myeloid leukaemia-specific antigen binds to normal haemopoietic progenitor cells and inhibits colony formation in vitro

Leukemia Research Vol. 12, Nos. 11/12, pp. 929-939, 1988.

0145-2126/88 $3.00 + 0.00 Pergamon Press plc

Printed in Great Britain.

A M O N O C L O N A L A N T I B O D Y TO A H U M A N MAST C E L L / M Y E L O I D L E U K A E M I A - S P E C I F I C A N T I G E N BINDS TO N O R M A L H A E M O P O I E T I C P R O G E N I T O R CELLS A N D INHIBITS COLONY F O R M A T I O N IN V I T R O ANTONY C. CAMBARERI, LEONIE K. ASHMAN, STEPHEN R. COLE and A. BRUCE LYONS* Department of Microbiology and Immunology, The University of Adelaide, Adelaide, South Australia

(Received 20 April 1988. Revision accepted 5 August 1988) Abstract--An antigen identified by murine monoclonal antibody YB5. B8 has previously been detected only on acute non-lymphoblastic leukaemia (ANLL) cells and tissue mast cells. We now report that the YB5.B8 antigen is present on a minor population (up to 3%) of normal bone marrow mononuclear cells which overlaps the set of progenitor cells capable of forming haemopoietic colonies in vitro. The results indicate that the antigen is a normal haemopoietic progenitor cell marker which is selectively retained on mast cells during maturation, and that leukaemias which express the antigen are not necessarily committed to the mast cell lineage. Furthermore, the antibody was capable of partially inhibiting the formation of haemopoietic colonies in vitro, indicating an important functional role for the antigen. This is consistent with the observation, reported in the accompanying paper, that expression of the YB5.B8 antigen is strongly correlated with poor response to therapy in patients with ANLL.

Key words: Acute lymphocytic leukemia, chronic mylocytic leukemia, chromosome, haploidy, hyperploidy, oncogene, bcr.

In our earlier work we were unable to detect binding of YB5.B8 to normal bone marrow populations [1]. We now report that a small proportion (up to 3%) of mononuclear cells from normal bone marrow express the YB5.B8 antigen at low intensity, and that at least some of these cells are haemopoietic progenitors (CFU-C) capable of giving rise to colonies in vitro. In addition we present data showing that inclusion of the antibody in the C F U cultures results in inhibition of colony formation indicating an important functional role for this antigen in haemopoietic differentiation.

INTRODUCTION MONOCLONAL antibody YB5.B8 [1] was raised against peripheral blood blast cells from a patient with A N L L of i m m a t u r e p h e n o t y p e according to the F A B classification [2]. The antibody identifies a 150kD (glyco)protein [3, 4] which is present on blast cells f r o m approximately 30% of patients with A N L L at presentation. In the accompanying p a p e r [5] we report that expression of the YB5.B8 antigen is strongly associated with p o o r prognosis. The antigen is absent from normal peripheral blood cells [1] and in tissues has been detected only on mast cells [4] of both mucosal and connective tissue types [6]. *Present adress: Division of Human Immunology, Institute of Medical and Veterinary Science, Adelaide, South Australia. Abbreviations: ANLL, acute non-lymphoblastic leukaemia; FAB, French-American-British; MoAb, monoclonal antibody; CFU-C, colony-forming unit in culture; MNC, mononuclear cells; HPCM, human placental-conditioned medium; PHA-LCM, phytohaemagglutininstimulated lymphocyte conditioned medium; PBS, phosphate buffered saline; FCS, foetal calf serum; G, granulocyte; M, monocyte; Eo, eosinophil. Correspondence to: Dr L. K. Ashman, Department of Microbiology and Immunology, The University of Adelaide, GPO Box 498, Adelaide, 5001 South Australia.

MATERIALS AND METHODS Normal bone marrow cells were obtained from allogeneic transplant donors at the Royal Adelaide Hospital. Umbilical cord blood specimens were obtained at normal delivery from patients of the Queen Victoria Hospital, Adelaide. Specimens were obtained with informed consent, and approval of the relevant ethics committees. Mononuclear cell fractions were prepared and, in some instances, cryopreserved as previously described [7]. A leucocyte fraction was prepared from a normal bone marrow aspirate by sedimentation of the erythrocytes with 10% v/v of a 6% solution of Dextran T70 (Pharmacia) in isotonic saline for 1 h at room temperature. 929

930

ANTONYC. CAMBARERIet al.

