Expression of MY7 Antigen on Myeloid Precursor cells

Transcription

1 International Journal of Cell Cloning 1: (1983) Expression of MY7 Antigen on Myeloid Precursor cells James D. Grifin, Jerome Ritz, Richard P. Beveridge, Jefrey M. Lipton, John F. Daley, Stuart F. Schlossrnan The Division of Tumor Immunology and Pediatric Oncology, Sidney Farber Cancer Institute; the Division of Hematology-Oncology, Children s Hospital Medical Center; the Departments of Medicine and Pediatrics, Harvard Medical School, Boston, MA, USA Key Words. CFU-C antigen. Monoclonal antibody Abstract A murine monoclonal antibody (anti-my7) has been developed that detects an antigen expressed by 6% of normal human bone marrow cells, including approximately 40% of myeloid colony-forming cells (CFU-C). The number of bone marrow cells and CFU-C expressing MY7 is significantly increased in regenerating bone marrow, but less than 5% of peripheral blood CFU-C express the MY7 antigen. Erythroid precursors are MY7 negative from peripheral blood and bone marrow. Thymidine suicide studies indicate that CFU-C in S-phase tend to be MY7 positive while CFU-C not in S-phase are MY7 negative. MY7 expression thus appears to identify a fraction of CFU-C that is actively proliferating. Introduction Human monocytes and granulocytes are derived from a small population of bone marrow precursor cells which differentiate in response to specific stimuli [l, 21. The development of an in vitro colony assay for myeloid progenitor cells (granulocyte-monocyte colony forming unit, CFU-C) [3, 41 has allowed determination of certain physical characteristics of these cells, and also definition of the factors regulating their growth and differentiation in vitro [5-91. However, the biochemical events associated with differentiation of CFU-C s are largely AlphaMed Press, Inc /83/$2.00/0

2 Griffin/Ritz/Beveridge/Lipton/Daley /Schlossman 34 Identification of lineage-specific cell surface antigens has recently provided insights into differentiation patterns of T lymphocytes, B lymphocytes, and myeloid cells In some cases, the relationship between leukemic cells and the normal counterpart cell has also been clarified [ We have recently generated a series of murine monoclonal antibodies to human acute myeloblastic leukemia cells for the purpose of examining the cell surface antigens of normal and malignant myeloid cells [14]. One of this series of antibodies, anti-my7, is an IgG1 antibody which detects an antigen expressed by the leukemic blasts of a majority of acute myelocytic leukemia (AML) patients, but not by lymphocytic malignancies or normal lymphocytes [14]. About 6% of lightdensity normal bone marrow cells express MY7 antigen, including some, but not all, CFU-C. Evidence is presented here that MY7 antigen is preferentially expressed by bone marrow CFU-C that are susceptible to thymidine suicide. Materials and Methods Production of Anti-MY7 Monoclonal Antibody Anti-MY7 was produced as previously described [I41 by immunizing a 6- week-old female BALB/c mouse (The Jackson Laboratory, Bar Harbor, ME) with cryopreserved leukemic cells from a single patient with acute myelomonocytic leukemia. Primary cultures were recloned by limiting dilution, and subsequently maintained by injection of 1 X 106 cells i.p. into BALB/c mice previously primed with pristane (Aldrich Chemical Co., Milwaukee, WI). Antibodycontaining ascites fluid was used in all subsequent experiments. Human Cell Fractions Mononuclear cells from the peripheral blood of normal volunteer donors were obtained by Ficoll-Hypaque sedimentation of whole blood [20]. Bone marrow was obtained from healthy volunteers by aspiration into syringes containing preservative-free heparin. Light density bone marrow cells were used in all experiments, after removing erythrocytes and mature neutrophils by Ficoll-Hypaque sedimentation (1.077 g/cc). For some experiments, peripheral blood and bone marrow were obtained from the same donor. Fluorescence Activated Cell Sorter (FA CS) Analysis FACS was used to sort Ficoll-Hypaque separated normal bone marrow mononuclear cells or peripheral blood mononuclear cells into fractions which were MY7 positive (+ve) and MY7 negative (-ve) by previously described techniques [ 141. The control antibody was a monoclonal IgG2 previously shown not to be reactive with bone marrow cells. Fluorescence intensity was displayed on a logarithmic scale which facilitated separation of antigen positive and negative

