Leukemia Research, Vol. 2, No. I, pp. 115.-126. 0145-2126/78/0301-0115502.00/0

~) Pergamon Press Ltd. 1978. Printed in Great Britain

ACUTE LYMPHOBLASTIC LEUKAEMIA ASSOCIATED ANTIGENmII.

ISOLATION AND PARTIAL CHARACTERISATION

ROBERT SUTHERLAND, JOHN SMART, PATRICK NIAUDET and MELVYN GREAVES

Imperial Cancer Research Fund, Lincoln's Inn Fields, London (Received 20 December 1977. Accepted 3 January 1978)

Abstract--An acute lymphoblastic ieukaemia (ALL) associated antigen has been isolated and purified from leukaemic cells and established ieukaemic cell lines. Under reducing conditions the antigenic determinants are identified on a single glycosylated polypeptide with an apparent molecular weight in 10% SDS polyacrylamide gels of IOO,OO0 daltons.

The cellular specificity of this molecular species parallels that of the ALL membrane antigen pre- viously defined by antibody binding criteria on whole cells (see previous paper). Leukaemic cell lines release the 100K molecule into the culture medium in a soluble form and one line (MOLT-4) produces these molecules but has no detectable cell surface expression.

Key words: Acute lymphoblastic leukaemia (ALL), ALL membrane antigen isolation

INTRODUCTION

IN THE past few years, a number of well-defined immunological markers have been developed to characterise the cell surface phenotypes of human leukaemias (reviewed in [I-3]). Heterologous trabbit) antisera have been raised against common (non-T, non-B) acute lymphoblastic leukaemia (cALL) [4-8] which react specifically with cALL cells, some cases of chronic myeloid ieukaemia in blast crisis, and a few undifferentiated acute leukaemias and lympho- mas (see previous paper).

In most circumstances the cALL associated anti- gen is operationally leukaemia specific and can be used as a sensitive and discriminating single cell detector of leukaemic cells: however, in foetal, neonatal and regenerating marrow a non-T, non-B lymphoid cell type can be detected which has a weak but definite expression of the ALL antigen [9, 10]. It is, therefore, possible that the antigen is a normal gene product of the haemopoietic precursor cells which are the cellular targets for ALL, and that the cALL antigen bearing structure plays a role in early haemopoiesis [10, Il] .

In view of the clinical and biological interest in this membrane antigen it was clearly of some

Correspondence to: Dr. M. F. Greaves, Membrane Immunology Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX.

Abbreviations: cALL, Common acute lymphoblastic leu- kaemia; FACS, Fluore~scence activater cell sorter; PAGE, Polyacrylamide gel electrophoresis; PBL, Peripheral blood lymphocytes; Ph ~, Philadelphia chromosome.

importance to attempt to isolate it and determin e its molecular nature. Previous preliminary data l~ad suggested that it was at least associated with glyco- protein [6]. Furthermore, the availability of purified cALL antigen might be expected to assist in ~he analysis of its function as well as providing an ideal immunogen for producing a monospecific diagnostic reagent.

In pursuing this aim we have taken advantage i of the recent availability of non-T, non-B ieukaemic lymphoblastoid cell lines which have a stable expression of the cALL antigen [12-15]. Some[ of the lines release a cALL antigen bearing molecule into the culture supernatant in a soluble form which substantially facilitates its purification.

MATERIALS AND METHODS Cells

Normal lymphocytes were obtained from heparini~e~l blood using Ficoll-isopaque density centrifugation Nat 1.077 g/cm ~, 400 g, 30 min at 20°). Tonsils were obtair~ed from routine tonsillectomies and thymuses from child(en (aged 2-8 years) undergoing cardiac surgery. Ribs w~re used as a source of normal adult bone marrow; th~se were obtained from patients receiving thoracic surgery who we have assumed to be haematologieally normal. Leukaemic cells were obtained from many different hospitals in the U.K. as listed in the previous paper.

Lymphoblastoid cell lines

Leukaemic cell lines were established, maintained and characterised (karyotypically, immunologically and enZy- matically) as previously described in detail [12-16]. The origin of the lines used in this study and their overall phenotypes are given in Table 1.

115

116 ROBEgT SUTHERLAND et al.

TABLE 1. CHARACTERISTICS OF LYMPHOBLASTOID CELL LINES USED

Designation Origin Phenotype §

NALM-I* Ph ~ positive CML blast crisis

Reht Non-T, non-B ALL, relapse

MOLT-4* T-ALL, relapse BRI-7:[: Normal

* Courtesy of Dr. J. Minowada. i" Courtesy of Dr. C. Rosenfeld. :[ Searle Laboratories. § See refs. [12-15] and previous paper for details.

