytoplasmic: nuclear ratio and Separation oflymphocyte populations azurophilic granules) and it is apparent that these large Cells were separated into T-cell-enriched (E+) or granular lymphocytes (LGL) are the principal effector T-cell-depleted fractions (E-) by formation ofrosettes cells of natural killing. In spite of such advances the with SRBC. SRBC were washed three times in PBS lineage ofNK remains in doubt. A high proportion of and resuspended in FCS (2 x 108/ml). Equal volumes NK cells form rosettes with sheep red blood cells oflymphocytes and washed SRBC were mixed, centri- (SRBC) and express T-cell antigens (West, Cannon, fuged (100 g 5' 40) and allowed to stand overnight at Kay, Bonnard & Herberman, 1977; Ortaldo et al., 40. Pellets were gently resuspended and rosetting and 198 la) but evidence suggestive of a myelo-monocytic non-rosetting cells separated by centrifugation (900 g association has also been presented (Lohmann- 15 min 40) on Lymphocyte Separation Medium. Mattes, Domzig & Roder, 1979; Zarling & Kung, Preparations were freed of SRBC by osmotic shock. 1980; Ortaldo et al., 1981a). Rosetting cells were washed a further three times in Certain lectins can be used as probes to identify RPMI, resuspended in RPMI or PBS and counted. distinctive cell surface markers on lym- phocyte subsets (Fowlkes, Waxdal, Sharrow, Thomas, Densityfractionation Asofsky & Mathieson, 1980). In a previous paper we Cells (2 5 x 107) enriched or depleted ofT lymphocytes screened several lectins for differential binding to by rosetting with SRBC and separation on Lympho- T-cell-enriched and depleted human lymphocyte sub- cyte Separation Medium gradients were suspended in populations (Blackledge, Gallagher, Swindell & 1 ml 35% BSA (Pathocyte 5, Miles Laboratories) in 6 Crowther, 1980a). We have now extended this ml polycarbonate centrifuge tubes (MSE 34411-113). approach to examine binding of lectins to supopula- Dilutions of BSA in RPMI 1640 corresponding to tions ofT cells isolated by SRBC rosetting and density 29%, 27%, 23% and 10% ( 1 ml) were layered onto this separation. Our findings show distinct patterns of and tubes centrifuged at 40 in a MSE 3 x 6 ml swing binding among different subsets. In particular, con- out rotor at 15,000 g for 30 min. Cells which accumu- siderable enrichment of NK cells from SRBC-roset- lated at each interface and the pellet were taken, ting cells was possible using a Fluorescein-activated washed twice in RPMI 1640 and resuspended in cell sorter (FACS) by selection of cells with relatively RPMI+10% FCS for NK and stimulation high binding of Lens culinaris agglutinin (LCA). It is assays or PBS/BSA for lectin binding studies. The suggested that expression of certain oligosaccharide method for fractionation is essentially that of Kondo, sequences may be important for NK activity. Slockert & Smith (1973). Interfaces were numbered from the top in order of increasing density between 1 and 4 and the pellet designated 5. MATERIALS AND METHODS Monitoring of cell population Donor Lymphocytes with the Fc receptor for IgG (FcR) were Heparinized blood samples (150 ml) were taken from assessed by rosette formation with ox-RBC coated six healthy donors (age 28-36 years) and from one with the maximum non-agglutinating dilution of the patient with Hodgkin's disease. Spleens were obtained IgG (DEAE purified) fraction of rabbit anti-ox RBC. from one road traffic accident victim and two patients Lymphocyte: RBC ratio was 40: 1. Cells were centri- with gastric carcinoma. Lymphocytes were separated fuged (50 g 5 min) and incubated on ice for 30 min by centrifugation (1000 g 20 min 40) on Lymphocyte before resuspension and counting. The presence of Separation Medium (Flow Laboratories, Irvine). cells binding the monoclonal antibodies Orthoclone Mononuclear cells were taken from the interface with OKT4 IND and OKT8 SUP against human inducer/ a pasteur pipette, washed twice in RPMI and adherent helper and suppressor cytotoxic T lymphocytes (Ortho cells depleted by incubation (30 mins 370) in 75 cm2 Pharmaceutical Corp., Rariton, N.J.) and OKMI Lectin binding ofhuman NK 621 (human blood monocytes, granulocytes and LGL) g 5 min) and incubated for 4 hr at 37°. Aliquots (0-2 and T28 (Pan T reagent: a gift from Dr PCL Beverley, ml) of the suspension were removed and radioactivity University College, London) were counted after stain- in the supernate samples and remaining pellet counted ing with (FITC)-labelled rabbit anti-mouse IgG (Nor- in