3 CFU-C Antigen 35 cells. Bone marrows from patients with acute lymphocytic leukemia (ALL) in remission recovering from maintenance chemotherapy were similarly analyzed. Cells were collected into 50% fetal calf serum (FCS) and cytocentrifuge preparations made for staining with Wright-Giemsa and cytochemical stains. Cells were collected sterilely and assayed for colony-forming cells as described below. CFU-C, BFU-E, CFU-E Assays CFU-C cells were analyzed by plating 105 mononuclear cells/ml in Iscove's modified Dulbecco's MEM (IMDM), 20% fetal bovine serum (FBS), over a feeder layer of 1 x 106 normal peripheral blood leukocytes in 0.5% agar [ 141. After 7 and 14 days of culture (37"C, 5% C02), colonies (>40 cells) and clusters (8-40 cells) were enumerated using an inverted microscope. Colony morphology was determined by a modification of the method of Kubota et al. [21]. The agar overlayer plug was separated from the underlayer and dried onto a glass slide using Whatman 4 filter paper. The slide was then fixed in acetone-methanol fixative (60% acetone; 10% methanol; 30 mm sodium citrate, ph 5.4) for 1 min at room temperature, washed in tap water, and then stained sequentially for napthol acetate esterase (nonspecific esterase, NSE), and napthol AS-D chioroacetate esterase (specific esterase, SE) [22]. Staining was done at 37 C for 0.5 and 1 h respectively. After washing, cells were counterstained with hematoxylin (Gill Formulation, Fisher Scientific) for 2 min. Monocytes stained heavily with NSE, while granulocytes stained brightly with SE and weakly with NSE. Eosinophil colonies were detected separately by staining fixed agar overlayers with Luxol Fast Blue (Eastman Kodak, Rochester, NY) [23]. Slides were stained for 4 h at room temperature in Luxol Fast Blue, 100 mg in 100 ml 70% ethanol saturated with urea, and counterstained with hematoxylin. Eosinophil colonies were identified by the characteristic bright green cytoplasmic staining. One hundred colonies were counted where possible. The assays for erythroid burst forming units (BFU-E) and colony forming units (CFU-E) were determined in the plasma clot system described by McLeod and coworkers 1241, as modified by CZark and Housman After washing in alpha media minus nucleosides (GIBCO Laboratories), appropriate cell numbers were added in 0.1 ml of alpha media minus nucleosides plus 5% FBS to 0.8 ml of erythropoietin (Connaught Step 111, Connaught Medical Research Laboratories, Willowdale, Ontario, Canada). The erythropoietin was used at a final concentration of 2 U/ml plasma clot. Final cell concentrations were 1 X 106/ml and 5 x lo5/ ml for peripheral blood and bone marrow, respectively. 10% v/v of conditioned medium from the human T cell line Mo (provided by Dr. David GoZde) was used as a source of burst-promoting activity. Clotting was initiated by the addition of 0.1 ml NCTC 109 (Microbiological Associates, Bethesda, MD) containing 1 U of grade 1 bovine thrombin (Sigma Chemical Co., St. Louis, MO). The 1.0 ml clotting mixture was dispensed in 0.1 ml portions into 0.2 ml microtiter culture wells (Linbro plates - Linbro Scientific Co., New Haven, CT) and incubated under 5% C02 in high humidity. The plasma clots were incubated for up to 14 days. CFU-E colonies were counted at 7 days in culture. The time of maximal BFU-E colony formation was