[mi~1~Joflaorescence Single cell fluorescence on viable cells in suspension

was carried out using standard fluorescence micro- scopy and the Fluorescence Activated Cell Sorter (FACS- 1, Becton Dickinson, as described in refs. [2, 17] and in the previous paper). Leukaemic and normal cells were also smeared onto glass slides using a cytocentrifuge (Shandon Ltd.), fixed (in 90% ethanol, 10% glacial acetic acid for 10 min at 4°C) and stained with R76 f(ab') s anti-ALL or control f(ab'p (30 rain, room tem- perature) followed by goat anti-rabbit IgG f(ab'P anti- bodies which had been purified on affinity absorbents (Sepharose 4B-Rabbit f(ab'p IgG) and labelled with fluorescein isothiocynate.

Antisera preparation Anti-ALL. R75 anti-ALL was produced by immunisa-

tion of rabbits as described previously [4-6]. After heat inactivation of complement (60°/30 min) 5 ml was absorbed (at 1:3 vol of cells to serum for 2 h at 4 '~) with the following tissues: AB red cells (x 7), tonsil iympho- cytes ( x 5), CML ( x I), AML ( x 2), thymocytes ( x 3), normal adult bone marrow ( x 5). Data on the specificity of anti-ALL sera have been published previously [4--6]. R75 behaved the same as previous sera and its specificity is summarised in Table 2. The absorbed antiserum was desalted on medium grade Sephadex G,25 (Pharmacia) pre-equilibrated with 0.02M sodium phosphate pH 7.5 and the first peak collected. The serum proteins were passed through a column of DEAE-celluiose (DE 52 Watman) pre-equilibrated with 0.02M phosphate pH 7.5 and the unbound fraction collected. This procedure removes much of the soluble 'debris' from the antiserum which accumulates due to the extensive absorption it receives. A crude IgG fraction is thus produced, the main contaminants being haemoglobins and some euglobulins.

R76 anti-ALL. 5 x 108 common (non-T, non-B, R75 reactive) ALL cells were washed three times in RPMI 1640 (GIBCO) and resuspeoded in 100 ml of RPM[ supplemented with 2mM P.M.S.F. (Phenyl Methyl Sulphonyl Fluoride--a broad spectrum protease in- hibi tor-Sigma). The cells were incubated in a Falcon flask at 37°C for 18 h with gentle rocking. The cells were centrifuged at 500 g and the supernatant retained. The supernatant was concentrated to 10 ml and washed twice with PBS by pressure filtration using an Amicon PMI0

ALL +, Ia +, T-, E-, Smlg-

ALL*, Ia +, T-, E-, SmIg-

ALL-, Ia-, T +, E-~, Smlg- ALL-, ia% T-, E-, Smlg*

filter. The concentrated supernatant was emulsified in complete Freund's Adjuvant and a rabbit immunisep (s.c., i.m.) with half the sample. After 21 days the animal was boosted with the remainder. After 28 days a test bleed was taken and after heat inactivation of the serum, it was made specific for cALL by absorption with A M M L (x3) , CLL (x2) , AML (x i), Thymus (x I). This anti- serum thus requires far less absorption than, but appears to have the same cellular specificity as, R75 anti-ALL (see Table 2).

For some experiments a f(ab'P dimer was prepared from the lgG fraction of R76 anti-ALL and normal rabbit sera by pepsin digestion followed by absorption with Staphylococcal aureus (Cowans strain i) to remove residual undigested IgG and Fc fragments [20].

Anti-Ia serum [19] was prepared by immunising rabbits with precipitin lines formed in agarose gels between a detergent extract of human spleen and an anti-Ia (p28,33) serum which had itself been previously raised against isolated B cell glycoproteins (see refs. [20-22] for further details).

Pooled normal rabbit sera (as controls) were absorbed with tonsil lymphocytes prior to preparation of the IgG fraction.

Radio-labelling of leukaemic cell proteins. Cells were metabolically labelled with s6S methionine and 8H leucine and membrane proteins 'extrinsically' labelled with t~[ and 8H potassium borohydride (KBH,) (iso- topes from Radiochemicals, Amersham, England).