Demonstration of YB5.B8-positioe cells in normal bone marrow Fresh mononuclear cells from normal bone marrow were labelled with YB5.B8, or control antibodies 1B4 (antiHLA Class I [3]), BI-3C5 (progenitor cell specific antibody [8, 9]), or Sal-2 (negative control, cf [10]), then fluoresceinlabelled affinity purified F(ab')2 sheep antibody to mouse immunoglobulins (Silenus, Australia) as previously described [10]. All monoclonal antibodies are of IgG1 type and all were used as culture supernatants except BI-3C5 which was a 1/500 dilution of ascites. Labelled cells were examined using a FACS analyser at Hinders Medical Centre, Adelaide.

Growth of hemopoietic colonies in vitro The method employed was essentially that of Metcalf [11]. MNC from fresh or cryopreserved cord blood or bone marrow were plated in a final volume of 1 ml in 0.3% agar containing HPCM or PHA-LCM as a source of colony stimulating factors. Details are given elsewhere [12]. Bone marrow cultures contained 1.5 x 104 cells/plate and cord blood cultures contained 2.5 x 104 or 5 x 104 cells/plate as indicated. After 14 days, culture plates were fixed and the agar discs dried onto slides. Colonies were stained with Luxol Fast Blue MBS (eosinophils), a~-napthyl acetate esterase (macrophages) and chloroacetate esterase (neutrophils) [cf 12]. In most cases total colony counts were determined prior to fixation using a dissecting microscope with darkfield illumination.

Isolation of YB5.B8-positive cells by immune rosetting Human erythrocytes were coated with affinity-purified goat antibody to mouse immunoglobulins (prepared in this laboratory) using chromic chloride as described by Goding [13]. Immune rosetting was carried out essentially as described by Parish and McKenzie [14]. MNC from cord blood or bone marrow were incubated with monoclonal antibody (100 Itl of sterile culture supernatant/7.5 × 105 cells, total volume 200 ~tl) for 60 min on ice, then washed 3 x in ice cold RPMl-1640 medium (Gibco) + 10% FCS. Coated erythrocytes (100~tl/7.5 x 105 cells) were then added and the cells pelleted by centrifugation at 200 g, followed by incubation for 60 rain at 4°C. The cells were gently resuspended and, for analytical purposes, stained with crystal violet [cf 14] and aliquots placed on a haemocytometer for counting. For preparative purposes, the rosetted cells were layered onto Ficoll-Paque (Pharmacia) gradients and centrifuged at 400 g for 25 min at room temperature. Unrosetted cells were recovered from the interface and rosetted cells were recovered from the pellet. Cells from both fractions were washed 3 x with RPM11640 + 10% FCS at room temperature and were plated in semi-solid agar as described above for the enumeration of CFUs. Fifteen replicate plates were used per fraction; thus each plate contained the rosetted or unrosetted cells derived from 5 x 104 cord blood MNC or 1.5 × 104 bone marrow MNC. Where sufficient material was available, additional cells were rosetted to enable the yield of cells in the two fractions to be determined.

Inhibition of colony formation by YB5.B8 Monoclonal antibodies YB5.B8, 1B4 (anti-HLA class 1) and Sal-2 (negative control) were purified from ascites or culture supernatant by affinity chromatogaphy on Protein A-Sepharose [15]. Azide was removed by extensive dialysis

against PBS then serum free medium. FCS was added to 10% V/V and the solutions were sterilized by filtration through 0.2 p.m filters (MiUipore Millex-GV). Antibodies were added to give 0.5-10 Ixg/ml final concentration (10-100 ixl/plate) to CFU assays (cf. above). Cord blood MNC were depleted of monocytes by adherence to plastic tissue culture dishes for 1 h at 37°C, and in some cases, as indicated, were also depleted of T cells by rosetting with sheep erythrocytes [16]. Approximately 50% of the cells were recovered in the non-adherent, unrosetted fraction. These were plated in 5 or 6 fold replicate at 2.5 × 104/plate in the CFU assay. RESULTS

