From www.bloodjournal.org by guest on February 2, 2015. For personal use only. Ontogeny of Hematopoietic Stem Cell Development: Reciprocal Expression of CD33 and a Novel Molecule by Maturing Myeloid and Erythroid Progenitors By Carolyn Brashem-Stein, David A. Flowers, Franklin 0. Smith, Steven J. Staats, Robert G. Andrews, and Irwin D.Bernstein W e have identified a molecule expressed by human marrow granulocyte/monocyte colony-forming cells (CFUGM), erythroid colony-forming cells (CFU-E), and erythroid burst-forming units (BFU-E), but not their precursors detectable in long-term bone marrow culture. This antigen, detected by flow microfluorimetry using monoclonal antibody 789, is coexpressed with CD33 on many CD34+ CFCs, although only the 7B9 antigen was detected on a portion of BFU-E and CFU-E, whereas only CD33 was found on a portion of CFU-GM. Antibody 7B9 appears to be useful for isolating subsets of progenitors based on their common or selective expression of 7 8 9 antigen and CD33. 0 1993 by The American Society of Hematology. D (Burroughs-Wellcome, Research Triangle Park, NC) at 2 pg/mL, for 7 days.23 To prepare marrow samples for immunization, normal bone marrow cells were enriched for CD34+ cells using an immunoadsorbant column as previously described,24or by magnetic bead enrichment. Using this latter method, cells were incubated with 12-8 antibody (anti CD34) at 25 pg/mL for 20 minutes, washed twice with phosphate buffered saline (PBS) plus 2% human AB serum (GIBCO, Grand Island, NY), and then incubated with biotin conjugated goat-antimouse IgM (p chain specific) (Tago, Burlingame, CA), at a dilution of 1530. After washing twice, magnetic streptavidin (Advanced Magnetics, Inc, Cambridge, MA) was added to cells suspended at 107/mL,at a ratio of 5 beads/cell. Cells and beads were rocked gently for 20 minutes at 4°C and bound cells were separated from unbound cells in a 25 cm2tissue culture flask using a magnet. When analyzed by immunofluorescence, CD34+-enriched marrow cells were shown to contain at least 70% CD34-positive cells. Leukemic cell lines were maintained in continuous culture in RPMl I640 with 10%Bovine Calfserum (BCS) (Hyclone Laboratories, Logan, UT). The KG-1 and KG- 1 a cell lines were supplied by Dr David Golde, University of California, Los Angeles. The other cell lines, HL-60, HEL, K-562, U-937, Jurkat, HSB-2, Raji, Nalm1 , and Nalm-6 were kindly provided by Dr John Hansen, Fred Hutchinson Cancer Research Center, Seattle, WA. The cell line FMY 9S5 clone 7 is a transfected mouse L-cell line that expresses the CD33 antigen.25It was kindly provided by Dr Thomas Look, St Jude Children’s Research Hospital, Memphis, TN. Antibodies. The IgM antibodies 5F1 (CD36), lGlO (CD15), L4F3 (CD33), 12-8 (CD34), and H I2C12 (antimouse Thy 1.2), and the IgG antibodies p67-6 (CD33), 7B9, and 31.A (antimouse Thy 1.1) were prepared as previously d e s ~ r i b e d . ~ , ~The ~ , ~anti-CD34 ~,~’ antibody MY-10 was kindly provided by Dr Curt Civin, Johns Hopkins Oncology Center, Baltimore, MD. Antibody 1F5 (CD20) was provided by Dr Paul Martin, Fred Hutchinson Cancer Research Center. All the above antibodies were purified from ascites fluid, and were used at a concentration of 10 pg/mL, except for antibody 12-8, which was used at 20 pg/mL. The anti-CD2 antibody (35.1), and the anti-CD4 antibody (66.1) were used in the form of ascites fluid at a I: 1000 dilution, and were kindly provided by Dr Paul Martin. The directly conjugated antibodies fluorescein isothiocyanate (F1TC)-1F5 and Cy-5-7B9 were prepared in our laboratory and were used at 50 pg/mL. The fluorochrome Cy5 was kindly provided by Dr Swati Mujumdar, Carnegie Mellon University, Pittsburgh, PA, and antibody was conjugated with Cy-5 as described.” For flow cytometry studies, the following conjugated antisera were used: FlTC goat-antimouse IgG and IgM (Tago, Inc), FlTC goat-antimouse IgG (7chain specific, Kirkegaard & Perry, Gaithersburg, MD), and phycoerythrin (PE) goat-antimouse IgM ( p chain-specific, Calbiochem, La Jolla, CA). All of the above antisera were used at a dilution of 1 :40. Monoclonal antibod-v screening and production. Balb/c mice were immunized via the intraperitoneal route (IP) with 0.2 to 1 .O X URING EARLY hematopoiesis, pluripotent stem cells give rise to progenitor populations that display progressively decreased proliferative potentials, accompanied by an increased commitment to differentiate along a single hematopoietic lineage.’,’ Progenitor cells have been distinguished in vitro based on their proliferation in response to specific growth factors and the nature of their mature p r ~ g e n y . ~More - ~ recently, it has been possible to characterize human hematopoietic progenitors on the basis of cell surface antigen expression.6-2’ Progenitor cells and their precursors express the CD34 antigen, whereas most progenitors express the CD33 antigen.’ In this report, we describe a novel 72 Mr molecule (detected by antibody 7B9) that is also expressed by hematopoietic progenitors but not their precursors. A population of progenitors, including a portion of granulocyte/monocyte colony-forming cells (CFU-GM), erythroid colony-forming cells (CFU-E), and erythroid burst-forming units (BFU-E), express both the CD33 and 7B9 antigens. Those cells expressing only detectable amounts of the 7B9 antigen are mainly committed to erythroid differentiation, whereas those expressing only detectable amounts of CD33 are mainly GM progenitors. MATERIALS AND METHODS Cells. Samples of blood and bone marrow were obtained from normal healthy volunteers following informed consent under an Institutional Review Board approved protocol. Peripheral blood lymphocytes (PBL), monocytes, and granulocytes were isolated as described by Ficoll-Hypaque density centrifugation (density of 1.077) (Pharmacia, Inc, Piscataway, NJ).” Normal thymocytes were obtained following informed consent from patients undergoing cardiac surgery at Children’s Hospital and Medical Center, Seattle, WA. Activated T lymphocytes were obtained as previously described by incubating PBL in phytohemagglutinin (PHA) From the Programs in Pediatric Oncology, Fred Hutchinson Cancer Research Center, and the Department of Pediatrics, University of Washington, Seattle. Submitted November 2, 1992; accepted April 6 , 1993. Supported by the National Cancer Institute Grant No. CA39492. Address reprint requests to Irwin D. Bernstein. MD, Pediatric Oncology Program, Fred Hutchinson Cancer Research Center, 1124 Columbia St, Seattle, W A 98104. The publication costs of this article were defayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1993 by The American Society of Hematology. 0006-4971/93/8203-0016$3.00/0 792 Blood, Voi 82,No 3 (August l ) , 1993:pp 792-799 From www.bloodjournal.org by guest on February 2, 2015. For personal use only. 793 CD33 AND 709 EXPRESSION ON PROGENITOR CELLS Table 1. Expressionof 7 8 9 Antigen on CFCs in Human Bone Marrow Cells Sorted (%) Experiment 1 7B9+ 709Unseparated Experiment 2 709+ 709Unseparated 13 85 13 86 CFU-GM BFU-E CFU-E ND 564 zt 89 (54)’ 74 IT 7 (46) 133 ? 7 ND 1095 f 28 (65) 89 i 8 (35) 132 i 10 687 f 127 (78) 29 2 6 (22) 60 f 7 ND and SchleyerMand screened for the ability to support the growth in vitro of the desired types of CFCs. Long-term marrow cultures. Separated and unseparated marrow cells were placed in a long-term marrow culture system (LTMC) as previously de~cribed.