85S methionine labelling. 1 x 108 cells were washed three times with methionine-free Eagles E, medium and re- suspended in 15 ml of methionine-free Eagles plus 2 ml of dialysed foetal calf serum and 250 ~Ci 38S methionine. The cells were incubated at 37°C for 16 h and then centrifuged at 500x g for 10 rain. The culture super- natant was collected and the labelled cells were washed three times in cold methionine free E,. The cells were resuspended in 4 ml of extraction medium; 0.01M Tris pH 8, 150raM NaCI containing 0.5% of the non-ionic detergent Nonidet NP40 and 0.2ram P.M.S.F. After 15 min at + 2°C cell debris was removed by centrifugation at 500x g for 10 rain. The labelled culture supernatant and cell extract were ultracentrifuged at 100,000x g for I h at + 4°C and the supernatants collected and stored at - 20°C.

in most cases, the NP40 extracts were further fraction- ated by lentil lectin (Lens culinaris haemagglutinin, LcH)

ALL--antigen isolation ! 17

TAnLE 2. CELLULAR SPECIFICITY OF R75, R76 ANaa-ALL SERA

Normal cells

Thymocytes t Blood T cells, B cells, non-T, non-B 'lymphoid' cells

Tonsil T cells, B cells Blood monocytes, polymorphs, erythrocytes Bone marrow myeioblasts, normoblasts

Malignant cells AMML, AMonL, AML, CGL "/ Erythroleukaemia, Myeloma ~ B-ALL, CLL cALL + Thy-ALL - or + CGL in blast crisis - or + "AUL' - or +

See previous paper and refs. [4, 5, 7] for details.

affinity chromatography. LcH was coupled to Sepharose 4B (Pharmacia) by the technique of Hayman and Crumpton [23]. LcH-Sepharose binds glycoproteins containing exposed or terminal mannose and to a lesser degree glucose residues. The bulk of the radioactively labelled material in the NP40 lysates passes through the column with the extraction buffer. The bound material can be subsequently eluted with extraction buffer supplemented with 4 o~ alpha methyl D-glucoside (Sigma). The eluted fractions were dialysed three times against extraction medium containing 0.02 % NAN3.

SH leucine labelling. 2 × 10 ~ Reh cells were cultured in 50 ml of fresh medium containing leucine at 10% of its normal concentration, 5 ml dialysed foetal calf serum and 15 mCi H31eucine. After 24 h the cells were spun down and the supernatant retained and clarified at 10,000 x g for 20 rain.

aZSl labelling. 2 x 107 cells were labelled with 300 t~Ci 126I by the lactoperoxidase method [24]. After washing five times with cold PBS the labelled cells were extracted as above and the 100,000 × g supernatant retained.

3H potassium borohydride(KBH~). Cell surface glyco- proteins were labelled by the Galactose oxidase (G.O.) method [25, 26]. In principal the galactosyl residues of exposed glycoproteins are oxidised by G.O. and then reduced with tritiated potassium borohydride. In practise neuraminidase is added together with G.O. which removes sialic acid residues to expose more galactosyl residues

10' ALL cells were washed three times with cold PBS (containing Ca ~+ and Mg 2+) plus 2ram PMSF and re- suspended in 5 ml 150 units of neuraminidase and 25 units of galactose oxidase were added to the cell suspen- sion and incubated at 37~C for 45 rain. After washing three times in PBS+PMSF, galactosyl residues were reduced with 5 mCi of KB3H, for 30 min at 20~C. The cells were washed four times in PBS and extracted in I ml of extraction buffer and ultracentrifuged as above.

Immune complex formation and precipitation

All 3bS-methionine labelled culture supernatants and cell extracts were ultracentrifuged at 100,000 × g for I h immediately before use. 2 ml of 3sS labelled culture super-

natant (containing 2mM PMSF) were incubated o~,er- night with 10 ~1 R75 rabbit anti-ALL or 10 ~1 NR |gG at + 2°C. Immune complexes were isolated on Staphylo- coccus aureus (Cowan 1 strain). The cell wall of [this bacterium (SaCI) contains protein A which binds tol the Fc region of most IgG sub-classes [18]. It has been sh0wn that immune complexes are strongly adsorbed by ShCI even in the presence of excess IgG [27]. A 10% v/v suspension of SaCI was prepared and stored in aliq~aots at -70~C as described previously [27, 28]. FreShly thawed SaCI were washed three times in NET buffer!J27] containing 0.25 % Gelatin and 0.5 % NP40 in an Eppen- doff Microfuge (5412). Thirty p,l of this 10% suspension were added to each tube containing the immune cDm- plexes and rotated for 20' at 4°C. The bacteria Were [

pelleted and washed three times in NET containing 0i5 % NP40 and 0.25% gelatin. The bound complexes Were then eluted from the bacteria by boiling for 5 mir~ in 70 ~l of gel sample buffer 0 0 % glycerol, 2% sodium dodecyl sulphate, 0.05M Tris pH 6.8) plus 10 ~1 I M Dithiothreitol in 0.1% Bromophenol Blue indicator. The bacteria were pelleted again and the supernatant subjected to SDS-Polyacrylamide Slab Gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE), auto-reMio- graph.v and fhlorograpby

