A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells

Leukemia Research Vol. S, No. 4, pp. 521-534, 1984. Primed in Great Britain.

0145-2126/8453.00 +0.00 © 1984 Pergamon Press Lid.

A MONOCLONAL ANTIBODY REACTIVE WITH NORMAL AND LEUKEMIC HUMAN MYELOID PROGENITOR CELLS* JAMES D. GRIFFIN, DAVID LINCH, KERT SABBATH, PETER LARCOM and STUARTF. SCHLOSSMAN Division of Tumor Immunology and Pediatric Oncology, Dana-Farber Cancer Institute, the Division of Hematology and Oncoiogy, Children's Hospital Medical Center and the Department of Medicine, Harvard Medical School, Boston, MA 02115, U.S.A. (Received 15 November 1983. Revision accepted 15 December 1983) AbstraclmAnti-MY9 is an IgG2b murine monoclonal antibody selected for reactivity with immature normal human myeloid cells.The MY9 antigen is expressed by blasts, promyelocytes and myelocytes in the bone marrow, and by monocytes in the peripheral blood. Erythrocytes, lymphocytes and platelets are MY9 negative. All myeloid colony-forming cells (CFU-GM), a fraction of erylbroid burst-forming cells(BFU-E) and multipotent progenitors (CFU-GEMM) are • MY9 positive. This antigen is further expressed by the leukemic cellsof a majority of patients with AML and myeloid CML-BC. Leukemic stem cells (leukemic colony-forming cells,L-CFC) from most patients tested were also MY9 positive. In contrast, MY9 was not detected on lymphocytic leukemias. Anti-MY9 may be a valuable reagent for the purification of hematopoietic colonyforming cellsand for the diagnosis of myeloid-lineage leukemias.

Key words: Myeloid surface antigen, leukemia diagnosis, CFU-GM, leukemic colony-forming cells.

INTRODUCTION H U M A N monocytes and granulocytes are derived from a small population of bone marrow progenitor cellswhich can be assayed by the formation of colonies of mature cellsin semisolid medium (CFU-GM) [24, 30]. In vitro studies have partially defined growth factors required for rnyeloid colony formation [3], but relatively little is known about the progenitor cells themselves. In particular, it has been difficultto purify progenitor cells because of their scarcity and lack of distinguishing physical characteristics. Monoclonal antibodies which recognize surface antigens unique to different types of progenitor ceils would be ideal reagents to identify and purify these cells [8]. A small number of monoclonal antibodies identifying myeloid-associated antigens have been described that recognize myeloid progenitor cells, most often produced following immunization with acute myeloblastic leukemia (AML) cellsor celllines [I, 2, I0, 14, 20, 22, 40]. The majority of such antibodies, however, also react with most mature myeloid ceilsor with other cellssuch as activated lymphocytes. In an effort to produce monoclonal

*This work was supported in part by NIH grants CA 36167-01, CA 19389 Project 3, and RR 005526. Abbreviations: CFU-GM, granulocyte and monocyte colony-forming cell; AML, acute myeloblastic leukemia; CML-BC, chronic myeloid leukemia in blast crisis; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; E + ceils,sheep erythrocyte rosetting ceils;FACS, fluorescence-activated cell sorting; BFU-E, erythroid burst-forming unit; CFU-E, erythroid colony-forming unit; IMDM, Iscove's modified Dulbecco's minimal essential medium; CFU-GEMM, mixed colony-forming ceil; C ', complement; A ,VLL, acute nonlymphoblastic leukemia; TdT, terminal deoxynucleotidyl transferase; CALLA, common acute lymphoblastic leukemia antigen; L-CFC, leukemic colony-forming ceil. Correspondence to: Dr J. Griffin, Division of Tumor Immunology, Dana-Faber Cancer Institute,44 Binney Street, Boston, MA 02115, U.S.A. 521

522

JAMES D. GR1FFINet aL

a n t i b o d i e s r e a c t i v e w i t h a m o r e r e s t r i c t e d p o p u l a t i o n o f i m m a t u r e h e m a t o p o i e t i c cells, we h a v e i m m u n i z e d m i c e w i t h cells f r o m a p a t i e n t w i t h the blast crisis p h a s e o f c h r o n i c myeloid leukemia (CML-BC), and selected for antibodies which react with immature m y e l o i d cells, b u t n o t w i t h n o n - m y e l o i d h e m a t o p o i e t i c cells in the p e r i p h e r a l b l o o d o r b o n e m a r r o w . T h e C M L - B C cells w e r e s e l e c t e d f o r i m m u n i z a t i o n b e c a u s e t h e y a p p e a r to lack m a n y o f t h e cell s u r f a c e a n t i g e n s c o m o n l y e x p r e s s e d by m a t u r e m y e l o i d cells a n d A M L cells [13]. Anti-MY9