~’ Briefly, irradiated adherent cell layers from 2 to 4-week-old LTMC were used as feeder layers for isolated marrow cells. Cells were inoculated onto irradiated layers and cultured for 5 to 7 days at 37”C, after which time cultures were maintained in a 33°C incubator. At weekly intervals, the cultures were fed by removing half of the culture supernatant and replacing it with fresh medium. The nonadherent cells removed with the culture supernatant were assayed for the presence of CFCs. Immunofluorescence and sorting. Cells were stained usingprevi- A Results are the mean ? standard error of colonies expressed per 1O5 cells. Abbreviation: ND, not done. In parentheses are the percentage of all CFCs sorted that are in that population. Bone marrow cells were stained and separated by indirect immunofluorescence and fluorescence activated cell sorting, as described in Materials and Methods. IO6 CD34+ normal bone marrow cells on days 0,7, 14 and 21, and with 1.5 X IO6 cells intravenously(IV) on day 32. The spleens were removed 4 days after the last immunization, and spleen cells were fused with SP2/0 cells, as described.” Supernatants from fusion wells were tested IO to 14 days later by enzyme-linked immunoadsorbent assay (ELISA). Supernatants were diluted 1:5 and tested using the SBA Clonotyping System 111 (Southern Biotechnology Assoc, Inc, Birmingham, AL) to select wells that were producing IgG or IgM antibodies. Supernatants were then tested by two-color indirect immunofluorescenceon normal human bone marrow by staining the test antibody with FITC goat-antimouse IgG, and counterstaining with antibody 12-8 followed by PE goat-antimouse IgM for the IgG-producing wells. For the IgM-producingwells, test antibodies were stained with the anti-IgM reagent, and CD34 stained with MY-10 followed by the anti-IgG second step. Hybridomas were cloned by limiting dilution techniques and selected for further study if they secreted antibody that bound to a portion of the CD34+cells, but not a large percentage of the CD34- cells. Cells from the hybridoma line producing 7B9 were expanded in vitro and injected into the peritoneal cavities of pristane-primedBalb/c mice. Ascites fluid was collected from these mice and 7B9 was purified from the ascites fluid by high pressure liquid chromatography (HPLC). Colony-forming assays. CFU-GM, CFU-E, and BFU-E were identified by culturing cells in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO) supplemented with 20% fetal bovine serum (FBS), (Hyclone), 20% human placental conditioned medium (HPCM), 2 IU/mL human erythropoietin (Amgen, Inc, Thousand Oaks, CA), mol/L 2-mercaptoethanol(2-ME)(BioRad Laboratories, Richmond, CA), and 0.3% agar (Seaplaque; FMC Corp, Rockland, ME), or in methylcellulose (0.88% Terry Fox Cancer Research Center, Vancouver, BC, Canada) with 20% FBS, 2% Bovine Serum Albumin (Intergen CO, Purchase, NY), HPCM, erythropoietin, and 2-ME. All cultures were incubated at 37°C with 5% CO2in air, in a humidified incubator. CFU-E were scored after 7 to 9 days, and CFU-GM and BFU-E were scored after 14 to 16 days of culture using an inverted microscope as previously de~cribed.2~ HPCM was prepared using the methods of Schlunck 5 n E 3 z Fluorescence Intensity Fig 1. Fluorescence histogram of 7 8 9 staining on bone marrow cells showing log fluorescence intensity (x axis) versus cell number (y axis). W i d line is 789, dashed line is 31.A, an isotype-matched nonspecific control antibody. The vertical lines show the sorting windows used, with all cells to the left of the left line sorted as the 7 8 9 population and the cells to the right of the right line sorted as the 7B9+ population. (A) One color 7B9 staining. (B) Two-color staining with 7B9 and anti-CD34 antibody 12-8, showing 7 8 9 staining on the CD34+ population. - From www.bloodjournal.org by guest on February 2, 2015. For personal use only. BRASHEM-STEIN ET AL 794 . .. . 31.A 31.A N 7 0 I 789 789 Log FIuoresce nce ously described indirect immunofluorescent antibody staining techn i q u e ~ . ~ , ~For , * * ,single-color ~~ fluorescence studies, I O7 cells/mL were incubated with antibody for 20 minutes at 4"C, washed twice, and then incubated in FITC-conjugated goat antimouse IgG plus IgM antiserum for 20 minutes at 4°C. Cells were then washed twice and kept at 4°C until analysis and cell sorting. For two-color staining to study the coexpression of 7B9 antigen and CD33 or CD34, cells were incubated with a mixture of 7B9 (IgG1) and the anti-CD33 antibody L4F3 or the anti-CD34 antibody 12-8 (IgM). Cells were then washed and incubated with a mixture of FITC goat-antimouse IgG and PE goat-antimouse IgM antisera. All stainingwas done with cellsresuspended in PBS supplemented with 2% human AB serum. Sorting techniques for one- and two-color fluorescence have been previously described.'