Analysis of the binding of YB5.B8 to normal bone marrow cells by flow cytometry A leucocyte fraction (obtained by dextran sedimentation) and a M N C fraction were prepared from a fresh normal bone marrow specimen. The cells were labelled with YB5.B8, BI-3C5, 1B4 (anti-HLA Class I) or Sal-2 (negative control IgG1) monoclonal antibodies, then with a fluoresceinated second antibody. Samples were analysed by flow cytometry at high sensitivity, such that the fluorescence of the sample labelled with the anti-HLA antibody was off scale (of. Fig. 1A). No specific binding of YB5.B8 could be detected when the whole leucocyte fraction was analysed (data not shown). However, comparison of the fluorescence histograms for the M N C fraction labelled with YB5.B8 and the control antibody Sal-2 indicated that a small proportion of the cells were weakly positive but these were not resolved from the negative p e a k (Fig. 1A). A small proportion of the cells bound BI-3C5. These were more clearly separated from the negative peak than the YB5.B8positive cells (Fig. 1A). BI-3C5, like MY-10 [17], is an anti-CD34 antibody which is known to bind to myeloid progenitor cells [18]. In order to examine whether the cells binding YB5.B8 could also be the myeloid progenitors, we gated the population based on 2-dimensional light scatter characteristics, to enrich for the BI-3C5 positive cells as shown in Fig. lB. The gated population contained 27% BI-3C5 positive ceils and 26% YB5.B8 positive cells, which m a d e up respectively 2.1% and 2.0% of the total M N C population.

Identification of YBS. B8-positive cells in normal bone marrow by immune rosetting The bone marrow specimens were examined for YB5.B8-positive cells by analytical immune rosetting. Typical rosettes obtained with YB5.B8 and the anti-Class I antibody 1B4 are shown in Fig. 2. The m a r r o w specimen examined by flow cytometry gave 2.8% of the M N C population cells rosetting with YB5.B8, in good agreement with the immuno-

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FIG. l. Identification of YB5.B8-positive cells in normal bone marrow by flow cytometry. (A) Fluorescence histograms for ungated bone MNC labelled with YB5.B8 and control antibodies. (B) Scatter characteristics of the bone marrow MNC with the gated population outlined, and fluorescence histograms for this population.

932

ANTONY C. CAMBARER1 et al.

TABLE 1. ENRICHMENT OF C F U s BY IMMUNE ROSETrING

A.

MNC from bone marrow (1 expt) or cord blood (2 expts) were separated into positive and negative fractions by immune rosetting using MoAbs as indicated. Results shown are mean ± S.E. from three independent experiments. HPCM was used as growth factor source. % yield of CFU in positive fraction (mean -+ S.E.; 3 expts) G

M 37 89 83 12

Eo

--- 5 ± 9 ± 8 ± 11

48 87 88 13

-+ 21 -+ 10 ± 3 -+ 11

GM

Total

64 -+ 7 82 ± 14 88--+ 7 13 ± 10

46 --+ 6 85 --- 12 86--- 6 13 ± 12

YB5.B8 BI-3C5 FMC4 Sal-2

49 ± 11 83 -+ 14 90- 7 18 --- 16

B.

Frequency data for various types of CFU in the positive fraction derived from two of the above experiments where total cell recoveries were determined. Frequency of CFU in positive fraction (%) G

M

Eo

GM

Total

Expt 1 YB5.B8 FMC4 Sal-2 unfrn

0.38 0.27 0.10 0.10

0.69 0.67 0.17 0.21

0.34 0.21 0.04 0.05

0.50 0.27 0.10 0.08

1.9 1.4 0.4 0.4

Expt 2 YB5.B8 BI-3C5 FMC4 Sal-2

0.23 0.29 0.27 0.10

0.33 0.55 0.48 0.14

0.07 0.07 0.07 0.02

0.17 0.18 0.16 0.05

0.8 1.1 1.0 0.3

Enrichment for haemopoietic progenitor cells by immune rosetting with YB5. B8

fluorescence result. T h e o t h e r specimens gave 1% a n d < 1 % of M N C s rosetting with YB5.B8. N o rosettes were o b t a i n e d w h e n the negative control a n t i b o d y , Sal-2, was used a n d 75% of cells were r o s e t t e d in the p r e s e n c e of 1B4. I n o n e e x p e r i m e n t with cord b l o o d M N C , 0.7% of the cells rosetted with Y B 5 . B 8 .