is a n I g G 2 b m o n o c l o n a l

a n t i b o d y g e n e r a t e d by this m e t h o d w h i c h r e a c t s

w i t h i m m a t u r e m y e l o i d cells, b u t n o t w i t h l y m p h o c y t e s , p l a t e l e t s o r e r y t h r o c y t e s . In a p r e l i m i n a r y r e p o r t this a n t i b o d y w a s listed as M Y 9 0 6 [12]. W e d e s c r i b e t h e d i s t r i b u t i o n o f M Y 9 a n t i g e n o n h e m a t o p o i e t i c c o l o n y - f o r m i n g cells, o n m y e l o i d a n d l y m p h o i d l e u k e m i a s a n d o n m y e l o i d l e u k e m i c c o l o n y - f o r m i n g cells. A n t i - M Y 9 is a n o v e l m o n o c l o n a l a n t i b o d y w h i c h will be u s e f u l f o r t h e p u r i f i c a t i o n o f s u b s e t s o f c o l o n y - f o r m i n g cells a n d f o r the diagnosis of myeloid-lineage leukemias. MATERIALS

AND METHODS

Production of monoclonal antibodies. A 6-week-old female Balb/c mouse was immunized with cryopreserved leukemic cells from a single patient with myeloid CML-BC as previously described I1 I]. Six months later, two i.v. injections of the same cells were given, and the splenocytes fused 3 days later with 3.0 x 10' P3/NSI/I-Ag4-1 myeioma cells by the method of Kohler and Milstein [191 with modifications [181. The fused cell mixture was suspended in hypoxanthine-aminopterin-thymidine-containing medium and distributed to microtiter wells. The hybidoma clones were grown at 37°C in 5% CO2 and 14 days later screened for the presence of immunoglobulin reactive with the immunizing cells by an indirect immunofluorescence assay previously described [14]. Fluorescent cells were detected on a cytofluorograph (FC200/4800A, Ortho Instruments, Westwood, Mass). Clones producing reactive antibody were screened for reactivity with B-cell lines, T-cell lines, erythrocytes, platelets, granulocytes and T cells. Clones lacking reactivity with these cell types were recloned twice and passaged as ascites tumors in Balb/c mice. All subsequent experiments were performed using diluted ascites fluid (l/500-1/1000). Ig class of anti-MY9 was determined by indirect immunofluorescence testing using fluoresceinated isotype-specific second antibodies (Meloy Laboratories, Springfield, Virginia). Isolation of human cell fractions. Granulocytes, monocytes, platelets, erythrocytes, sheep erythrocyte rosetting cells (E + cells, T cells) and B cells were prepared by standard techniques as previously described [ll]. In all cases, purity was assessed by either morphology (granulocytes, erythrocytes, platelets) or by reactivity with lineage-specific monoclonal antibodies Mo2 (monocytes) [38], T3 (T cells) [31], BI (B cells) [35]. In some experiments, E + cells were activated by culture for 7 days with phytohemaggultinin (Difco, 4 lag/ml in RPMI 1640 medium with 10% fetal bovine serum). The degree of activation was assessed with anti-HLA-DR monoclonal antibody 12 [28]. Human bone marrow was obtained from healthy volunteers by aspiration. Mononuclear cells were prepared by Ficoll-Hypaque density gradient sedimentation (I.077 g/cm~). Human leukemia cells. Leukemia cells from peripheral blood or bone marrow from 271 patients with AML, acute lymphoblastic leukemia (ALL), CML-BC or chronic lymphocytic leukemia (CLL) were tested for reactivity with MY9 antibody. Diagnoses were established by standard clinical and morphological criteria and confirmed in most cases by surface marker analysis [27]. All leukemia samples contained greater than 50070 leukemic cells and usually greater than 75070. Reactivity was determined by indirect immunofluorescence. A positive reaction was considered as greater than 20070 of test cells more fluorescent than background (control antibody non-reactive with test cells). When testing samples that had residual non-leukemic cells present, several measures were taken to exclude the possibility that MY9 reactivity was with normal cells and not with leukemic cells. Light scatter windows on the flow cytometer were carefully selected to minimize inclusion of normal myeloid cells with the leukemic cells. These windows were selected by prior cell-sorting experiments. The reactivity of anti-MY9 with leukemic cells was confirmed by cell sorting or by using a fluorescent microscope to determine morphology of MY9 + cells (phase optics) as necessary. Anti-MY9 was also used in conjunction with a series of other monoclonai antibodies which facilitated determining the purity of the leukemic cells being tested. Since most leukemic cells express la antigen, and normal promyelocytes, myelocytes and other granulocyte precursors do not, la antigen was also a useful marker [1 11. While approx. 10070of normal bone marrow cells are MY7 +, the antigen density (and thus fluorescence) is quite low. Leukemic cells are generally more brightly fluorescent. Finally, both peripheral blood and bone marrow cells were tested for MY9 antigen expression wherever possible. Using these maneuvers, it was possible to confirm the expression of MY9 antigen on the leukemic cells of each of the samples reported as positive. Virtually all negative samples had less than 5% positive cells, and the majority of positive acute myeloblastic leukemia (AML) samples had greater than 7507o fluorescent cells, la, MY7 [I II, CALLA [331 and TII [321 antigens were run simultaneously as positive control antibodies where appropriate. Cell separation techniques. In various experiments, cells were separated into MY9 positive and MY9 negative cell fractions by fluorescence-activated cell sorting (FACS) [111. FACS was performed using indirect immunofluorescent staining and a Coulter Epics V Cell Sorter (Coulter Electronics, Healeah, Florida). Cells were