a gamma counter. Spontaneous 51Cr release was dic Immunological Laboratories, Maidenhead). measured from target cells incubated in medium and maximum 51Cr release obtained by lysis of cells with Triton X100. Percentage 51Cr release for each tube was Stimulation assays calculated and cytotoxicity derived from the formula: Mitogen responsiveness of lymphocyte fractions was cytotoxicity [(% release test - spontaneous release)/ determined in flat-bottomed Sterilin microtest plates (maximum release -spontaneous release)] x 100. with a 0-2 ml capacity. All assays were performed in Cytotoxic activity is also sometimes expressed as lytic the presence of adherent cells. Mononuclear cell units/ 107 cells, calculated from dose-response curves populations (0- 1 ml of 2 x 106/ml) prepared from [one lytic unit (l.u.) being defined as the number of peripheral blood by sedimentation on Lymphocyte effector cells required to produce 30% kill above the Separation Medium were dispersed into microtest base line]. wells in RPMI 1640 plus 10% FCS and incubated at 370 for 30 min. Non-adherent cells were washed from the wells and 0-1 ml medium added. Plates were Interferon treatment incubated overnight before stimulation assays. Separ- Human lymphoblastoid interferon (IFN-a), produced ated density subpopulations (0 1 ml 2 x 106/ml) were by Namalva cells, was prepared and purified by Dr K plated onto adherent cells and PHA ( 2 yg/ml H Fantes, Wellcome Research Laboratories, Becken- Wellcome purified grade) added. Cells were cultured ham. The preparation (batch 479/602) was of specific for 3 days at 370 with a pulse of 1 pCi/well of activity 2-2 x 108 i.u./mg. protein, contained human [3H]-thymidine (specific activity 2-5 uCi/mmol Radio- protein as a stabilizer and was stored at -700. chemical Centre, Amersham) over the last 6 hr. Cells Lymphocytes (3 x 106/ml in RPMI plus 10% FCS) were harvested onto paper discs using a Skatron were pretreated for 18 hours with 1000 i.u./ml IFNa. multiple harvester and radioactivity counted in 5 ml These conditions have previously been shown to be toluene scintillation fluid containing 01 % w/v optimal for augmentation of NK activity. Cells were 2,5-diphenyloxazole (PPO). washed three times in RPMI before use in NK or lectin binding studies.

Cell line Lectin binding The erythroleukaemic cell line K562 was used in Separated lymphoid cell populations were adjusted to assays of NK activity because of its exquisite sensiti- final concentrations of2 x 106 cells/ml in PBS contain- vity to lysis by human lymphocyte effectors. Cells were ing 0-1% w/v BSA (PBS/BSA). Fifty microlitres of maintained in suspension cultures in RPMI 1640 plus cells (105) were added to a series of microtitre plates 10% FCS and labelled (1 x 106 cells in 0 5 ml RPMI) by and varying amounts of FITC-lectins were added dropwise addition of 100 pCi sodium [51Cr] chromate from stock solutions of lectins of concentration 0-4 specific activity 100-350 pCi/pg (Radiochemical mg/ml in PBS/BSA. Final volumes were made up to Centre, Amersham). After incubation (1 hr at 370) 150 M1 of PBS/BSA. All solutions and cell suspensions targets were washed three times in Hanks's balanced were cooled to 40 for 30 min with occasional agitation. salt solution, incubated for a further 30 min in RPMI Kinetic experiments showed that 30 min was an 1640 plus 10% FCS centrifuged resuspended in RPMI adequate time for attainment of the equilibrium of plus 10% FCS and counted. bound: unbound lectin. The degree of specificity of lectin binding was established by incubation with appropriate monosaccharide inhibitors. These sugar Cytotoxicity assays ligands were mixed with lectins for 5 min before Target cells (5 x 103) were dispensed into LP3 tubes in addition of cells. After incubation, the contents of 0.1 ml ofRPMI plus 10% FCS and lymphocytes added each microtitre well were suspended in 1 ml of to give an effector: target ratio of 20:1-5:1. Fluid PBS/BSA and cell fluorescence measured by flow volume was adjusted to 0-4 ml, tubes centrifuged (100 cytometry. Competition studies were also carried out 622 B. M. Vose et al. in which native lectins were assessed for their ability to RESULTS either inhibit the binding to cell surfaces ofFITC-con- jugated lectins (i.e. native lectins added first to cell Enrichment of NK Cells on bovine serum albumin suspensions followed by FITC-lectins) or to displace NK cells were enriched by density centrifugation on FITC-lectins already bound to the cell