PAGE was carried out in slabs where 12 samples could be run in parallel in the buffer system of Laemmli [29]. The gel was stained overnight with Coomassie Blue solution (520 ml H20, 405 ml methanol, 53 ml glacial acetic acid, containing 125 mg Coomassie Blue) and destained for 2 h in the same solvent.

When the gel contained a2Sl-labelled extracts the gels were dried down and auto-radiography performed with Kodak X-Omat RP54 X-ray film. When the gelscontained /~ emitters, the gels were processed for fluorography [30]. The gel was washed twice in 100 ml dimethyl sulphoxid¢ (DMSO) for 30 min and then with 22.5°0 PPO [2,5 Diphenyloxazole) in 100 ml DMSO for 2 h. The gel is washed with running tap-water for I h and then dried down. The fluorographs were made on Kodak X-Omat XH I film.

118 ROBERT S ~ L A N D et al.

RESULTS

Early attempts to isolate cALL associated antigen from internally labelled ALL NP40 cell lysates were thwarted by relatively high background radioactivity. Much of the high background radioactivity could be removed from these culture supernatants (and to a lesser degree the cell extracts) by uitracentrifuging them immediately before use. The background on cell extracts could be further reduced by pre- absorbing the extracts with 10 ~1 of NR IgG and 50 ~1 of 10% ScCI before addition of the antiserum. However, in most experiments involving meta- bolically labelled NP40 extracts, they were subject to LcH-Sepharose affinity chromatography and the glycoprotein fractions produced little or no back- ground radioactivity on the fluorographs.

In preliminary experiments 'ALL associated antigen' was isolated from a~S methionine labelled Reh cells. Using a LcH sepharose eluted fraction of an NP40 extract, the antigen was identified as a single band in 10% PAGE, with an apparent molecular weight of 100K daltons (slot a, Fig. 1). Normal rabbit lgG does not precipitate this band (slot b) and the non-specific binding of labelled proteins to SaCI alone is negligible (slot d). Anti-Ia (p28,33) antiserum precipitated two bands of approximate molecular weight 28,000 and 33,000 (slot c). Since we had previously been successful in raising a potent anti-ALL serum to a tissue culture supernatant of cALL (R76 see Methods) it seemed possible that the ALL associated antigen could be isolated from the culture supernatants in which the cells were labelled. The antigen, when isolated from this source, is again a single glycosylated (i.e. lentil bound mannoside eluted) polypeptide of ~100K

(Fig. 2). In slot a the bound complexes (labelled with aH leucine) were eluted from SaCI under reducing conditions and in slot b, under non reducing conditions; no significant molecular weight changes are apparent indicating that the molecule, as released by cells, has no significant disulphide- bond-stabilized secondary structure. Anti-Ia serum precipitates two bands (slot c) from LcH purified 3H leucine labelled culture supernatants of Reh cells. The p28,33 polypeptides are more highly labelled with aH leucine compared with 35S methionine (cf. slot c Fig. 1) which reflects the aminoacid compo- sition of these structures (see [33]).

We have investigated several normal lymphoid tissues, leukaemias, leukaemia and non-leukaemia derived lymphoblastoid cell lines for the presence of the 100K polypeptide using 35S methionine labelled culture supernatants. The results are shown in Figs 3 and 4. In Fig. 3 R75 anti-ALL precipitated the 100K polypeptide from Reh cell (slot a) and MOLT- 4 (slot d)' culture supernatant. R75 does not pre- cipitate this component from peripheral blood lymphocyte (PBL) (slot b) or Bri 7 cell line (slot g) culture supernatants. Normal rabbit IgG controls are shown for PBL (slot c), MOLT-4 (slot f) and Bri 7 (slot k).