Progenitor cell a t ~ t i ~ y

523

sorted at 3000/s and collected under sterile conditions into 50% fetal calf serum in lscove's modified Dulbecco's minimal essential medium (IMDM) for morphologic analysis (cytocentrifuge preparation) or colony assay. Human cell lines. The myeloid leukemia cell line KG-I was supplied by Dr David Goide, University of California - Los Angeles School of Medicine. All other cell lines were supplied by Dr Herbert Lazarus, DanaFarber Cancer Institute. Progenitor cell colony assays. CFU-GM were assayed in triplicate or quadruplicate by plating 1 x 10~(bone marrow) or 5 × 10~ (peripheral blood) cells/ml in IMDM containing 20% fetal bovine serum and 0.3% agar over a 0.5% agar underlayer containing 10% GCT medium (Gibco) as a source of CSF [10, 11]. Colonies (greater than 40 cells) and clusters (10--40cells) were enumerated on day 7 using an inverted microscope. Day 14 CFU-GM were counted as colonies greater than 40 cells. Erythroid colonies were grown in IMDM containing0.9% methylcellulose, 30% fetal calf serum, 0.9% bovine serum albumin, 2 × 10-'M 2-mercaptoethanol, 2 units/ml erythropoietin (Step Ill, Connaught Labs, Swiftwater, Pennsylvania) and 10% Mo cell-conditioned medium [16]. Cells were plated at 10~/ml. Well hemoglobinized colonies on day 7 were scored as CFU-E, and the larger, multicentric and late hemoglobinizing bursts scored as BFU-E at 14 days. Mo cell conditioned medium was added to the cultures as a potent source of burst-promoting activity and to reduce any effects on progenitor growth due to removal or enrichment of accessory cells during fractionation procedures [7]. In other replicate cultures the addition of erythropoietin was delayed for 3-5 days, and mixed colonies (CFU-GEMM) were enumerated in these cultures after 14-16 days [23]. Leukemic blast colonies [4, 25, 26] were grown by plating I x IIY to I × 10~ peripheral blood cells from untreated AML patients with high peripheral blood blast counts in IMDM containing 20% fetal bovine serum and 0.3% agar. An underlayer Of 0.5% agar in the same medium contained 20% GCT medium [9, 37]. This assay method for L-CFC appears to yield similar results to the methylcellulose-leukocyte conditioned medium assay of Buick [4]. However, it has the advantages that the frequency of T-cell colonies is very low and the use of agar allows cytochemical analysis of all colonies formed. After 7 days, the agar overlayers were removed from the underlayers, dried onto glass slides, fixed and stained for specific and non-specific esterase activity as previously described [29]. All patients' colonies stained positively for either specific or non-specific esterase, or both, confirming their myeloid origin. Colonies were considered as aggregates of greater than 20 cells. In order to determine the surface antigen phenotype of the colony-forming cells, aliquots of 2 × 10' leukemia cells were treated with complement alone, or with complement plus anti-la or anti-MY9. After treatment, the cells were resuspended in IMDM and plated in agar to determine residual L-CFC. Complement lysis. Aliquots of cells were incubated with monoclonal antibody (1:250) for 30 min at 4°C. Following one wash step, the cells were suspended in rabbit complement (Gibco) at a dilution of 1:4. After 90 rain at 37°C, the cells were washed twice, recounted and resuspended for colony assays. Anti-Ia [12] was used as a positive control. RESULTS

Expression o f M Y 9 antigen on normal peripheral blood and bone marrow cells T h e expression o f MY9 a n t i g e n o n n o r m a l h e m a t o p o i e t i c cells as d e t e r m i n e d by indirect i m m u n o f l u o r e s c e n c e is s h o w n in T a b l e 1. In the p e r i p h e r a l b l o o d M Y 9 a n t i g e n was expressed by 7.7 =[: 4.9070 o f the p e r i p h e r a l b l o o d m o n o n u c l e a r cells o f six n o r m a l individuals. P u r i f i e d erythrocytes, platelets, T l y m p h o c y t e s a n d B l y m p h o c y t e s did n o t express detectable MY9. M o n o c y t e s f r o m five n o r m a l i n d i v i d u a l s were u n i f o r m l y MY9 positive. M Y 9 fluorescence o f purified g r a n u l o c y t e s was extremely d i m a n d in m o s t cases was i n d i s t i n g u i s h a b l e f r o m b a c k g r o u n d (Fig. I). MY9 was expressed o n 27 =[: 9 % o f the m o n o n u c l e a r cells f r o m eight samples o f n o r m a l b o n e m a r r o w . Figure 1 shows typical F A C S h i s t o g r a m s o f g r a n u l o c y t e s , m o n o c y t e s , T cells a n d n o r m a l b o n e m a r r o w . N o r m a l tonsil, spleen a n d t h y m u s cells were MY9 negative. N o n - h e m a t o p o i e t i c cells have n o t been tested.