I m m u n e rosetting was used to separate the positive a n d n e g a t i v e cell fractions from cord b l o o d a n d b o n e m a r r o w for Y B 5 . B 8 a n d c o n t r o l a n t i b o d i e s (BI-3C5, Sal-2 a n d the a n t i - H L A class II M o A b F M C 4 [19]) prior to p l a t i n g in the C F U assay. T h e results of three

TABLE 2. T H E EFFECT YB5.B8 AND CONTROL ANTIBODIES WHEN INCLUDED IN THE C F U ASSAY

Antibody added None YB5.B8 1B4 Sal-2

Relative colony yield (%) Eo GM

G

M

100 66 -+ 2 100 -+ 22 90 - 14

100 58 -+ 8 101 ± 13 102 --- 5

100 42 -+ 18 116 - 25 78 -+ 19

100 54 ± 18 77 ± 26 91 --- 11

Total 100 54 - 7 95 -+ 5 96 --- 4

The results are the means ± S.E. derived from three separate experiments. All antibodies were used at a final concentration of 2 g,g/ml. Monocyte-depleted cord blood MNC were cultured at 5 x 104/plate (five replicates in each experiment) with PHA-LCM (two expts) or HPCM (one expt) as growth factor source. Total colony counts were determined prior to fixation and staining for individual colony types.

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C FIG. 2. Identification of YB5.B8-positive cells in normal bone marrow by immune rosetting. Bone marrow MNC were incubated with monoclonal antibody Sal-2 (negative control) (A); YB5.B8 (B); or 1B4 (anti-HLA Class I) (C); then with human erythrocytes coated with goat antibody to mouse immunoglobulins. Leucocytes were then stained with crystal violet.

933

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Haemopoieticprogenitor cell antigen separate experiments (two using cord blood, one using bone marrow) are summarized in Table 1. Comparable results were obtained irrespective of the cell source. In all cases the total yield of CFUs in the positive plus negative fractions was similar to that obtained with unfractionated cells, indicating that non-specific losses and cell interactions did not influence the results. In two of the three experiments (both using cord blood) sufficient cells were available for enrichment data to be calculated (Table 1B). An average of 46% of all CFUs was recovered in the YB5.B8 positive fraction compared with 85% and 86% with antibodies to CD34 and Class II antigens which are known to be present on the majority of CFUs [20]. YB5.B8 was more efficient in isolating CFU-GM (64% in the positive fraction) than single lineage progenitors. Significantly more CFUs of all types were recovered in the YB5.B8 rosetted fraction than in the "rosetted" fraction obtained with the negative control (Sal-2) antibody in all cases. The presence of CFUs in the rosetted fraction with Sal-2 was not due to true rosettes but was due to leakage through the gradient of non-rosetted cells from the two cord blood specimens which had been cryopreserved. This may also in part account for why the degree of enrichment of CFUs is less than might be expected from the analytical rosetting results (see below). Although the yield of CFUs prepared by rosetting with YB5.B8 was lower than that obtained with BI3C5 and FMC4, the level of enrichment was comparable (3-5 fold for total CFU). Enrichment of CFU-GM and CFU-Eo was greater than for CFU-G and CFU-M in both experiments (Table 1B).