As shown in Fig. 4, R75 anti-ALL precipitates the looK component from common ALL (slot a - - diffuse), NALM-I (slot c) and Reh (slot i) culture supernatants. R75 anti-ALL does not precipitate this polypeptide from AML (slot e) or Thy-ALL (slot g) culture supernatants. Normal rabbit IgG controls are shown for cALL (slot b) NALM-1 (slot d), AML (slot f) and Thy-ALL (slot h). In addition, we cannot detect this polypeptide in

FIG. 1. Fluorography of SDS-PAGE-separated =6S-methionine-labelled, NP40 extracts of Reh cells after LcH-affinity chromatography and immune precipitation on 30/~i of 10% v/v SaCI. Slot a: Immune precipitation with 10 #! R75 anti-ALL;

b: Immune precipitation with I0 ~1 NR lgG; c: Immune precipitation with 10 ~,1 R anti-Ia (p28,33); d: Binding of labelled glycoproteins to SaCI alone; e: Fluorography of Reh culture supernatant-ALL-associated antigen after R76-Seph-4b affinity

chromatography. Marker proteins (arrows) are: ~ galactosidase (135,000 daltons), Phosphorylase a (94,000 daitons),

Lactoperoxidase (80,000 daitons), Catalase (60,000 daitons), Alcohol dehydrogenase (41,000 daitons), Carbonic anhydrase (29,000 daltons), Myoglobin (I 7,200 daltons).

Fie. 2. Fiuorography of SDS-PAGE-scparated aH leucine labelled Reh culture supernatant after immune precipitation on SaCI.

slot a: 2 ml of Reh supernatant with 10 ~,1 R75 and immune precipitation on SaCI. slot b: as slot a except elution from SaCI under non-reducing conditions on 7.5% PAGE.

slot c: LcH affinity chromatography purified Reh culture supernatant+ 10 ~,1 R anti-la serum and immune precipitation on SaCI on 10% PAGE.

Marker proteins are as Fig. I.

~0

ALL--antigen isolation

culture supernatants of thymocytes or tonsillar lymphocytes, but R75 anti-ALL does precipitate it from HPB-ALL (a Thy-ALL cell line--ref. [13]) and a case of chronic myeloid leukaemia in ' lymphoid' (anti-ALL reactive) blast crisis culture supernatants (data not shown).

R75 anti-ALL also precipitates the looK poly- peptide from NP40 extracts of cALL cells which had been extrinsically labelled with either KB3H4 (Fig. 5) or 12sI (Fig. 6).

R75 anti-ALL absorbed with cALL cells was no longer able to precipitate the looK antigen .from a~S methionine labelled Reh cell culture super- natants, or NP40 extracts of 1251-1actoperoxidase labelled cALL cells or Reh cells. These results suggest therefore that the cell surface and culture supernatant antigen are cross-reactive and probably the same, or part of the same, molecular structure.

R76 anti-ALL, in contrast to R75 anti-ALL (see Methods), was not very efficient at precipitating the 100K polypeptide on SaCI. This may be due to the relevant antibodies in this serum having a low affinity for the Protein A binding sites on SaCI. To confirm that R76 anti-ALL does in fact react with the 100K polypeptide R76 lgG has been coupled to Sepharose-4B, and 3~S methionine labelled Reh cell line culture supernatant absorbed on to it. After elution with 2°0 SDS the 100K ALL associated antigen can in fact be easily detected (under reducing conditions), see Fig. 1, slot e. A minor component is also present and has a molecular weight of approximately 38,000 daltons.

121

DISCUSSION

The existence of a membrane antigen in human acute ieukaemia identified by antisera raised against the common, or non-T, non-B subtype of ALL (cALL) has been previously documented [4--9]. The antigen is shared by the majority of cALL and some Ph 1 positive CML in blast crisis (and Ph 1 positive acute leukaemias with no preceding chronic disease), acute 'undifferentiated' leukaemias and some lymphomas (see preceding paper). In this study we have been able to isolate a single glycosylated polypeptide, with an apparent molecular weight of IOO,000 daltons, from both whole cell extracts and from the culture supernatants of leukaemic cell lines which reacts specifically with anti-ALL antibodies. The molecule is metabolically labelled with ass methionine and aH leucine and is clearly a product of the leukaemic clone. The same polypeptide can be demonstrated on the cell surface by radi'o- iodination, or 3H galactose labelling.

Uncultured leukaemic cells and cells from estab- lished leukaemic cell lines appear to produce an antigen which is identical in terms of immunological cross-reactivity and general biochemical charac- teristics.