F A C S separation o f M Y 9 positive bone marrow cells In order to d e t e r m i n e the m o r p h o l o g y o f the a p p r o x . 2707o MY9 positive cells in n o r m a l b o n e m a r r o w , positive a n d negative cells were isolated by f l u o r e s c e n c e - a c t i v a t e d cell sorting ( F A C S ) a n d c y t o c e n t r i f u g e smears stained with W r i g h t - - G i e m s a s t a i n ( T a b l e 2). ldentific.ation o f m o n o c y t e s was c o n f i r m e d by s t a i n i n g with a l p h a - n a p t h y l acetate esterase [29]. T h e expression o f MY9 a n t i g e n i d e n t i f i e d a subset o f m y e l o i d - l i n e a g e cells in the b o n e m a r r o w r a n g i n g f r o m blasts to myelocytes, a n d cell s o r t i n g resulted in a several-fold e n r i c h m e n t for these i m m a t u r e m y e l o i d ceils. In c o n t r a s t , l y m p h o i d a n d e r y t h r o i d cells were e n r i c h e d in the MY9 negative fraction. M e t a m y e l o c y t e s a n d b a n d s were f o u n d in both fractions, suggesting that fluorescence s t a i n i n g o f these cell types was o f low intensity.

524

JAMES D. GRIFFIN et al.

TABLE

II Ill

1. EXPRESSION OF MY9 ANTIGEN ON NORMAL HEMATOPOIETIC CELLS

No. tested

MY9 expression

(070)

Peripheral blood* Granulocytes Monocytes T cells (E + ) PHA-activated T cells B cells

5 5 5

60+15

5 2

1± 2 2 ± 1

Erythrocytes Platelets

5 5

! 4- 1 1 4-'I

Bone marrow

8

27 -t- 9

Miscellaneous Tonsil Spleen Thymocytes

2 2 2

0 4- 0 1 4- 1 0 4- 0

6"i-4 l±l

*Cells were purified as described in Materials and Methods. MY9 antigen expression was determined by an indirect immunofluorescence assay using a flow cytometer.

Expression of MY9 antigen on normal hematopoietic precursor cells The finding of MY9 antigen on myeloid cells as immature as the myeloblast prompted us to examine the expression of this antigen on hematopoietic colony-forming cells. In the bone marrow of six individuals tested 90 4- 7.007o of day 7, and 74 4- 25°70 of day 14 CFUGM were recovered in the MY9 + cell fraction. Representative experiments are shown in Table 3. Recovery of CFU-GM in these experiments was similar to recovery of total cells sorted. In peripheral blood (n = 3), 67 4- 14070 CFU-GM were MY9 positive. Erythroid and mixed colony-forming cells were also assayed (Table 3). The majority of CFU-E were MY9 negative by cell-sorting experiments. The expression of MY9 antigen on BFU-E, however, was more variable, as shown in representative experiments in Table 3. Between 9 and 73070 of BFU-E were recovered in the MY9 + cell fraction (mean 4- S.D. of six experiments = 40 4- 3207o). The fraction of CFU-GEMM recovered in the MY9 + fraction ranged from 7 to 10007o (39 4- 43.607o in four experiments). It should be noted that cell-sorting experiments with control antibodies resulted in a recovery of less than 1°70 of BFU-E or CFU-GEMM in positive cell fractions. In order to determine if the apparent variability of the expression of MY9 antigen on BFU-E might be related to low antigen density, normal bone marrow cells were separated by FACS into the 25070 most brightly fluorescent MY9 positive cells (MY9 + + ) and the approx. 5007o least fluorescent MY9 positive cells (MY94-) in a series of experiments. The FACS cytograms for a typical experiment are shown in Fig. 2. Cell differentials, CFUGM, CFU-E and BFU-E were then assayed in each cell fraction (Table 4). Blasts and promelocytes were enriched in the MY9+ + cell fraction, while myelocytes were enriched in the MY94- cell fraction, suggesting that antigen density of MY9 decreases from the myeloblast to the myelocyte, and at the granulocyte stage is not detectable on

Progenitor cell antibody

525

MY9 ANTIGEN EXPRESSION i

A. Monocytes

~ ,

!

• Non-Adherent

Cells

Fluorescence I n t e n s i t y - - - FIG. 1. Expression of MY9 antigen on monocytes, granulocytes, non-adherent cells and normal bone marrow mononuclear cells. Cell fractions were purified as described in Materials and Methods and tested for MY9 antigen by indirect immunofluorescence and FACS analysis.

TABLE 2. DISTRIBUTION OF

MY9 POSITIVE CELLS IN NORMAL BONE MARROW BY CELL SORTING

Nucleated Cell fraction*

Blast

Pro

Myelo

Meta/band

Mono

Lymph erythrocytes

Other

Unseparated

1 4- 0.4

3 "t" 2

15 4- 8

37 4- 25

7 4- 4

9 5- 8

26 5- 7

2 -1- 2

MY9 positive

6 + 4

15 4- 3

35 4- 7

19 4- 15

18 4- 11

2 4- 2

3 4- 3

2 4- 2

MY9 negative

2 4- 2

1 4- 1

1 4- 1

9 4- 8

0 4- 0

30 4- 9

56 4- 8

1 4- 2

*Bone marrow mononuclear cells were separated into MY9 positive and negative cell fractions by FACS. Cytocentrifuge smears were stained with Wright-Giemsa, and 200 cell differentials were recorded. The data is expressed as the mean 4- S.D. of three experiments with separate donors. Pro, promyelocyte; myelo, myelocyte; recta, metamyelocyte; mono, monocyte; lymph, lymphocyte; other includes basophils, eosinophils, plasma cells and unidentifiable cells.