surface. At 40, gradients of bovine serum albumin (BSA; Table 1), cell bound FITC-lectins appeared to be located mainly fractions 1 and 2 (low density populations) containing at the cell surface with minimal intracellular fluores- the greatest lytic activity against the K562 cell line. cence as judged by fluorescence microscopy. F-LCA This pattern was found in nine of ten individual runs. saturation binding was achieved at 20 ug/ml, the The possibility that cytotoxic cells present in fractions concentration used for cell sorting (see below). other than 1 or 2 were suppressed by contaminating suppressor cells of high density was discounted, since Flow cytometry admixture of low NK fractions 4/5 with fractions 1/2 FITC-lectin binding was measured in a Biophysics did not inhibit lysis of K562 (data not shown). In Cytofluorograf 4800A interfaced to a desk top com- non-SRBC rosetting cells separated on BSA gradients, puter (Hewlet Packard 9845S). This has been pre- cells with greatest anti-K562 activity could also be viously described (Blackledge, Swindell, Hodgson & concentrated in fractions 1 and 2 from E- (non-SRBC Crowther, 1980b) and allows for analysis of cell rosetting cells). Isolated density fractions from E+ populations using two dimensional histograms. The populations were pretreated with interferon as de- mean fluorescence of the cell populations with lectin -scribed in Materials and Methods. Boosting of lytic bound was estimated using the expression: activity was apparent in separated E+ cells, fractions /Nc Nc\ 1 and 2 and to a much lesser extent in fraction 3 ( Sixni/Ncx ni - zero correction (Table 2). No augmentation of killing was found in \i=l i=l/ fractions 4 and 5. The cells boosted by interferon treatment thus have the same density profile as the where Nc is the total number of channels and ni is the untreated effector cells. Marker analysis of E+ sub- number of cells in channel i (modified from Bohn, fractions showed that low density 1/2 sets were also 1976). Values are corrected to allow for variations in enriched for cells bearing FcR compared with higher cell auto-fluorescence (Blackledge et al., 1980a). When density NK-depleted cells (Table 1). 'mean cell fluorescence' values for individual lectins are compared the data were corrected for the specific fluorescence of the lectin (number of FITC residues Monoclonal antibody analysis of density fractions per molecule). This allows a direct comparison of In order to characterize the BSA gradient fractions numbers oflectin-binding sites per cell but we have not further, cell populations were monitored for binding determined absolute values for numbers of sites. of monoclonal antibodies by fluorescence microscopy (Table 3). Fractions 1 and 2 (pooled because of low Cell sorting yields of cells) were depleted of cells staining with the Cell sorting on the basis of lectin-derived cell fluores- Pan T reagent T28. Unseparated E+ cells showed cence was carried out on a Becton Dickinson staining of =90% of cells with this antibody as did FACS-IV cell sorter kindly made available by Profes- fractions 3,4 and 5, but only 39% ofcells in fractions 1 sor R W Baldwin, Cancer Campaign Laboratories, and 2 showed mature T-cell characteristics. The Nottingham and performed by Dr R A Robbins. opposite effect was noted with OKMl with strong Sorting gates were set to sort to left and right the lower enrichment of stained cells in fractions 1/2 and few fluorescent and upper fluorescent cells, respectively. reactive cells in other fractions. Similar proportions of The results ofsorting were checked using conventional cells were stained in all fractions by OKT8 (suppres- flow cytometry. sor) but OKT4 staining was reduced in fractions 1 and The following FITC-conjugated lectins were 2 which would be compatible with the low number ofT obtained from Miles Chemicals: concanavalin A cells. (F-Con A, F: P 2-14) Lens culinaris agglutinin (F-LCA, F: P 1-8) and wheat germ agglutinin (F-WGA, F: P 1-3): peanut agglutinin (F-PNA, F: P Mitogen responsiveness 2 4) was purchased from Sigma Chemical Co. All density fractions except fraction 5 responded Lectin binding ofhuman NK 623

Table 1. Enrichment of NK activity by BSA gradient density separation

Fraction Cell recovery (%) Lysis of K562* FcR cells Unseparated 100 14+4 E+ 61+5 4 35-2+±73 12+3 1 2-2+1-5 47-8+8 5 17+4- 2 9-5+7 9 52 3+7 9 8+2 3 390+142 232+6-8 3+1 4 32-8+11 0 14-3+39 4+1 5 18-8+9-6 5-3+1-7 2+1

Mean of 10 determination + SE. * E:T=20: 1.