4 Griffin/Ritz/Beveridge/Lipton/Daley/Schlossman 36 for days. Erythroid colonies in three to six clots were counted and the results expressed as the mean and standard error of the mean of the number of erythroid colonies per 105 cells plated. CFU-E colonies were enumerated by counting all benzidine positive erythroid colonies of 4 cells or larger. BFU-E colonies were scored if they contained three or more subcolonies consisting of our or more hemoglobinized cells per subcolony: Throughout the experiments, the standard error of the means ranged between 4 and 15% of the mean CFU-E and BFU-E colony scores. Immune Red Cell Rosette Cell Separation Technique To facilitate identification of CFU-C surface antigens, a technique was developed to separate antigen positive cells from antigen negative cells on the basis of rosette formation with antibody-coated erythrocytes [26,27]. Separation was then effected by density gradient centrifugation, and colony-forming cells assayed in the positive and negative cell fractions. Preparation of antisera Rabbits were immunized with mouse Ig and immune serum passed sequentially over a human Ig affinity column (to remove crossreacting antibodies) and a mouse Ig affinity column. Purified rabbit anti-mouse Ig was then eluted from the second column with 1 M glycine buffer, ph 2.0, extensively dialysed against 0.16 M borate buffered saline, ph 7.4, and stored at -2OOC until use. Preparation of Sheep Erythrocytes Coated with Anti-Mouse Immunoglobulin. Rabbit anti-mouse Ig was attached to erythrocytes as follows: sheep erythrocytes (Microbiological Associates, Walkersville, NC) were washed 5 times with 0.9% NaC1, and 0.5 ml packed erythrocytes was added to 0.5 ml chromium chloride (Fischer Scientific, 1 mg/ml in 0.9% NaCl) and 0.5 ml rabbit anti-mouse Ig (1 mg/ml in BBS). All solutions were passed through a 0.45 micron filter unit (Millipore Corp., Bedford, MA) prior to use. The cell suspension was gently mixed by shaking for 7 min at 23 C. The reaction was terminated by addition of 10 ml cold phosphate buffered saline, and after centrifugation, the Ig-coated erythrocytes were washed 5 times with 0.9% NaC1. These cells were stable when stored as a 10% suspension at 4 C for up to 4 days. Cell preparation x 108 mononuclear cells (normal peripheral blood or normal bone marrow) were incubated for 30 min at 4 C with 1 ml of MEM- AB wash containing a 1:lOO dilution (as ascites) of anti-my7 or a control antibody. Anti-MY4 (negative control) is an IgG2a monoclonal antibody which detects an antigen expressed by human monocytes, but has not been detected on CFU-C's [ 141. Anti-I2 (positive control) detects a nonpolymorphic region of the human Ia-like antigen (HLA-DR) Unbound antibody was removed by 2 wash steps. To form immune rosettes, 1 X lo7 monoclonal antibody-treated cells were added to 0.1 ml of a 10% Ig-coated erythrocyte suspension, the mixture was pelleted (300 g, 10 min) and then incubated at 4OC for 30 min. The red cell-leukocyte mixture was then vigorously suspended with a pasteur pipette, and the frequency of rosetted leukocytes ascertained by microscopic examination. Rosetted cells were then separated from non-red cell bearing cells by Ficoll-diatrizoate density gradient sedimentation. Rosetted cells (antibody positive) were recovered

5 CFU-C Antigen 37 from the Ficoll pellet by suspending the pellet in 0.5 ml hypotonic buffer (0.17 M NH4C1, 0.01 M KHC03, O.OOO1 M ethylene diamine tetracetic acid) at 4 C for 5 min, followed by addition of 10 ml of cold wash medium, pelleting of the cells and 2 more wash steps. Cells were counted in a hemacytometer and cell viability assessed by trypan blue exclusion. Viability was greater than 95% in all cell fractions in these experiments. Interface (negative) cells were collected in a sterile pipette and washed 2 times prior to suspension at appropriate dilutions for plating in colony assays. In experiments with normal bone marrow cells, a small number of monocytes, mature myeloid cells, and nucleated erythrocytes, often agglutinated, could be found in the Ficoll pellet, regardless of the antibody used. All experiments included cell aliquots separated with anti-i2 (anti-hla-dr) [28] and anti-my4 antibodies as controls. Tritiated Thymidine Suicide Technique The proportion of CFU-C in S-phase of the cell cycle was determined by the 3H-thymidine (3H-TdR) suicide technique as described by Minden et al. [31]. Ten- 20 x 106 normal bone marrow mononuclear cells were suspended in RPMI 1640 medium with 10% dialysed FBS. 100 pci/ml of 3H-TdR (New England Nuclear) with a specific activity of 20 Ci/mM was added, and the cells incubated at 37 C for 30 min. A similar aliquot of bone marrow cells was incubated with 10-6 M unlabelled thymidine as control. The incorporation of 3H-TdR was stopped by the addition of 10 ml cold RPMI 1640 containing 10-4 M TdR. The cells were washed twice and then separated into MY7 positive and MY7 negative fractions by the rosette technique. CFU-C were assayed in each cell fraction including unseparated bone marrow. The percent of CFU-C in S-phase was determined as: [(Control - exposed)/control] x 100 where control = number of colonies formed in the presence of 10-6 M unlabelled TdR and exposed = number of colonies formed in the presence of 3H-TdR. There was no significant difference in the number of colonies formed in the simultaneous presence of 10-6 M TdR and 3H-TdR compared to 10-6 M TdR alone. Likewise, exposure to 10-6 M nonradioactive TdR did not reduce colony formation relative to no drug treatment. In two experiments, bone marrow cells were separated into and cell fractions prior to thymidine exposure, rather than after thymidine exposure. Results Expression of M Y7 Antigen on Normal Human Hernatopoietic Cells The distribution of MY7 antigen on normal peripheral blood cells has been previously reported [ 141. Most granulocytes and monocytes express detectable MY7, while T cells and B cells (resting and activated), null