27

19

35

070M¥9 positive

(97O7o)1" (3070)

58 79 4 (98070) (2070)

3.3 18 (66070) 2 (34%)

264 396 8

CFU-GM

149 40 (807o) 177 (920/o)

----

187 19 (12%) 94 (88%)

CFU-E

Colonies/10' cells

48 46 (26070) 33 (74%)

30 74 (8207o) 4 (18070)

102 106 (73%) 27 (27°70)

BFU-E

(!00070) (0070)

(78%) 0.8 (220/0)

7

5.2

8 12 (100070) 0 (0070)

35 40 0

CFU-GEMM

Colonies/I0' cells

ANTIGEN ON NORMAL HEMATOPOIETIC COLONY-FORMING CELLS BY CELL SORTING

*Bone marrow mononuclear cells were separated by FACS into MY9 + and MY9- cell fractions and colony-forming cells assayed as described in Materials and Methods. CFU-GM (colonies plus clusters) and CFU-E were enumerated on day 7, BFU-E and CFU-GEMM on day 14. Recovery o f total cells was 45--61°70 o f cells sorted. 1 Per cent of total colonies recovered after sorting.

Unseparated MY9* MY9-

Unseparated MY9 + MY9

Peripheral blood

Bone marrow

Unseparated MY9 ÷ MY9

Bone marrow

I.

2.

Cell fraction

MY9

Experiment

TABLE 3. EXPRESSION OF

"7

o~

I

6

MY9___

MY9 + +

29

2

I

2

Pro

28

55

2

21

Myelo

2

27

17

27

Meta/band

28

5

0

9

Mono

Cell differential

I

I

33

14

!

7

44

21

Nucleated L y m p h o erythrocytes

5

2

3

5

Other

256

124

!

98

58

75

!

23

CFU-GM Day 7 Day 14

4

91

385

218

CFU-E

5

58

146

130

BFU-E

Colony assays/IO s cells

MY9 POSITIVE NORMAl. BONE MARROW CELLS BY FLUORESCENCE INTENSITY

*See legend to Table 2. Normal bone marrow cells were separated by FACS. MY9 + + cells were selected as the 25% most brightly fluorescent cells (see Fig. 2). MY9+_ cells included the 50% least fluorescent MY9 positive cells. tColony assays were performed as described in Materials a n d Methods. See legend to Table 3. The results are shown as the m e a n of four replicate cultures, S . D . -<- + 2 5 % for all values.

I

0

Unseparated

MY9-

Blast

Cell fraction*

TABI.E 4. DISTRIBUTION OF

O" O

.-5

528

JAMES D. GRIFFIN et al. I

II

I

I

I

A.

B.

T .Q MY9-

E Z 4D

¢J

~-- Control

~

MY9 -+-

MY9

Fluorescence Intensity

,

FIG. 2. Separation of MY9 positive normal bone marrow cells by cell sorting. (A) Expression of MY9 antigen on normal bone marrow mononuclear ceils. (B) Aliquots of normal bone marrow mononuclear cells were separated into the 25070 most brightly fluorescent MY9 positive cells. (MY9 + + ), the 50070 least fluorescent MY9 + cells (MY9+), and MY9 negative (MYg--) cell fractions. After sorting, the samples were reanalysed and that data is shown here. Cells from each fraction were then analysed for morphology and colony assays (see Table 4).

most cells. Similarly, day 7 CFU-GM were enriched in the MY9 + + cell fraction, and BFU-E in the M Y g + cell fraction, suggesting that the MY9 antigen density is higher on CFU-GM than on BFU-E. CFU-GEMM were not assayed in these experiments. The FACS experiments were confirmed by complement lysis and by immune rosetting studies [8] with normal bone marrow. In three experiments, a mean of 84°70 of day 7 CFUGM, and 81 070 of day 14 CFU-GM, were lysed by treatment with anti-MY9 and complement (Table 5). In one experiment BFU-E were inhibited by 46070 and CFU-GEMM by 38070 compared to C ' alone or C ' plus control antibody. Similarly, when normal marrow cells were separated by immune rosettes [8, 10] in three experiments, 62 + 24070 of day 7 CFU-GM were recovered in the MY9 positive fraction. Expression of M Y 9 antigen on human leukemias Since studies with normal bone marrow showed that MY9 was expressed primarily by immature myeloid cells, it was of interest to examine the expression of MY9 on leukemic cells. Table 6 shows the results of testing cells from 271 patients with AML, ALL, CMLBC or CLL for expression of MY9 antigen. In each case, the diagnosis was established by standard clinical criteria and confirmed by surface marker analysis using panels of extensively characterized monoclonal antibodies [27]. The expression of HLA-DR (Ia) and MY7 antigens is shown for comparison. ANLL cases were subgrouped by FAB classification [3]. Of all acute nonlymphocytic leukemias (ANLL), 84070 were MY9 +. A positive reaction was considered as greater than 20070 of leukemic cells more fluorescent than background. However, in almost all cases which expressed MY9 antigen, 50-99°7o of leukemic cells were brightly fluorescent. MY9 was not preferentially expressed by any