." Cells considered positively stained displayed a fluorescence intensity greater than 96% to 99% of cells stained with an isotype-matched control antibody. All analysis and sorting was done on a modified Becton Dickinson FACS I1 flow cytometer (Becton Dickinson, San Jose, CA). Radiolabeling, immunoprecipitation. and gel analysis. Radioimmunoprecipitation studies were performed as de~cribed.~' Cells were labeled by cell surface lactoperoxidase iodination.33Cells were washed 3 times with PBS pH 7.0 and resuspended to 5 X lo7 cells/ mL in PBS containing glucose (J.T. Baker, Phillipsburg, NJ) (90 mg/ 100 mL PBS). Iz5I(2 mCi), 20 pL glucose oxidase (Calbiochem) (5 mg/mL) and 20 pL lactoperoxidase (Calbiochem) (70 IU/mL) were added, in order, to 1 mL of cells at room temperature. The cells were incubated for 20 to 25 minutes at room temperature and then washed three times with PBS. The radiolabeled cells were lysed with 50 mmol/L Tris-HC1 pH 7.6 (Sigma, St Louis, MO), 150 mmol/L NaC1, 2% Triton X-100 (Sigma), 2 mmol/L phenylmethylsulfonylfluoride (Boehringer Mannheim, Indianapolis, IN) and I % (wtivol) aprotinin. The lysate was centrifuged and the supernatant was precleared with irrelevant antibody (3 l .A) sepharose con- Fig 2 . Fluorescence scatter analysis of bone marrow cells stained with H12C12 (IgM control), 31.A (lgG control). 7B9. and L4F3 (anti-CD33) is shown as a two-dimensional representation of red (L4F3) y axis fluorescence versus green (7B9) x axis fluorescence. The windows used for sorting have been outlined and labeled. (1) 7B9-CD33+, (2) 7B9+ CD33+, (3) 7B9+ CD33-. (4) 7 8 9 - CD33-. jugate (100 p L of a I :1 suspension containing 50 pg antibody) for 20 minutes at 4"C.'4 The radiolabeled precleared lysate (200 pL) was then incubated with each monoclonal antibody sepharose conjugate for 2 hours at 4°C. The beads were washed twice with lysis buffer (50 mmol/L Tris, pH 8.0, 0.15 mmol/L NaCI, 20 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, 2% TX-100) and twice with 50 mmol/L Tris-HC1 containing 0.5% NP-40 (Sigma) and 150 mmol/L NaCl. The radiolabeled protein was released by addition of 60 pL sample electrophoresis buffer (0.125 mol/L Tris-HC1,2.5% sodium dodecyl sulfate (SDS) 25% glycerol, 0.002% bromophenol blue with or without 2.5% 2-ME) and was heated at 100°C for 5 minutes. Immunoprecipitated proteins were separated by electrophoresis in 8% polyacrylamide gels in the presence of SDS under reducing and nonreducing condition^.^^ The gels were dried and radiolabeled bands identified by radioautography. RESULTS Expression of 7B9 antigen by normal marrow hematopoietic progenitors. Table 1 shows two of three experiments in which normal bone marrow was separated by flow cytometry into populations that stained positively or negatively with 7B9, and tested for the presence of CFC. The 7B9+ population was found to contain 90% of the BFU-E in both experiments, and 78% of the CFU-E (experiment 2). This 7B9+ population was also found to contain 54% and 65% of the CFU-GM in experiments 1 and 2, respectively. A fluorescence histogram of the staining of 7B9 on bone marrow cells is shown in Fig 1A. Expression oj'7B9 antigen on CD34' normal marrow progenitor ceLls. To better determine the reactivity of 7B9 on the bone marrow progenitor population known to be vir- From www.bloodjournal.org by guest on February 2, 2015. For personal use only. CD33 AND 789 EXPRESSION ON PROGENITOR CELLS 795 Table 2. ExDression of 7B9 and CD33 on CFCs in Human Bone Marrow Experiment 1 Cells sorted (%) CFU-GM BFU-E CFU-E Experiment 2 Cells sorted (%) CFU-GM BFU-E CFU-E Experiment 3 Cells sorted (%) CFU-GM BFU-E CFU-E Experiment 4 