6 Griffin/Ritz/Beveridge/Lipton/Daley/Schlossman 38 lymphocytes, erythrocytes and platelets do not. In bone marrow cells prepared from normal volunteers, approximately 6% of light density cells are more fluorescent than background (mean 5.9% f 2.876, n-8). Also, the bone marrow cells are only weakly fluorescent, suggesting that antigen density may be low. However, in actively regenerating marrow samples (from ALL patients in remission recovering from maintenance chemotherapy), the fraction of light density cells that are MY7 positive is substantially increased (mean 27.5% f 3.696, n= 3). In order to identify MY7 positive cells, bone marrow cells from both normal marrow and recovery marrow were separated into MY7 positive and negative fractions by cell sorting, and the cell differential determined on cytocentrifuge smears (Table I), The cells included all stages of myeloid differentiation, with a predominance of immature cells (myeloblasts, promyelocytes, myelocytes) in both normal and recovery bone marrow. Two other experiments with normal bone marrow, and one other experiment with recovery marrow gave similar results. Thus, in normal bone marrow only a small fraction of myeloid cells at each level of differentiation express detectable MY7. This fraction increases substantially in an actively regenerating marrow. Lymphocytes and nucleated erythrocytes at all levels of differentiation did not express MY7 antigen. Expression of MY7 Antigen on Bone Marrow and Peripheral Blood CFU-C Expression of MY7 antigens on bone marrow and peripheral blood CFU-C was determined by separating normal or regenerating cells into and cell fractions by FACS (Table 11) as previously described [14]. CFU-C were counted after 7 days (colonies and clusters) and 14 days (colonies). Approximately 40% of the starting number of day 7 CFU-C were recovered in the cell fraction, and 30% of day 14 CFU-C were recovered in the fraction (Table 11, Exp. 1 and 2). Recovery of CFU-C after sorting was similar to recovery of total cells in each experiment, In regenerating marrow, both the percent of cells expressing MY7 and the percent of MY7 + ve CFU-C were increased in several experiments (Table 11, Exp. 3 shows one of three similar experiments). In contrast, when normal peripheral blood cells were separated by FACS, <2% of CFU-C were recovered in the MY7 fve cell fraction (Table 11, Exp. 4 shows one of two similar experiments). Thus, MY7 antigen is expressed on a subset of bone marrow CFU-C, but is detected on very few peripheral blood CFU-C by these methods.

7 Table I. MY7 positive cells in bone marrow Experiment" Cell fraction %MY7 cells Percent Blastb Pro Myelo Meta Bands- Mono Ly NRBC Plasma 6 Gran cells E c,? 1. Normal Unseparated Bone Marrow Recovery Unseparated Bone Marrow MY7+ve a Light-density bone marrow cells separated by FACS into and fractions. b Blast, myeloblast; Pro, promyelocyte; Myelo, myelocyte; Meta, metamyelocyte; Band, neutrophilic band; Gran, granulocyte; Mono, monocyte; Ly, lymphocyte; NRBC, nucleated erythrocyte. Cytocentrifuge smears stained with Wright-Giemsa stain; 200 cells counted. w W

8 Table 11. FACS separation of MY7 antigen positive cells Experimenta Cell. Percent CFU-C (%)b Colony Morphology (76) fraction MY7 (Day 14)c positive cells Day 7 Day 14 Gran Mono G-M Eosino 1. Normal Bone Marrow Unseparated (41) 29 (39) 193 (31) 14 (32) Normal Bone Marrow Unseparated (35) 29 (35) Recovery Bone Marrow Unseparated (33) 27 (7) (48) 6 (3.6) Normal Peripheral Blood Unseparated ' (1.4) 24 (41) a20 X 106 Ficoll-Hypaque bone marrow cells were sorted by FACS into a brightly fluorescent fraction () and a negative (background) fraction. bday 7 CFU-C/105 mononuclear cells (mean of 4 plates, S.D. I f 25%), day 7 CFU-C includes clusters. The percent of starting (total) CFU-C recovered in each cell fraction is indicated in parentheses. c Determined by cytochemical staining. Gran, granulocyte; Mono, monocyte; G-M, mixed granulocyte-monocyte; Eosino, eosinophil. dper 10s mononuclear cells (mean of 4 plates, S.D. 5 f 25%, except for fraction where S.D. = f 64%). 2 P kj C 9. a R._ \ Y ~