Progenitor cell antibOdy

529

TABLE 5. EFFECT OF TREATMENT WITH ANTI-MY9 AND COMPLEMENT ON COMMITTED MYELOID PROGENITORS

Treatment*

CFU-GM (colonies/l x 10' cells) Day 7 Day 14

Complement alone

192 ± 16

48 ± 6

Complement + anti-MY9

31 ± 14 (84070)3`

9 ± 7 (81070)

Complement + anti-la

36 ± 19 (81070)

6 ± 4 (88070)

*Bone marrow mononuclear cells were treated with antibody and complement and cultured as described. Results are the mean colonies ± S.D. for three experiments. 1"Per cent inhibition of colony formation.

TABLE 6. EXPRESSION OF

MY9 ANTIGEN ON ACUTE AND CHRONIC LEUKEMIAS Per cent of cases expressing antigen

Leukemia*

No. tested

la

MY7

MY9

54 6 31

89 0 97

78 67 81

85 100 81

A M o L (M5) EL(M6)

6 1

I00 0

50 0

83 0

A11 ANLL

98

86

76

84

59 2"/ 21 2 109

93 85 5 100 74

0 0 5 0 1

3. CML blast crisis Myeloid Lymphoid Erythroid Megakaryoblast

13 13 1 l

92 100 0 0

92 0 0 0

92 0 0 0

Undifferentiated

6

83

0

33

23 7

I00 14

0 0

0 0

AML (M1, M2) APL (M3) AMML (M4)

2.

4.

CALLA + ALL CALLA- ALL T-ALL B-ALL AI 1 ALL

CLL B-CLL T-CLL

2t" 0 0 0 0

*CALLA, common acute lymphoblastic leukemia antigen; ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; CLL, chronic lymphocytic leukemia. ~f30eToof blasts were MY9 positive.

m o r p h o l o g i c a l s u b t y p e o f A N L L . I n p a r t i c u l a r , expression o f M Y 9 p r o v e d to be a n excellent m a r k e r for u n d i f f e r e n t i a t e d A M L ( F A B type M1), a n d was o f t e n positive in the small n u m b e r o f MY7 negative u n d i f f e r e n t i a t e d A M L s . A p p r o x i m a t e l y 93~0 o f all cases of A N L L expressed either MY9, MY7 or b o t h . A single case o f e r y t h r o l e u k e m i a (surface g l y c o p h o r i n positive) was M Y 9 negative. I n c o n t r a s t to the results with A N L L , M Y 9 was

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JAMES D. GRIFFIN et al.

not detected on 108 of 109 cases of ALL of either T-cell, B-cell or null lineage. One case of CALLA + ALL expressed MY9 on 30°70 of blast cells. Similarly, none of 30 cases of CLL were MY9 positive. Thus, it appears that detection of MY9 antigen can be used as a reliable test to distinguish AML from lymphoid leukemias. This was further tested in CML-BC. In myeloid blast crisis (diagnosed by morphology, MY7 expression [13] and negative TdT), 92o70 of 13 cases expressed MY9. In contrast, none of 13 cases of lymphoid blast crisis (CALLA positive, TdT positive) expressed MY9 antigen. Erythroid and megakaryoblast (one case each) leukemias were also negative, while two of six undifferentiated cases were MY9 positive. In myeloid blast crisis, the fluorescence of MY9 staining is often brighter than MY7 staining and thus is a useful addition to the monoclonal antibodies already available for the analysis of blast crisis. Expression of M Y 9 antigen on leukemic colony-forming cells in A M L Since leukemic colony-forming cells may represent progenitor cells in AML [4, 25], the expression of MY9 antigen on L-CFC was determined in seven cases. Greater than 75°70 of leukemic cells in each case were Ia posit{ve and MY9 positive by FACS analysis. The expression of Ia and MY9 on the L-CFC was then determined by measuring the L-CFC surviving treatment with complement C ' alone, anti-la plus C ' and anti-MY9 plus C ' (Table 7). In six of seven cases, greater than 60070 of L-CFC were eliminated by anti-Ia. Also in six of seven cases, greater than 48°70 of L-CFC were eliminated by treatment with anti-MY9. In case six there was minimal (l I o70)loss of CFC. Sufficient cells were available in all patients for repeat analysis and similar results with anti-la and anti-MY9 were obtained in each case. Thus, L-CFCexpress MY9 antigen in a substantial proportion of AML patients.

TABLE 7. EXPRESSION OF MY9 ANTIGENON LEUKEMICCOLONY.FORMINGCELLS BY COMPLEMENT LYSIS

Patient No.