Effect of YB5. B8 on haernopoietic colony growth CFU cultures of cord blood, in the presence of HPCM or PHA-LCM yielded 20-110 colonies per 5 x 104 MNC at day 14 of culture depending on the cell specimen and the source and level of growth factor. The ratio of colony types, G : M : Eo : GM, was approx. 20 : 60 : 14 : 6 when PHA-LCM was used and 38 : 47 : 5 : 10 when HPCM was used. Thus PHALCM favoured M and Eo colony growth, while HPCM gave better yields of G and GM colonies. Both types of stimulus were used in these experiments, and gave similar results for YB5.B8 inhibition. In the initial experiments, purified YB5.B8 and control IgG1 MoAbs which bound (1B4) or did not bind (Sal-2) to the cells were included in the CFU assays at a final concentration of 2 Ixg/ml. Monocytedepleted cord blood MNC were plated with slightly sub-optimal levels of PHA-LCM or HPCM (sufficient to give approx. 70% of the maximum colony

935

number). The results (PHA-LCM, two experiments; HPCM, one experiment) are shown in Table 2. The inclusion of YB5.B8, but not 1B4 or Sal-2, in the assay led to a reproducible reduction in the yield of all types of colonies. In some cases the size of colonies, especially GM colonies was reduced. The effect of varying the concentrations of antibody and of growth factors was examined. Table 3 summarizes the results of three experiments in which different amounts of MoAbs were added to the cultures. For simplicity only total colony data are shown but similar results were obtained for all colony types. At least 1 ~tg/ml purified YB5.B8 was required for inhibition to be reproducibly observed and the level of inhibition never exceeded 50%. Earlier experiments (not shown) using YB5.B8 culture supernatants indicated that up to 50% inhibition of colony formation could be obtained with as little as 0.4 ~tg antibody/ml, and it is possible that some inactivation of the antibody occurred on purification. Results of a preliminary experiment in which the level of growth factor (HPCM) was varied, using antibodies at 2 ~tg/ml, are summarized in Table 4 and indicate that the extent of inhibition of colony formation by YB5.B8 was not simply related to the degree of saturation with growth factor. To determine whether the effect of YB5.B8 was due to its direct action on CFUs or was mediated by T cells in the cord blood MNC population, the cells were fractionated by E-rosetting as well as adherence prior to plating. A significant inhibitory effect of YB5.B8 was still observed with populations depleted of T cells as well as monocytes (cf Table 4). However it appeared to be reduced to approximately 30% inhibition compared with up to 50% inhibition in cultures containing T cells. In another experiment a direct comparison of T-depleted and non-depleted cultures was carried out (Table 5). In T-depleted cultures YB5.B8 (1 ~tg/ml; a slightly suboptimal level) brought about a 22% reduction in total colony yield, compared with 37% for the T-containing cultures. Whether the inhibition in T-depleted cultures can be attributed to a small number of residual T cells remains to be determined. DISCUSSION The antigen indentified by MoAb YB5.B8 has a highly unusual distribution, having been detected only on myeloid leukaemic cells and tissue mast cells [1, 4, 6]. The antigen appears to define a distinct biological type of ANLL, since patients whose cells expressed this marker in general responded poorly to standard ANLL therapy [5]. Since mast cells are bone-marrow derived [21] it was possible that

936

ANTONY C. CAMBARERI et al. TABLE 3. DOSE-DEPENDENCE OF THE INHIBITION BY YB5.B8 OF COLONY FORMATION

Total colony yield (% of control) Conc. (/~g/ml) Expt 1

10 5 2

Expt 2

2 1 0.5

Expt 3

5 1

0.5

YB5.B8

1B4

Sal-2

56 63 50

88 103 99

66 86 90

65 79 89

90 95 82

106 94 89

7(1 78 104

108 103 103

98 100 102

YB5.B8 (purified from ascites---Expts 1, 2, or from culture supernatant--Expt 3) and control MoAbs (purified from ascites) were included in the CFU assay at the indicated concentrations (6 replicate plates/treatment). Cord blood MNC were depleted of monocytes (Expts 1, 2) or monocytes and T cells (Expt 3) before plating with HPCM (40 #1/ plate) as growth factor source.

TABLE 4. EFFECT OF GROWTH FACTOR CONCENTRATION ON THE INHIBITION OF COLONY FORMATION BY Y B 5 . B 8

No. of colonies/plate and (% of control)

Antibody None YB5.B8 1B4 Sal-2

HPCM added (/d/plate) 50

20 85.1 64.7 78.3 83.8

-+ 2.8 - 3.4 4- 2.8 - 2.6

(100) (76) (92) (98)

97.4 70.6 94.6 99.2

-+ 3.3 -+ 1.0 4- 5.0 -+ 3.3

(100) (72) (97) (102)

100 102 82.6 99.3 102.8

4- 3.3 4- 2.0 - 1.3 - 4.1

(100) (81) (97) (100)

Cultures were established in 6-fold replicate using 2.5 × 104 monocyte and T-cell depleted cord blood MNC per plate. All antibodies were used at 2/~g/ml final concentration.