The specificity of the expression of the anti-ALL serum defined 100K polypeptide parallels that described for cell surface immunofluorescence assays (see previous paper). An unexpected exception to this was, however, afforded by the line MOLT-4, a Thy(or T)-ALL cell line [16]. Several Thy-ALL cell

FIG. 3. Fluorography of SDS-PAGE separated a~S-methionine labelled cell culture supernatants after immune precipitation on SAC1.

slot a: 2 ml Reh cell culture supernatant with 10 t,l R75 and immune precipitation on SaCi. b: 2 ml P.B.L. supernatant +R75.

c: 2 ml P.B.L. supernatant + N R IgG. d: 2 ml MOLT..4 supernatant+R75.

f~. 2 ml MOLT-4 supernatant+ NR IgG. g: 2 ml Bri-7 su0ernatant+R75.

h: 2 ml Bri-7 supernatant + NR IgG. Marker proteins are as Fig. 1.

FIG. 4. Fiuorography of SDS-PAGE separated 3~S methionine labelled cell culture supernatants after immune precipitation on SaCI.

Slot a: 2 ml of cALL culture supernatant with 10 t~l R75 and immune precipitation on SaCI. b: 2 ml cALL supernatant + NR Ig. c: 2 ml NALM-I supernatant+R75.

d: 2 ml NALM-I supernatant+NR IgG. e: 2 ml AML supernatant +R75.

f: 2 ml AML supernatant +NR IgG, g: 2 ml Thy-ALL supernatant + R75.

h: 2 ml ThyoALL supernatant + NR lgG. i: 2 mi Reh supernatant + R75

Markers as Fig. 1.

.122 R O ~ T Strrrmm.ar~na et al,

5 B

4 - -

T 0 3 m

x

E 0 . (J

2

5 I0 15 20 25 :30 35 40 Froct ion number

Fxo. 6. Electrophoretic mobility of ALL-associated Ag on a 10~ PAGE-SDS gel after LPO ~"I labelling and extraction with i ~ NP40. Immune precipitation with R75 anti-ALL II, or NR lgG and 30/~1 of SaCI. Gel was cut into 2 mm slices and counted in LKB 1280 gamma counter. Markers

(arrowed) are as Fig. 1.

lines have a weak but definite expression of the ALL antigen (e.g. HPB-ALL) [14, 15]. MOLT-4 releases the 100K polypeptide into the culture medium but has no demonstrable cell surface ALL antigen [14]. These cells do, however, show cytoplasmic fluores- cence (unpublished observations, MFG).

The molecule derived from the culture super- natant runs in the same position in 10% PAGE under both reducing and non-reducing conditions suggesting that it is a single molecule with no disulphide bonded subunits and probably minimal or no intrachain disulphide bonds. This does not necessarily mean that the cell surface associated looK molecule is also in its native state a single molecule. Indeed, in preliminary experiments, we find that an unreduced cell surface glycoprotein bearing the ALL antigen runs at approximately 135K. Thus, the ALL antigen bearing molecule may be disulphide linked to a smaller polypeptide on the cell surface. This observation plus additional studies on iectin binding activity of isolated proteins from

ALL ceils is the subject of a later report. ALL cells excepting those within the Thy-ALL

subgroup react with anti-Ia serum [19, 31, 32]. The cellular selectivity of la-like antigen and cALL antigen in leukaernia are very different (see previous paper) and it is clear from the studies reported here that the two antisera react with separate molecules. Anti-Ia serum identifies on B cells a non-covalently linked complex consisting of two polypeptides of 28,000 and 33,000 daltons [33, 34]. The anti-Ia serum identifies two polypeptides also of 28,000 and 33,000 daltons in ALL cells (see Results). The ' la- like' antigen on acute leukaemic cells (ALL, AML) and B cells is completely cross-reactive immuno- logically [19, 22] which further suggests that very similar molecules are produced by these different normal and malignant cell types. The 33,000 dalton polypeptide appears to be coded for by chromosome 6 and is probably the product of HLA, D locus genes [33, 35]. The apparent identity of the qa ' structure on ALL, AML and B cells could, however,

123

Fro. 5. Fluorography of SDS-PAGE separated SH (Galactose oxidase) labelled NP40 ~xtracts of cALL cells after immune precipitation on 30 ~,1 of 10% SaCI.

Slot a: Immune precipitation of 250 ~1 of NP40 extract with 10 ~1 N R IgG. b: 50 ~1 of NP40 extract.

c: as slot a except 10 ~1 R75 ami.ALL. Marker proteins are as in Fig. 1.