Cells sorted (%) CFU-GM BFU-E CFU-E Experiment 5 Cells sorted (%) CFU-GM BFU-E CFU-E Experiment 6 Cells sorted (%) CFU-GM BFU-E CFU-E 769-033- 7B9-CD33' 18 0 0 18 2 8 (5) 66 23 f 3 (25)' 0 3 f 3 (3) 14 0 2+2(1) BO I 12 (13) 70 81 f 13 (55) 1 i l(1) 3 t 1 (2) 10 0 0 0 7B9TD33- 7B9+CD33+ Unseparated 2 0 300 f 100 (8) 900 2 200 (30) 14 317 i 6 7 (75) 500 f 29 (92) 267 f 33 (62) 5924 35 f 6 33 2 3 2 140 F 76 (3) 842 k 182 (34) 3,333 rt 333 (75) 15 291 f 3 (42) 209 f 91 (64) 58 f 21 (10) 52 f 5 21 f 3 20 f 4 15 2,300 f 322 (93) 0 40 f 40 (22) 47 f 5 9 f 3 62 f 5 71 20 f 20 (4) 0 0 4 260 k 78 (3) 200 f 71 (100) 520 rt 136 (78) 17 7 2 7 (2) 20+0(15) 7 f 7 (10) 71 13f 13 (15) 0 0 2 250 rt 50 (8) 933 rt 17 (85) 517 60 (90) 9 500 f 60 (74) 0 0 96 f 5 4 9 f 16 17f7 11 60 f 23 (2) 27 f 7 (10) 293+47(17) 69 40 i 23 (10) 0 0 2 333 I 101 (2) 1,267 I 101 (90) 6,317 f 368 (68) 20 1,200 f 231 (85) 0 133 f 88 (14) 196 i 12 3 B i 10 179 f 10 7 0 13 i 7 (2) 220f 31 (8) 67 13 f 1 3 (7) 0 0 17 rt 17 (0.2) 683 f 109 (16) 2,900f 1,102(21) 22 533 f 186 (93) 233 f 88 (83) 633 k 120 (71) 130f2 48 2 0 136 f 12 1 Bone marrow cells were stained and separated by indirect immunofluorescence and fluorescenceactivated cell sorting, as described in Materials and Methods. Results are the mean f standard error of triplicate cultures, expressed as colonies per 1O5 cells. The percentage of sorted CFC detected in each population is shown in parentheses. tually all CD34+, we analyzed the expression of 7B9 on CD34+ bone marrow cells by two-color immunofluorescence (Fig 1B). In each of two experiments, approximately half of the CFU-GM were in the 7B9+CD34+group (52% and 49%), whereas the majority of BFU-E were in the 7B9TD34' group (90% and 82%). Expression of CD33 and 7B9 antigens by normal marrow progenitor cells. Because CD33 is also known to be expressed by most progenitors, we compared the expressionof CD33 and 7B9 antigens on these cells using two-color immunofluorescence. Cells were separated into groups that Table 3. Long-Term Bone Marrow Cultures of 7B9+ and 789- Cells Start of Culture Unseparated 789+ 789- Week 5 of Culture GM BFU-E GM BFU-E 132210 1,095f28 89 f 8 52f5 815f67 142 1 42f19 0 29511 7f5 0 I f 1 Results are the mean +_ standard error of colonies expressed per 1O5 cells plated, cultured in triplicate. The data shown were from the same experiment as 2 in Table 1. did or did not stain with 7B9 and/or anti-CD33 antibody (Fig 2). The majority of sorted BFU-E (85% to 100%)and CFU-E (82%to 100%)was found in either the 7B9+CD33+ or 7B9+CD33- groups. In six experiments, a range of 0%to Table 4. Reactivity of 7B9 W i t h Normal Bone Marrow Cells Determined by Indirect Immunofluorescence and Cell Sorting Myeloblast Immature myeloid Mature myeloid Erythroblast Immature erythroid Monocyte Eosinophil Basophil Lymphocyte 7B9+ 789. 8.7 i 5.5 43.3 rt 18.8 8.3 f 3.2 4.0 1 .O 4.7 f 4.6 9.7 f 10.3 19.7f 13.9 1 .o rt 1 .o 0.7 f 0.6 1 .o f 1 .o 53.3 f 11.5 30.7 i 4.0 0.3 0.6 9.0 i 6.9 1 .o i 1 .o 1.3 1.5 0 4.0 f 5.3 * * * Numbers represent the mean and standard deviation of three different sorts in which 100 cell differentials were performed on Wright-Giemsa stained cytocentrifuge preparations. The immature myeloid group contains promyelocytes, myelocytes, and metamyelocytes; mature myeloid is bands and PMN's; and immature erythroid is all nucleated red blood cell precursors except erythroblasts. From www.bloodjournal.org by guest on February 2, 2015. For personal use only. BRASHEM-STEIN ET AL 796 KG-I I HL-60 Fluorescence Intensity 92% of the BFU-E was in the 7B9+CD33+group, with 8% to 100%ofthe BFU-E in the 7B9+CD33- group. Similarly, 0% to 7 1% of the CFU-E sorted into the 7B9+CD33+group, and 21% to 90% of the C N - E sorted into the 7B9TD33group. A minor portion of BFU-E and C N - E expressed neither the 7B9 or CD33 antigens (Table 2). The majority of sorted CFU-GM (89% to 100%)was found in the 7B9-CD33+ or the 7B9+CD33+groups, with only 0% to 10%of CEU-GM found in