Table 2. Boosting of natural killer activity in Table 4. Response of density fractions of E+ cells to density subfractions of E+ cells by interferon PHA

Cytotoxic activity against K562 Incorporation* of [3H]-thymidine in stimu- lated cultures Untreated Interferon treated* Fraction Donor 1 Donor 2 Donor 3 Donor 4 E+ 32t 89 Fraction 1 57 180 E+ 18,964 16,147 13,719 5020 2 57 160 1 15,550 14,100 4167 3 13 25 2 20,220 21,943 23,996 5519 4 8 7 3 21,084 5421 23,447 4379 5 <1 <1 4 15,712 8014 12,233 773 5 7995 3125 1478 893 * Four hour pretreatment 1000 u./ml a inter- feron. Stimulation was performed in the presence of con- stant numbers of adherent mononuclear cells with 2 t Lytic units (30% kill/107 cells). pg/ml PHA. * Counts per minute. Table 3. Composition ofdensity fractions of E + cells determined by reactivity with monoclonal antibodies declined with increasing cell density. Dose-response curves were similar (optimal PHA concentration, 2 Mg Cells (%) showing fluorescent ml-') for the cells of all fractions. Spontaneous staining with [3HJ-thymidine incorporation in each fraction was less T28 OKT8 OKT4 OKM I than 1000 c.p.m. Unseparated 71 18 47 15 E + 90 26 68 8 Lectin-binding studies Fractions 1+ 2 39 15 18 66 Four lectins, peanut agglutinin (F-PNA, Lens 3 87 20 45 8 A F-Con A and wheat 4 87 25 55 3 culinaris F-LCA, Concanavalin 5 88 17 48 4 germ agglutinin (F-WGA) were chosen for the study E- 26 31 of density subfractions because they showed differen- tial binding to E+ and E- cells (Blackledge, Vose, Crowther, Morris & Gallagher, submitted for publica- significantly to addition of PHA (2 ug/ml), although tion). Interaction of F-LCA (10 Mg/ml) with E+ cells the degree of stimulation varied (Table 4). Fraction 1 distinguished two populations (Fig. 1). The three cells responded similarly to unseparated E+ cells, dimensional display allows examination of the fraction 2 cells showed the greatest response, while number ofcells (vertical axis) showing a particular size the mitogenic responses of the remaining fractions (as assessed by light scattering) and fluorescence. 624 B. M. Vose et al. Approximately 90% of cells showed low binding and of density isolation is shown. Fractions 4 and 5 the remainder were in the higher fluorescent popula- showed identical low level binding and only results tion. Separation on the basis of density concentrated with fraction 4 are presented. By contrast with the cells for high F-LCA binding to low density fractions. differential reaction with F-LCA, F-Con A and Cells in these fractions were invariably larger in size F-PNA did not reveal two populations in E+ cells, and than fractions 3,4 and 5 (Fig. 1) but correction for cell high density fractions had similar fiuorescence to size indicated that high F-LCA binding was not unseparated E+ cells. Both F-LCA and F-Con A have attributable to this variable. Increasing cell density as the sugar target but lectin-binding sites was associated with reduced cell fluorescence. It is within glycoprotein structures are different (Black- important to realize in examining these displays that ledge et al., submitted for publication). Binding of they represent data from 5000 cells. Since fractions 1 F-WGA o (2 pg/ml) to E+ cells showed similar trends and 2 together represent only 2% and 9%, respectively to F-LCA but populations were less well defined (data oftotal E+ cells, the considerable concentration effect not shown).