9 CFU-C Antigen 41 With the method for determining CFU-C used in these experiments, several types of colonies are grown: granulocytic, monocytic, mixed G-M and eosinophil[23, 321. In order to determine if the type of colonies in the fraction were different than the type of colonies in the MY7 +ve fraction, the proportion of each colony type was determined in each cell fraction using cytochemical staining as described in Materials and Methods. The distribution of colony types was similar in the unseparated marrow, and cell fractions from normal bone marrow and peripheral blood (Table 11). Thus, the expression of MY7 on only a fraction of CFU-C cannot be explained by differential expression on different types of CFU-C. Expression of M Y7 Antigen on Erythrocyte Progenitors In order to facilitate determination of antigen expression on CFU-C cells in several samples simultaneously, a technique of cell separation using antibody-coated erythrocytes was employed. Table I11 shows the results of experiments in which aliquots of normal bone marrow and peripheral blood mononuclear cells were separated into MY7 antigen positive and negative cell fractions by the immune rosette technique. Separated cell fractions and unseparated cells were then assayed for CFU-C, CFU-E, and BFU-E. The colony count and the percent of starting colonies recovered in each cell fraction are shown in Table I11 for bone marrow (Exp. 1, one of four experiments with similar results is shown), peripheral blood (Exp. 2, one of three experiments), and bone marrow and peripheral blood from the same donor (Exp. 3, one of two experiments). As in previous experiments, MY7 positive CFU-C were detected in the bone marrow, but not in the peripheral blood. In contrast, CFU-E and BFU-E were MY7 negative in both bone marrow and peripheral blood. In two experiments with both bone marrow and peripheral blood, cell aliquots were also separated by the same technique using a negative control antibody, anti-my4, which detects an antigen expressed on normal monocytes but not on CFU-C [ 141, and a positive control antibody, anti-i2 (anti-ia) [28]. In both peripheral blood and bone marrow, I l % of CFU-E and BFU-E were recovered in the MY4 positive cell fraction, and 250% of BFU-E were recovered in the I2 positive cell fraction. DNA Synthesis in CFU-C The expression of MY7 antigen on only a fraction of bone marrow CFU-C, and the lack of expression of MY7 antigen on peripheral blood

10 Table III. Expression of MY7 antigen on erythrocyte progenitors Experiment Cell fractiona CFU-Cb (7%) CFU-Eb BFU-Eb Day 7 Day Bone Marrow Unseparated 98 f 14 (-) 72f4 (-) 106f26 (-) 16f5 (-) 115 f21(29) 64f 18 (22) 124 f 23 (32) 77 f 11 (27) 6f2 (1.4) 0.5 f 0.8 (0.8) 272f31 (64) 46f8 (72) 2. Peripheral Blood Unseparated 24f4 (-) 18f7 (-) 0 (0) 36f8 (44) 0 (0) 50f9 (82) 3. Bone Marrow Unseparated 36f6 (-) 32f3 (-) 72 f 14 (26) 41f8 (17) 28+2 (27) 22f4 (24) 51f7 (-) 1152 (-) 0.2 f 0.2 (0.1) 0.2 f 0.2 (0.2) 102f26 (70) 17f4 (54) 4. Peripheral Blood Unseparated 46f8 (-) 2f2 (0.3) 56f3 (50) 24f5 (-) 1 f 1 (0.3) 29f6 (49) a cells separated by immune rosettes (see Materials and Methods). b colonies per 105 mononuclear cells (bone marrow) or 106 mononuclear cells (peripheral blood), mean of 3-6 plates f S.D. The percent of starting (total) colonies recovered in each fraction is indicated in parentheses. Day 7 CFU-C includes clusters and colonies. A R