TABLE 5. EFFECT OF T-CELL DEPLETION ON THE INHIBITORY EFFECT OF YB5.B8 ON COLONY FORMATION BY CORD BLOOD CELLS I N V I T R O

T-depletion

Added antibody

No No Yes Yes

none YB5.B8 none YB5.B8

G 45 30 49 34

4- 2 4- 3 4- 3 -+ 1

No. of colonies/plate (Mean - S.D.) M Eo GM 44 27 44 41

_+ 2 -+ 3 4- 3 - 3

6 3 5 4

44-+ 4-

1 1 1 1

10 6 11 7

4- 1 -+ 2 - 1 4- 1

Total 105 66 110 86

4- 4 4- 2 4- 3 -+ 2

Cord blood MNC were depleted of monocytes by adherence to plastic and, where indicated, of T lymphocytes by rosetting with sheep erythrocytes. The recovery of ceils in unrosetted (nonT) fraction was 50%. Cells were plated in 6-fold replicate at 5 x 104/plate (not T-depleted) or 2.5 x 104/plate (T-depleted) with HPCM as the source of growth factors. Purified YB5.B8 was included, where indicated, at a final concentration of 1/~g/ml.

Haemopoietic progenitor cell antigen expression of the YB5.B8 antigen in ANLL indicated commitment to the mast cell lineage. However, several factors argued against this conclusion. Firstly, approx. 30% of ANLL cases have some YB5.B8positive blasts, which is higher than would be expected for mast cell leukaemias. Secondly, these leukaemias lack mast cell characteristics and are mostly of poorly differentiated myeloblastic type (M1 or M2), although some myelomonocytic (M4) ANLLs express the antigen. Thirdly, some blast rich, YB5.B8-positive ANLL specimens differentiate in vitro to give rise to monocytes and neutrophils (L. K. Ashman, unpublished data). An alternative hypothesis is that the YB5.B8 antigen is a normal haemopoietic progenitor cell marker which is retained during mast cell maturation, but lost from other haemopoietic cell lineages. To test this hypothesis, we re-examined the binding of MoAb YB5.B8 to normal bone marrow cells using sensitive analytical and functional assays. The results indicate that the YB5.B8 antigen is present at a low level on a small proportion (up to 3%) or normal bone marrow MNC, and that some of these cells are haemopoietic progenitors capable of giving rise to colonies in vitro. YB5.B8 and the anti-CD34 antibody BI-3C5 bound to a similar proportion of bone marrow MNC with similar 2-dimensional light scatter characteristics. Nevertheless, they clearly identify different antigens. The CD34 antigen has a mol. wt of 115 kD (compared with 150kD for the antigen identified by YB5.B8) and BI-3C5, unlike YB5.B8, binds to some acute lymphoblastic leukaemia specimens and to vascular endothelial cells [8, 18]. YB5.B8 binds to mast cells whereas anti-CD34 antibodies have not been reported to do so. Whether BI3C5 and YB5.B8 bind to the same cell population in bone marrow remains to be determined. The CFU data indicate that the populations are overlapping if not identical. Forty-six percent of CFUs were recovered in the YB5.B8-positive fraction by immune rosetting, and these represented all lineages assayed (G, M, Eo and GM). The highest yield was of GM mixed colonies of which 64% were recovered in the positive fraction. These results could indicate that CFUs are heterogeneous with respect to YB5.B8 binding (perhaps in a cell-cycle related manner) or that the rosettes are fragile because of the low level of expression of the antigen. In contrast, rosetting with FMC4 or BI3C5 (which bind to the cells in greater amounts) resulted in recovery of most CFUs in the positive fraction. Despite the lower recovery of CFUs by immune rosetting with YB5.B8, the level of enrichment in the positive fraction was comparable with that obtained with BI-3C5 and FMC4. The degree of