ALL--antigen isolation 125

mask an impor tan t microheterogenei ty if different Ia (D) loci genes were active in different lymphoid and myeloid cell types.

Acknowledgements--We are very grateful to Drs. J. Minowada and C. Rosenfeid for giving us leukaemic

cell lines which were established in their respective laboratories. We thank W. Verbi for his technical assistance and Dr. K. Welsh for supplying the anti-la (p28,33) serum. This work was supported by the Leukae- mia Research Fund (R.S.) and the Imperial Cancer Research Fund and DGRST Paris (P.N.).

REFERENCES

1. THIERFELDER S., RODT H. & THIEL E. (Eds.) (1976) Immunological Diagnosis o f Leukaemias and Lymphomas. Springer, New York.

2. GREAVES M. F. (1975) Clinical applications of cell surface markers. Prog. Haemat. 9, 255. 3. COOPER M. D. ~¢ SELIGMANN M. (1977) B and T lymphocytes in immunodeficiency and iymphoproliferative

diseases. In B and T Cells in Immune Recognition (LOOR F. & ROELANTS G. E., Eds.) p. 377. John Wiley, London. 4. GREAVES M. F., BROWN G., RAPSON N. & LISTER T. A. (1975) Antisera to acute lymphoblastic ieukaemia cells.

Clin. Immun. lmmunopath. 4, 67. 5. BROWN G., CAPELLARO D. ~ GREAVES M. F. (1975) Leukaemia-associated antigens in man. J. Rain. Cancer Inst.

55, 1281. 6. BROWN G., HOGG N. & GREASES M. F. (1975) Candidate leukaemia specific antigen in man. Nature 258, 454. 7. GREAVES M. F. (1978) Immunodiagnosis of leukaemia. In lmmunodiagnosis o f Cancer (HERaERMAN R., Ed.),

Marcel Dekker, New York (in press). 8. GREAVES M. F., JANOSSY G., ROBERTS M., RAPSON N. T., ELLIS R. B., CHESSELLS J., LISTER T. A. & CATOVSKY D.

(1976) Membrane phenotyping: diagnosis, monitoring and classification of acute 'lymphoid' leukaemias. In Immunological Diagnosis o f Leukaemias and Lymphomas (THIER~LDER S., RODT H. & THIEL E., Eds.) p. 61. Springer, New York.

9. GREAVES M. F., JANOSSY G., FRANCIS G. & MINOWADA J. (1978) Membrane phenotypes of human leukaemic cells and leukaemia cell lines: clinical correlates and biological implications in Cell Proliferation 5 (CLARKSON g., TILL J. & MARKS P. A., Eds.) (in press).

10. GREASES M. F. & JANOSSY G. (1978) Patterns of gene expression and the cellular origins of human leukaemias. Biophys. Bioehim. acta (Reviews in Cancer) (in press).

11. GREAVES M. F. (1978) Cell surface structures, differentiation and malignancy in the haemopoietic system. In Cellular Interaction (CURTIS A., Ed.) Cambridge Univ. Press (in press).

12. MINOWADA J., TSUBOTA T., GREAVF_.S M. F. & WALTERS T. R. (1977) A non-T, non-B human leukaemia cell line (NALM-I): establishment of the cell line and presence of icukaemia-associated antigens. J. natn. Cancer Inst. 59, 83.

13. MINOWADA J., JANOSSY G., GREAVES M. F., TSUBO'rA T., SRIVASTXVA B. I. S., MORIKAWA S. & TA'rSUMI E. (1978) The expression of acute lymphoblastic leukaemia antigen in human leukaemia-lymphoma cell lines. J. natn. Cancer Inst. (in press).

14. JANOSSY G., GREASES M. F., CAPELLXRO D., MINOWADA J. & ROSENVELD C. (1977) In Proc. 25th Colloquium on Protides o f the Biological Fluids, Brugge, p. 591 (PETERS H., Ed.) Pergamon Press, Oxford.

15. ROSENFELD C., GOUTNER CHOOUET C., VENUAT A. M., KAYIBANDA, Plco J. L. & GREASES M. F. (1977) Pheno- typic characterisation of a unique non-T, non-B acute lymphoblastic leukaemia cell line. Nature 267, 841.

] 6. MINOWADA J., OHNUMA T. & MOORE G. E. (1972) Rosette-forming human lymphoid cell lines--I. Establishment and evidence for origin of thymus-derived lymphocytes. J. hath. Cancer Inst. 49, 891.