11 ~~ ~ CFU-C Antigen 43 CFU-C cells suggested that expression of this antigen may in part be related to the proliferative state of the precursor cell. It has previously been shown that a lower percentage of peripheral blood CFU-C cells are in S- phase compared to bone marrow CFU-C in the mouse [33] and in humans [34]. To examine the cell cycle status of CFU-C, aliquots of normal bone marrow cells were exposed to high specific activity 3H-thymidine or nonradioactive thymidine, separated into and MY7 -ve cell fractions and assayed for surviving CFU-C (Table IV). Two of three similar experiments from three separate donors are shown. Exposure of unseparated bone marrow to 3H-TdR resulted in a mean decrease of 26% in CFU-C (day 7), indicating that 26% of CFU-C were actively synthesizing DNA during the 30 min exposure to 3H-TdR. The 3H-TdR Table IV. Thymidine suicide of normal bone marrow Experiment Cell fractiona Day 7 CFU-Cb % of CFU-Cc per 105 cells in S-phase Unseparated Control 3H-TdR Control 3H-TdR Control 3H-TdR Unseparated Control 3H-TdR Control 3H-TdR Control 3H-TdR 191 f f f21 119f f & f f f21 23 f f32 117f6 13 a Normal bone marrow was separated into and cell fractions followed by exposure to 3H-thymidine (3H-TdR) or 10-6 M non-labelled thymidine (control). b mean of 3 plates f S.D. c 100 X (control - 3H-TdR)/control.

12 Griffin/Ritz/Beveridge/Lipton/Daley/Schlossman 44 susceptible CFU-C were almost entirely in the cell fraction, indicating that virtually all the CFU-C traversing S-phase expressed MY7. In contrast, CFU-C showed much less sensitivity to TdR suicide. In two other experiments, low density bone marrow cells were first separated into and cell fractions, followed by exposure to 3H-TdR or control TdR. Equivalent results to those presented in Table IV were obtained (data not shown). Discussion Maturation of myeloid cells from committed precursor cells in vitro proceeds in response to certain colony-stimulating factors produced by monocytes, activated T cells [35,36], and other cells. The initial biochemical events associated with myeloid differentiation are largely unknown. Likewise, little is known about the cell surface structure of progenitor cells or their malignant counterparts, the cells of acute myeloblastic leukemia. In this report, we further describe a cell surface antigen detected by a monoclonal antibody, anti-my 7, that was generated after immunization with acute myelomonoblastic leukemia cells [ 141. About 6% of normal light density bone marrow mononuclear cells express MY7 by immunofluorescence, but this fraction is increased substantially in a regenerating normal bone marrow (Tables I and 11). When bone marrow cells are separated by FACS, a wide range of differentiation stages are found. However, it is clear that as only 6% of total cells are positive, the majority of myeloid cells at each level of differentiation either do not express MY7 antigen, or express undetectable levels. In this respect, MY7 antigen is different from many differentiation antigens studied in our laboratory [14] and others, which are expressed by all of the cells at a given level of differentiation. It is possible, therefore, that expression of MY7 antigen by myeloid cells is not related to differentiation, but to some other aspect of the cells that is increased substantially by bone marrow regeneration. In the peripheral blood, both monocytes and granulocytes express low levels of MY7 antigen, although higher levels of fluorescence are detected on these cells than on bone marrow myeloid cells. This further supports the possibility that MY7 is actually a pan-myeloid antigen, but in the absence of some yet undefined stimulus, antigen density is low, particularly in the bone marrow. This type of antigen expression may in some ways be analogous to certain activation antigens of lymphocytes which

13 CFU-C Antigen 45 are present in very low levels in resting cells, but can be induced after exposure to a mitogenic lectin [37]. In contrast to normal myeloid cells, MY7 antigen can be detected on the majority of AML cells in relatively high antigen density. It was therefore of particular interest to investigate the expression of MY7 antigen on early normal myeloid cells, such as the CFU-C, in more detail. Cell sorting experiments showed that the bone marrow cells include a substantial fraction (approximately 30%) of day 14 CFU-C cells from normal bone marrow, and a slightly higher fraction (40%) of day 7 CFU-C from normal bone marrow (Table 11). However, less than 5% of CFU-C from normal peripheral blood expressed MY7, as determined by cell sorting or a positive selection technique using antibody-coated erythrocytes (Tables I1 and 111). The lack of expression of MY7 on normal peripheral blood CFU-C could not be explained by selective lack of reactivity with eosinophil colonies, as the percent of eosinophil colonies was equal in and cell fractions in both bone marrow and peripheral blood. The peripheral blood CFU-C may represent a less differentiated cell, while the bone marrow contains a more heterogeneous population of CFU-C, some of which are actively proliferating, presumably in response to colony-stimulating factors. Evidence was therefore sought to determine if MY7 antigen was expressed predominantly by proliferating (and possibly more differentiated ) myeloid progenitor cells. The thymidine suicide studies shown in Table IV support this hypothesis. The thymidine suicidesensitive CFU-C were almost entirely in the MY7 + ve fraction. In contrast to myeloid precursor cells, MY7 antigen was not detected on erythroid precursors (CFU-E and BFU-E) from peripheral blood or bone marrow. This provides evidence that CFU-C can be distinguished by cell surface markers from CFU-E and BFU-E, and thus supports the hypothesis that the cell which is assayed in vitro as a myeloid colony-forming cell is indeed unipotent [2,38], having already acquired on its surface an antigen expressed by myeloid, but not erythroid, lineage cells. The function of the surface structure defined by anti-my7 has not yet been identified. The studies presented here suggest that MY7 antigen may be expressed at low antigen density throughout myeloid differentiation, from late CFU-C to granulocyte, and that the level of MY7 antigen may be influenced by factors other than differentiation stage. Further studies will be required to identify the function of this structure in normal and malignant myeloid cells.