..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.--A.-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... -:...... -.. Figure 1. Two dimensional histrograms of E+ PBL incubated with F-LCA (20 g/ml). The histogram on the left represents the usual E+ pattern, with most of the cells having a low.~~~~~~~~~~~~~~~~~~.fluorescence....:...... and a small-s; percentage ofcells having a higher fluorescence. The histograms labelled Fr 1-4 represent F-LCA binding to increasing density fractions ofE+ PBL. The lowest density fraction (Fr 1) consists almost exclusively of cells with high fluorescence. The highest density fraction (Fr 4) consists primarily of low fluorescent cells. The intermediate fractions show a progression from cells in the upper fluorescent population to the low fluorescent population. ~~~~~~~~~~~~~~~~~~~~~~~~~~~..X...... Lectin binding ofhuman NK 625

Table 5. Separation ofNK cells by FACS ofE + cells stained (Ortaldo et al., 1981a). These patterns of reactivity with fluorescent-conjugated lens culinaris suggest either that human NK cells belong to a lineage separate from those oflymphoid and myelomonocytic Cytotoxicity against K562 Cells (%) cells, or that the cells originate from a precursor cell common to both lineages. E+ 50* 100 High lens binding 125 15 The differential binding of lectins to lymphocyte Low lens binding < 1 85 subsets offers an effective alternative means to mono- E+ lectin bound 54 clonals whereby cells of defined in vitro activity can be purified for further study oftheir lineage and function. *Lytic units (30% kill per 107 cells). In addition, the data have important implications for the nature of the effector: target cell interaction. NK showed enhanced F-LCA binding compared FACS sorting with other subsets. It was possible to isolate NK cells The association between high F-LCA binding and NK on the basis of F-LCA binding using FACS. These activity was confirmed by cell sorting on a FACS. E+ lectin binding characteristics may reflect a high con- cells were labelled with F-LCA (20 ug/ml) and separ- centration of cell surface oligosaccharides containing ated using the Becton Dickinson FACS IV into high mannose in the case of LCA (Allen, Desai & Neu- and low fluorescent groups. The presence of F-LCA berger, 1976) and sialic acid or N-acetyl glucosamine on the cell surface did not influence K562 lysis. NK with respect to WGA (Allen, Neuberger & Sharon, activity was markedly enriched in high F-LCA frac- 1973; Bhavanandan & Katlic, 1979). Differences tions which represented 15% of the total E+ popula- between subsets could result from the presence of tions and was completely absent from low binding certain glycoproteins of relatively high carbohydrate populations (Table 5). Low F-LCA cells had a content which may either be enriched in, or unique to protective effect on 51Cr release usually associated with these functionally active populations. Some distinc- inactive 'filler' cells such as ox-RBC. tion between the surface glycoproteins of mature T and NK cells is implicit from certain animal studies. For example some, but not all, alloreactive murine DISCUSSION cytotoxic T cells have been found to express character- istic with distinctive lectin The phenotypic characterization of NK cells is one of binding (Kimura, Wigzell, Holmquist, Ersson & the central issues in contemporary study of natural Carlsson, 1979; Kaufmann & Berke, 1981). NK immunity in man and other species. Data accumulated activity but not allosensitised killing can be blocked by before the introduction of monoclonal reagents dis- addition ofsimple sugars such as D-mannose, D-galac- closed a degree of heterogeneity which impeded the tose, N-acetylglucosamine and to a lesser extent D-glu- preparation of these cells for further characterization cose (Stutman, Dien, Wisun & Lathime, 1980). Since of their nature and function. The enrichment of the the binding of F-LCA did not influence cytolytic morphologically identifiable subset of human NK activity, it is suggested that sites involved in mannose- cells-the large granular lymphocytes-by density directed LCA attachment and recognition of target gradient separation on Percoll (Timonen & Saksela, cell structures are different. 