17. GREAVES M. F., CAPELLARO D., BROWN G., REVESZ T. & JANOSSY G. (1976) Analysis of human leukaemic cells using cell surface binding probes and the Fluorescence Activated Cell Sorter. In Modern Trends in Human Leukaemia I I (NET8 R., GALLO R. C., MANNWEILER K. & MOLONEY W. C., Eds.) p. 243. J. F. Lehmanns, Munich.

18. KRONVALL G., SEAL U. S., F1NSTAO J. & WILLIAMS R. C. (1970) Phylogenetic insight into evolution of mam- malian Fc fragment of ~'G globulin using staphylococcal protein A. J. Immun. 104, 140.

19. SCHLOSSMANN S. F., CHESS L., HUMPHREYS R. E. & STROMINGER J. L. (1976) Distribution of la-like molecules on the surface of normal and leukaemic ceils. Proc. hath. Acad. Sci. U.S.A. 73, 1288.

20. WELSH K. ~ TURNER M. J. (1977) Preparation of antisera specific for human B cells by immunisation of rabbits with immune complexes. Tissue Antigens 8, 197.

21. JANOSSY G., GREAVES M. F., SUTHERLAND R., DURRANT J. & LEWIS C. (1977) Comparative analysis of membrane phenotypes in acute lymphoid leukaemia and in lymphoid blast crisis of chronic myeloid leukaemia. Leukae- mia Res. 1,289.

22. JANOSSY G., GOLDSTONE A. H., CAPELLARO D., GREAVES M. F., KULENKAMPFF J., PIPPARD M. & WELSH K.

126 ROBERT SUTHERLAND e/' a/.

(1977) Differentiation linked expression of p28,33 (Ia-like) structures of human leukaemia cells. Br. J. Haemat. 37: 391.

23. HAYMAN M. J. & CRUMPTON M. J. (1972) Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culinaris phytohaemaglutinin. Biochem. biophys. Res. Commun. 47, 923.

24. MARCHALONXS J. J., CONE R. E. & SANTER V. (1971) Enzymic iodination. A probe for accessible surface pro- teins of normal and neoplastic lymphocytes. Biochem. J. 124, 921.

25. GAHMSERG C. G. & HAgOMORI S. (1973) External labelling of cell surface galactose and galactosamine in glycolipid and glycoprotein of human erythrocytes. J. biol. Chem. 248, 431 I.

26. ANDERSON L. C., GAHMBERG C. G., NILSSON K. & WIGZELL H. (1977) Surface glycoprotein patterns of normal and malignant human lymphoid cells--l. T cells, T blasts and leukaemic T cell lines, b~t. J. Cancer 20, 702.

27. KESSLER S. W. (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. [mmtm. 115, 1617.

28. CULLEN S. E. & SCHWARTZ B. D. (1976) An improved method for isolation of H~ and Ia alloantigens using immunoprecipitation induced by Protein A bearing staphylococci. J. lmmun. 117, 136.

29. LAEMMLt U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T~. Nature 227, 680.

30. BONNER W. M. & LASKEY R. A. (1974) A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Fur. J. Biochem. 46, 83.

31. WtNCH~TER R. J., RoSS G. D., JAROWSKI C. I., WANG C. Y., HALPER J. • BROXMEYER t'[. (1977) Expression of la-like antigen molecules on human granulocytes during early phases of differentiation. Proc. hath. Acad. Sci. 74, 4012.

32. BILLING R., RAFIZADEH B., DREW I., HARTMAN G., GALE R. & TERASAKI P. (1976) Human B lymphocyte antigens expressed by lymphocytic and myelocytic leukaemia cells--l. Detection by rabbit antisera. J. exp. Med. 144, 167.

33. SPRINGER T. A., KAUFMAN J. F., TERHORST C. & STROMINGER J. L. (1977) Purification and structural charac- terisation of human HLA-linked B cell antigens. Nature 268, 213.

34. SNARY D., BARNSTABLE C. J., BODMER W. F., GOODFELLOW P. N. & CRUMPTON M. J. (1977) Cellular distribution, purification and molecular nature of human Ia antigens. Scand. J. lmmun. 6, 439.

35. GIPHART M. J., KAUFMAN J. F., FUKS A., ALBRECHTSEN D., SOLHEIM B. G., BRUNING J. W. ~¢. STROMINGER J. L. (1977) H LA-D associated alloantisera react with molecules similar to Ia antigens. Proc. natn. Acad. Sci. U.S.A. 74, 3533.