Diagnosis*

L - C F C / I O ' cellst (C ' alone)

I 2 3 4 5 6 7

AMML AMML AML AML AMML AML AML

212 88 380 84 156 105 162

L-CFC (°70 o f control)~: after antibody + C ' la MY9

34e70 28 3 72 l 7 12

29 8 52 5 2 89 9

* A M M L , acute myelomonocytic leukemia (FAB M4); A M L , acute myeloblastic leukemia (FAB MI). t L - C F C were assayed in a double-layer agar system described in Materials and Methods. Colonies (greater than 20 cells) were counted on days 7 and lO, mean (~f quadriplicate cultures (S.D. + / - 20o70). ~:Aliquots of leukemic cells were treated with C ' alone (control), C ' plus anti-la or C ' plus anti-MY9. Residual L-CFC after antibody treatment are expressed as per cent o f control (C ' alone).

Expression o f MY9 antigen on human leukemic cell lines The expression of MY9 antigen on a variety of cultured cell lines was tested by indirect immunofluorescence. The myeloid leukemia cell lines HL60, KG-I, U937 and K562 were positive (greater than 90°/o fluorescent cells). All other cell lines tested were negative (less

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than 5°70 fluorescent cells). These negative cell lines included T-cell lines CEM, Molt-4 and HSB; B-cell lines SB, Raji, Daudi, Laz 156, Laz 221, Laz 388 and Laz 471; ALL cell lines Nalm-1 and Nalm-6. DISCUSSION Monoclonal antibodies can be used to define discrete stages of differentiation of human hematopoietic cells [11,27]. In the present study we have described the characterization of an IgG2b murine monoclonai antibody, anti-MY9, which identifies a cell surface antigen expressed by only monocytes in the peripheral blood, and a subset (27%) of bone marrow myeloid cells including myeloblasts, promyelocytes and myelocytes (Table 2). Erythroid and lymphoid cells (including activated lymphocytes) do not appear to express detectable MY9 antigen. The distribution of MY9 antigen on normal progenitor cells is of particular interest. CFU-GM were MY9 positive in blood and bone marrow (both day 7 and day 14 as tested by cell sorting, complement lysis or immune rosettes); while CFU-E were generally less than 1007oMY9 positive. In contrast, the expression of MY9 antigen on BFU-E and CFUGEMM was more variable. Significant numbers of BFU-E and CFU-GEMM were recovered in both the MY9 positive and the MY9 negative cell fractions in most experiments (Table 3) with a mean of 40 _ 32°7o of BFU-E recovered in the MY9 positive fraction, and 39 _ 44o7o of CFU-GEMM in the MY9 positive fraction. In some experiments, less than 10% of recovered BFU-E were MY9 positive, despite the detection of MY9 antigen on greater than 95°7o of CFU-GM in the same experiment. A likely explanation for this variability in cell-sorting experiments is that MY9 antigen density (and thus fluorescence intensity) is low on BFU-E compared to CFU-GM (Table 4). Minor variations in setting the positive and negative windows for cell sorting may thus have considerable effect on the subsequent recovery of the cells. Alternatively, there may be variations in the expression of MY9 antigen from individual to individual, although this was not noted when studying monocytes. Furthermore, there are other possibilities to explain this variability. MY9 antigen expression on BFU-E and CFU-GEMM could be affected by cell cycle status, level of differentiation, or other parameters, and thus identify subsets of these progenitor cells. As an example, the expression of MY7 antigen on CFU-GM appears to be related to cell cycle [10]. Further experiments to investigate these possibilities with anti-MY9 are underway. The expression of MY9 antigen on progenitor cells suggest that this antibody may be useful in a number of biological applications. There are only a small number of wellcharacterized monocional antibodies reactive with progenitor cells. Ia antigen, although expressed by mature cells of multiple lineages (B cells, activated T cells and monocytes) has proven to be a useful marker of progenitor cells, and is expressed by at least a subset of CFU-GM, BFU-E and CFU-GEMM [6, 17, 21, 31, 39]. Like Ia antigen, MY9 is lost gradually during granulocyte differentiation, but is expressed throughout monocyte differentiation. MY9 is distinguished from Ia antigen by the lack of expression of MY9 on B cells and activated T cells, and also by the lack of expression of Ia antigen on myelocytes and most promyelocytes. Several antigens restricted to myeloid cells have b ~ n described which also react with CFU-GM and other progenitors [1, 2, 10, 14, 20, 22, 40]. MY7 is expressed by granulocytes and monocytes in the peripheral blood, and by 6-20~70 of bone marrow mononuclear cells, including about 40% of C F U - G M [10]. MY7 is not expressed by BFU-E, CFU-E or by peripheral blood CFU-GM. Other monoclonal antibodies reactive with progenitor cells have been described by Linker-Israeli [20], Young [40], Hanjan [14], Ball [2], Mannoni [22], Civin [36] and Andrews [1]. The tool. wt of the antigens identified by these antibodies have generally not been reported. Attempts to obtain a tool. wt of MY9 antigen using standard immunoprecipitation techniques have