1980) or BSA, as in this study, has improved the Consistent with the enrichment of NK cells by situation, though the lineage of the cells remains binding ofLCA was the demonstration that these cells obscure. Thus, NK cells are unreactive with the alone among the various gradient fractions, would be markers of mature T lymphocytes T28, OKT3 or amplified by pretreatment with exogenous interferon. OKT4, (Ortaldo et al, 1981a) but express other This agent is a major immunodulator of NK function markers such as the SRBC receptor (West et al., 1977) in man (Trinchieri, Santoli & Koprowski, 1978; and at least a proportion of the cells bind the Einhorn, Blomgren & Strander, 1978; Herberman, monoclonals OKT8 (suppressor/cytotoxic, 20% posi- Ortaldo & Bounard, 1979) and it is well established tive), OKT1O (early thymus antigen; 90% positive) and that only low density large granular lymphocytes are Lyt3 (SRBC receptor, 40% positive). In addition, boosted (Timmonen, Ortaldo, Stadler, Bonnard & LGL-mediating NK bind OKM1 (monocytes and Herberman, submitted for publication) under the PMN, 80% positive) and anti-asialo GMI (M0-PMN) conditions of these experiments. Other (unpublished) 626 B. M. Vose et al. data suggest that binding of F-LCA and F-WGA is and a comparison of its properties with lectins of increased on IFN-a treated cells (B. Vose, G. Black- similar specificity. Biochem. J. 155, 127. ALLEN A.K., NEUBERGER A. & SHARON N. (1973) The ledge & J. Gallagher, unpublished observation). Study purification, composition and specificity of Wheat Germ of the nature of this effect might shed further light on Agglutinin. Biochem. J. 131, 155. the role of glycoprotein expression in NK function. BHAVANANDAN V.P. & KATLIC, A.W. (1979) The interaction Enrichment of T and B cells on the basis ofbinding to of wheat germ agglutininin with . J. biol. Chem. 254, 4000. Helix pomatia was described by Hellstrom, Dilner, BLACKLEDGE G., GALLAGHER J.T., SWINDELL R. & Hammarstibm & Perlmann, 1976a, and to Lens CROWTHER D. (1980a) Flow cytometric analysis of culinaris by Blackledge et al., submitted for publica- fluorescent lectin binding to surface membranes of tion. A population ofT lymphocytes highly responsive tumour lymphoid and myeloid cells. Flow Cytometry, IV, to PHA and Con A was also separated by virtue of 222. BLACKLEDGE G., SWINDELL R., HODGSON B.W. & CROWTHER their high affinity receptors for WGA (Hellstrom, D. (1980b). Computerised acquisition and analysis of Hammarstrom, Dillner, Perlmann & Perlmann, flow cytometric data. Int. J. biomed. Comp. 11, 41. 1976b), a finding consistent with the present study in BOHN B. (1976) High sensitivity cytofluorometric quanti- so far as mitogen-responsive and high F-WGA binders tation of lectin and hormone binding to surfaces of living co-existed in the low density T-cell fractions. cells. Exp. cell res. 103, 39. EINHORN S., BLOMGREN H. & STRANDER H. (1978) Interferon It was also noteworthy that populations enriched and spontaneous cytotoxicity in man. I. Enhancement of for NK activity, staining with OKM 1, and containing spontaneous cytotoxicity of peripheral lymphocytes by very few mature lymphocytes, as assessed by staining human leukocyte interferon. Int. J. Cancer, 22, 405. with the T28 monoclonal antibody, exhibited a high FOWLKES B.J., WAXDAL N.J., SHARROW S.O., THOMAS III C.A., ASOFSKY R. & MATHIESON B.J. (1980) Differential degree of T-cell-mitogen responsiveness. It has binding of fluorescein-labelled lectins to mouse thymo- recently been shown (Ortaldo, Sharrow, Timonen & cytes: subsets revealed by flow microfluorometry. J. Herberman, 198 lb; Timonen et al., submitted for Immunol. 125, 623. publication; Vose & Bonnard, manuscript in prep- HELLSTROM U., DILNER M.-L, HAMMARSTROM S. & PERL- MANN P. (1976a) Fractionation of human T lymphocytes aration) that purified LGL can be grown in interleuk- on wheat germ agglutinin-sepharose. J. exp. Med. 144, in-2 produced in the presence of mitogen. The obser- 1381. vation that growth of LGL under these conditions is HELLSTROM U., HAMMARSTROM S., DILLNER M-L., PERL- accompanied by the emergence of cells with mature MANN H. & PERLMANN P. (1976b). Fractionation of T-cell characteristics (as defined by the appropriate human blood lymphocytes on Helix pomatia A hemag- glutinin coupled to sepharose beads. Scand. J. Immunol. 5 monoclonal antibodies) would be consistent with the (Suppl. 5), 45. T-cell lineage of NK cells. The validity of this HERBERMAN R.B. (1980) Natural cell-mediated immunity provisional conclusion is currently under investigation against tumours, pp. 1365. Academic Press, New York. in these laboratories. HERBERMAN R.B. & HOLDEN H. (1979) Natural killer cells as antitumour effector cells. J. natn. Cancer Inst. 62, 441. HERBERMAN, R.B., ORTALDO, J.R. & BONNARD, G.D. (1979). Augmentation by interferon of human natural and ACKNOWLEDGMENTS antibody-dependent cell-mediated cytotoxicity. Nature (Lond.), 277, 221. 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