14 Griffin/ Ritz! Beveridge / Lipton/ D aley / Schlossman 46 Acknowledgements This work was supported by National Institutes of Health grants CA-25369, CA Project 3, CA-09172, and CA Dr. Gn@n is supported in part by the Johanna C. Wood Foundation (Philadelphia, PA) and the Medical Foundation (Boston, MA). Dr. Ritz is a Special Fellow of the Leukemia Society of America. References Pike, B.L.; Robinson, W.A.: Human bone marrow colony growth in agar-gel. J Cell Physiol 76: 77 (1970). Metcalf, D.: Hematopoietic colonies. In vitro cloning of normal and leukemic cells. (Springer-Verlag, Berlin-Heidelberg 1977). Till, J.E.; McCulloch, E.A.: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14: 213 (1961). Bradley, T.R.; Metcalf, D.: The growth of mouse bone marrow cells in vitro. Aust J Exp Biol Med Sci 44: 287 (1966). Moore, M.A.S.; Williams, N.; Metcalf, D.: Purification and characterization of the in vitro colony-forming cell in monkey hemopoietic tissue. J Cell Physiol 79: 283 (1970). Dicke, K.A.; van Noord, M.J.; Maat, B.; Schaeter, U.W.; van Bekkum, D.W.: Identification of cells in primate bone marrow resembling the hemopoietic stem cell in the mouse. Blood 42: 195 (1973). Worton, R.G.; McCulloch, E.A.; Till, J.E.: Physical separation of hemopoietic stem cells from cells forming colonies in culture. J Cell Physiol 74: 171 (1969). Haskill, J.S.; McNeil, T.A.; Moore, M.A.S.: Density distribution analysis of in vivo and in vitro colony forming cells in bone marrow. J Cell Physiol 75: 167 (1970). Burgess, A.W.; Metcalf, D.: The nature and action of granulocyte-macrophage colony stimulating factors. Blood 56: 947 (1980). Chess, L.; Schlossman, S.F.: Human lymphocyte subpopulations; in Dixon, Kunkel, Advances in Immunology, pp (Academic Press, New York 1977). Reinherz, E.L.; Schlossman, S.F.: The differentiation and function of human T lymphocytes: A review. Cell 19: 821 (1980). Stashenko, P.; Nadler, L.M.; Hardy, R.; Schlossman, S.F.: Characterization of a human B lymphocyte specific antigen. J Immunol125: 1678 (1980). Todd, R.F.; Nadler, L.M.; Schlossman, S.F.: Antigens on human monocytes identified by monoclonal antibodies. J Immunol126: 1435 (1981). Griffin, J.D.; Ritz, J.; Nadler, L.M.; Schlossman, S.F.: Expression of myeloid differentiation antigens on normal and malignant cells. J Clin Invest 68: 932 (1981).