937

enrichment (3-5 fold) was less than that theoretically possible (25 fold; based on 2% YB5.B8-positive cells; 50% of CFUs YB5.B8-positive). Similarly, the enrichment obtained using BI-3C5 was lower than predicted from analytical data. These results were probably due to the inefficiency of the gradient separation technique employed, together with the use of cryopreserved material, since up to 20% of the total cells were recovered in the high density ("rosetted") fraction in the absence of antibody. These cells would have diluted the small numbers of cells rosetted with YB5.B8 and BI-3C5. Thus, the separation system needs to be refined before the possibility of using YB5.B8 to purify CFUs for practical purposes can be assessed. In additon, it will be important to determine whether the antigen is present on earlier progenitor cells which give rise to multilineage colonies (CFU-GEMM; [22]) and blast cell colonies with high self-renewal capacity [23-25]. The unusual distribution of the YB5.B8 antigen, together with its expression at low intensity and its prognostic significance, suggest that this molecule may have an important function in cell proliferation and/or differentiation, e.g. as a growth factor receptor, perhaps for 1L-3 or 1L-4. To assess the likelihood of this, we included YB5.B8 and control antibodies in the CFU assay which measures progenitor cell growth and differentiation in response to growth factors. MoAb YB5.B8, but not anti-HLA Class I or negative control IgG1 antibodies, caused reproducible inhibition of the yield of all four types of colonies examined (G, M, Eo, GM). Near maximal inhibition occurred at an antibody concentration of 2 ~tg/ml, but the level of inhibition did not exceed 50% even at 10 ~tg YB5.B8/ml. Higher levels of antibodies were not tested due to non-specific effects observed with control antibodies. A preliminary experiment was carried out to determine whether the inhibition by YB5.B8 could be overcome by increasing the level of growth factors, as would be expected if YB5.B8 competes for a receptor. No evidence for this was found. However, the growth factor source used (HPCM) contains several factors and, since complex receptor interactions occur, e.g. [26], this hypothesis must be tested using defined, e.g. recombinant, growth factor preparations. Similarly, the effect of T cells which, although not essential, appeared to enhance the inhibition also needs to be examined using defined growth factors. It should be noted that YB5.B8 does not bind detectably to T cells from peripheral blood [1; unpublished data]. In conclusion, the experiments reported here show that MoAb YB5.B8 is expressed at low levels by normal haemopoietic progenitor cells in bone marrow and cord blood. It binds to up to 3% of MNC in

938

ANTONYC. CAMBARERIet al.

normal b o n e m a r r o w , and these have 2-dimensional scatter characteristics consistent with their being blast cells. F u r t h e r m o r e , the YB5.B8-positive population contains at least 50% of the h a e m o p o i e t i c progenitor cells which give rise to colonies in the standard C F U C assay. In addition, the antibody specifically caused partial inhibition of colony formation when included in the C F U - C assay. W h e t h e r Y B 5 . B 8 identifies a growth factor r e c e p t o r or an associated peptide (such as the 150 k D a m e m b r a n e protein associated with the murine 1L-3 receptor [27]) must be tested in further experiments using defined growth factors. These experiments are in progress. Acknowledgements--This work was supported by grants from the Rotary Peter Nelson Leukemia Research Fund and the National Health and Medical Research Council of Australia. We wish to thank Dr C. A. Juttner, Division of Haematology, Institute of Medical and Veterinary Science, Adelaide, for the provision of bone marrow specimens and Professor B. Robinson, Department of Obstetrics and Gynaecology, University of Adelaide, for the cord blood specimens. We also thank Joseph Webster of Flinders Medical Centre, South Australia, for operating the flow cytometer, and Dr R. W. Tindle, Department of Medicine, Princess Alexandra Hospital, Queensland, Australia, and Dr H. Zola, Flinders Medical Centre, for the gift of MoAbs BI-3C5 and FMC4 respectively. HPCM for these experiments was kindly provided by Mr A. Spurling, Oncology Unit, Adelaide Children's Hospital.

7. 8.

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11. 12.

13. 14.

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