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not revealed a discreteband on SDS-polyacrylarnide gels. It is possible that M Y 9 antigen, like M Y I [15] and IGI0 [l] antibodies, recognize a carbohydrate determinant. It is clear, however, from the cellular distribution of M Y 9 antigen that anti-MY9 is likely to be distinct from previously reported antibodies. The lack of reactivity of anti-MY9 with granulocytes and mature bone marrow myeloid ceils in particular separates anti-MY9 from antibodies such as M M A [14], P M N 6 [2], P M N 2 9 [2], AML-2-23 [2],80H. I [22], 8OH.3 [22], 80H.5 [22], IGI0 [I] and M Y I [15]. Further studies will be necessary to compare anti-MY9 with previously reported antibodies. Anti-MY9 may have considerable utilityin the purificationof hematopoietic progenitor ceils, especiallywhen used to positivelyselect C F U - G M following a depletion step using other antibodies [8]. W e have previously described the use of anti-la for the immunological purification of C F U - G M by positive selection [II]. Anti-MY9 may provide similar purification, with the additional advantage of not selecting activated T lymphocytes. Highly enriched populations of progenitor cells (essentially free of potential regulatory cellssuch as monocytes and activated T cells)are likelyto be extremely useful in the investigation of stem cell regulation. Expression of M Y 9 antigen also appears to distinguish C F U - E from BFU-E, and it is possible that purification strategies can be developed using M Y 9 and other antibodies to enrich highly for C F U - E ceils. A n important clinicalapplication of anti-MY9 is the identificationof myeloid lineage leukemias (Table 6). M Y 9 was detected on 8 4 % of 98 A M L cases, and was generally brightly fluorescent using indirect immunofluorescence. The minimal reactivity with lymphoid leukemias (I/I09 A L L and 0/30 C L L cases) suggests that M Y 9 antigen expression can be used reliablyto distinguishA M L from ALL. This can be of particularvalue in the less differentiated forms of A M L (FAB M O and MI), and in cases where other discriminatory testsare negative or unclear. Used in conjunction with anti-MYT, approx. 93We of A M L cases are identified.M Y 9 antigen does not appear to be expressed preferentiallyon any subtype of A M L , unlike antigens such as M Y 4 and M o 2 which are expressed primarily in acute myelomonocytic and monocytic leukemias [II]. A preliminary review of cases does not suggest that presence or absence of M Y 9 antigen is of prognostic significance. The expression of M Y 9 on leukemic colony-forming ceilswas also investigated.A M L cells will form colonies of leukemic cellswith a plating efficiencyof 10-2-10-` [4], and it has been suggested that the leukemic colony-forming cells(L-CFC) may act in vivo as progenitor cells to maintain the rest of the leukemic cell population [4, 7]. There is little known about L-CFC, however, and it has not been possible to distinguish L-CFC from other leukemic ceilsexcept by their higher rate of proliferation.Previous studies with the monocyte surface antigen M Y 4 in acute myelomonoc~,tic leukemia demonstrated that the surface antigens of the L-CFC ( M Y 4 negative) may be distinct from those of the majority of the leukemic'cells( M Y 4 positive)[9].W e therefore determined the expression of M Y 9 antigen on a seriesof seven A M L patients all of w h o m expressed M Y 9 on greater than 5 0 % of leukemic cells (Table 6). In six of seven cases, the majority of L-CFC were M Y 9 positive, suggesting that M Y 9 (like la, but in contrast to MY4) is frequently expressed on leukemic progenitor cells.Further characterizationof the L-CFC from a larger series of patients is underway using M Y 9 and other A M L antibodies. These studies may have particularrelevance when considering the possibilityof using monoclonal antibodies to clean up residual leukemic cells prior to autologous bone marrow transplantation. If L-CFC do represent the leukemic counterpart of a progenitor ceil, then a better understanding of their surface antigen structure relative to that of the majority of leukemic cellsis necessary to design therapeutic strategies.The expression of M Y 9 antigen on normal progenitor cells would make this antibody an unsuitable candidate for therapeutic use. However, anti-MY9 may contribute significantlyto our understanding of

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the b i o l o g y o f n o r m a l a n d l e u k e m i c m y e l o i d cells b y d e f i n i n g d i f f e r e n t i a t i o n s t a g e s o f i m m a t u r e cells a n d i m p r o v i n g p u r i f i c a t i o n o f p r o g e n i t o r cells f o r f u n c t i o n a l s t u d i e s . REFERENCES 1. ANDREWSR. G., TOROK-SToRaB. & BERNSTEINI. D. (1983) Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies. Blood 62, 124. 2. BALLE. D. & FANGERM. W. (1983) The expression of myeloid-specific antigens on myeloid leukemia cells: Correlations with leukemia subclasses and implications for normal myeloid differentiation. Blood 61,456. 3. BENNETTJ. M., CATOVSKYD., DANIELM-T., FLANDRING., GALTOND. A. G., GRALNICKH. & SULTANC. (1976) Proposals for the classification of the acute ieukemias. Br J. Haemat. 33, 451. 4. Buick R. N., TILL J. E. & MCCULLOCHE. A. (1977) Colony assay for proliferative blast cells circulating in myeloblastic leukemia. 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