15 CFU-C Antigen Aisenberg, A.C.; Bloch, K.J.: Immunoglobulins on the surface of neoplastic lymphocytes. N Engl J Med 287: 272 (1972). Borella, L.; Sen, J.: T cell surface markers on lymphoblasts from acute lymphocytic leukemia. J Immunol111: 1257 (1973). Brouet, J.C.; Seligmann, M.: The immunological classification of acute lymphoblastic leukemias. Cancer 42: 817 (1978). Reinherz, E.L.; Kung, P.C.; Goldstein, G.; Levey, R.H.; Schlossman, S.F.: Discrete stages of intrathymic differentiation: Analysis of normal thymocytes and leukemic lymphoblasts of T cell lineage. Proc Natl Acad Sci 77: 1588 (1980). Nadler, L.M.; Ritz, J.; Griffin, J.D.; Todd, R.F.; Reinherz, E.L.; Schlossman, S.F.: Diagnosis and treatment of human leukemias and lymphomas utilizing monoclonal antibodies. Prog Hematol12: 187 (1981). Boyum, A.: Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 21 (Suppl. 97): 77 (1968). Kubota, J.; Mizoguchi, H.; Miura, Y.; Suda, T.; Takaku, F.: A new technique for the cytochemical examination of human hematopoietic cells grown in agar gel. Exp Hematol8: 339 (1980). Yarn, L.T.; Li, C.Y.; Crosby, W.H.: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol55: 283 (1971). Verma, D.S.; Spitzer, G.; Zander, A.R.; Fisher, R.; McCredie, K.B.; Dicke, K.A.: The myeloid progenitor cell: A parallel study of subpopulations in human marrow and peripheral blood. Exp Hematol8: 32 (1980). McLeod, D.L.; Shreeve, M.M.; Axelrod, A.A.: Improved plasma culture system for production of erythrocyte colonies in vitro: Quantitative assay method for CFU-E. Blood 44: 517 (1974). Clarke, B.J.; Houseman, D.: Characterization of an erythroid precursor cell of high proliferative activity in normal human peripheral blood. Proc Natl Acad Sci 74: 1105 (1977). Indiveri, F.; Ng, A.K.; Russo, C.; Quaranta, V.; Pellegrino, M.A.; Ferrone, S.: Isolation of Ia-like antigen-bearing cells from human peripheral lymphocytes through the use of a monoclonal antibody to framework determinants of Ia-like antigens. J Immunol Methods 39: 343 (1980). Griffin, J.D.; Beveridge, R.P.; Schlossman, S.F.: Isolation of myeloid progenitor cells from peripheral blood of chronic myelogenous leukemia patients. Blood 60: 30 (1982). Nadler, L.M.; Stashenko, P.; Hardy, R.; Pesando, J.M.; Yunis, E.J.; Schlossman, S.F.: Monoclonal antibodies defining serologically distinct HLA-D/DR related Ia-like antigens in man. Hum Immunol I: 77 (1980). Winchester, R.J.; Ross, G.D.; Jarowski, (2.1.; Wang, C.Y.; Halper, J.; Broxmeyer, H.E.: Expression of Ia-like antigen molecules on human granulocytes during early phases of differentiation. Proc Natl Acad Sci 74: 4012 (1977). Janossy, G.; Francis, G.E.; Capellaro, D.; Goldstone, A.H.; Greaves, M.F.: Cell sorter analysis of leukemia-associated antigens on human myeloid precursors. Nature 276: 176 (1978).

16 Griffin/Ritz/Beveridge/Lipton/Daley / Schlossman Minden, M.D.; Till, J.E.; McCulloch, E.A.: Proliferative state of blast cell progenitors in acute myeloblastic leukemias. Blood 52: 592 (1978). Chervenick, P.A.; Boggs, D.R.: In vitro growth of granulocytic and mononuclear cell colonies from blood of normal individuals. Blood 37: 13 1 (1971). Iscove, N.N.; Till, J.E.; McCulloch, E.A.: The proliferative states of mouse granulopoietic progenitor cells. Proc SOC Exp Biol Med 134: 33 (1970). Dresch, D.; Faille, A.: Subpopulations of human granulomonocyte CFC in bone marrow and blood. Exp Hematol6: 80 (1978). Golde, D.W.; Cline, M.J.: Identification of the colony-stimulating cell in human peripheral blood. J Clin Invest 51: 2981 (1972). Parker, J.W.; Metcalf, D.: Production of colony-stimulating factor in mitogen-stimulated lymphocyte cultures. J Immunol112: 502 (1974). Hercend, T.; Ritz, J.; Schlossman, S.F.; Reinherz, E.L.: Comparative expression of T9, T10 and Ia antigens on activated human T cell subsets. Hum Immunol3: 247 (198 1). Quesenberry, P.; Levitt, L.: Hematopoietic stem cells. N Engl J Med 301: 819 (1979). Received: September 13, 1982; accepted: December 2, 1982 Dr. James D. Griffin, Division of Tumor Immunology, Sidney Farber Cancer Institute, 44 Binney Street, Boston, MA (USA)