the 7B9+CD33- or 7B9-CD33- groups. The CFU-GM were predominantly in the 7B9+CD33+group in the six experiments (42% to 93%). In these six experiments, we also found that most of the CFU-GM were 7B9+ (45% to 96%). In one experiment, we also assessed the morphology of the CFU-GM by picking colonies from the methylcellulose plates and performing differentials on Wright-stained cytospin preparations. Colonies derived from both the 7B9-CD33+ and 7B9+CD33+ groups showed 5 of 10 colonies picked contained granulocyte precursors, with the remaining cells being monocytes and macrophages (data not shown). Lack of expression of 7B9 antigen by LTMC initiating cells. We tested the ability of isolated 7B9+ and 7B9marrow cells to generate or maintain CFC in a long-term culture system. Sorted cells were cultured in the presence of previously established irradiated marrow stromal cell layers. The entire layers were harvested after 5 weeks of culture and tested for CFC activity. The cultures of 7B9- cells were found to contain CFU-GM and BFU-E after 5 weeks in culture, whereas the 7B9+-sorted cells did not, suggesting the presence of precursors of both CFU-GM and BFU-E in the 7B9- but not the 7B9+ population (Table 3). Normal peripheral blood and bone marrow cells. In four experiments looking at peripheral blood mononuclear cells by immunofluorescence and gating on cells with high forward light scatter, 68.4% k 2.9% ofthe cells reacted with the anti-CD36 antibody 5F1, and 81.3% +- 5.4% of the cells reacted with 7B9. In contrast, no reactivity with granulocytes was found in two-color studies with 7B9 and the anti- Fig 3. Fluorescence histogram of 7B9 staining on hematopoietic cell lines, showing fluorescence intensitv (x axis) versus cell number I.. v axis). . Solid line is .769, dashed line is 31.A. an isotypematched nonspecific control antibody. CD15 antibody 1GIO. Platelets, which stained positively with the anti-CD42b antibody C7E10, also all reacted with 7B9 (data not shown). Reactivity of 7B9 with a portion of T lymphocytes was shown by gating around the lymphocyte sized population based on forward and right angle light scatter characteristics. In four experiments, 82.7% f 0.5% of this population consisted of T cells, as determined by staining with antiCD2 antibody 35.1. In this same population, 36.9% f 5.4% of the cells reacted with 7B9; therefore, at least 19.6%of this population must coexpress 7B9 and 35.1. In two color studies, no reactivity of 7B9 was seen with CD4+ cells, detected by antibody 66.1. Antigen 7B9 was also detected on 80% of PHA-activated T cells, and 25% of thymocytes. Studies of B-lymphocyte reactivity, tested by using direct conjugates of an anti-CD20 antibody (FITC-IF5) and 7B9 (Cy-5-789) revealed low levels of 7B9 antigen on 34% of CD20+ cells (data not shown). In bone marrow, 7B9 stained 10% to 15% of mononuclear cells, of which 70% to 80% displayed high forward light scatter. Cytospin preparations of positively stained marrow cells selected by cell sorting showed mainly myeloid cells including myeloblasts, immature myeloid cells and eosinophils, erythroblasts, and small percentages of immature erythroid cells (Table 4). Leukemic cells and cell lines. Our panel of human hematopoietic cell lines was analyzed three times for 7B9 reactivity (Fig 3). The myeloid cell lines HEL, HL-60, and U-937, the B-lymphocyte lines Raji and Nalm-6, and the T-lymphocyte line HSB-2 all were reactive with antibody 7B9. In addition, the myeloid line KG-I showed a slight shift in its fluorescence intensity when stained with 7B9 compared with the negative control antibody, and a small number of the cells of the T-cell line Jurkat expressed 7B9 antigen. The cell line K562 was negative for 7B9. The myeloid cell line KG-la and the mouse L cell line FMY 9S5 Clone 7, which expresses CD33, were 7B9 negative (data not shown). From www.bloodjournal.org by guest on February 2, 2015. For personal use only. CD33 AND 789 EXPRESSION ON PROGENITOR CELLS 797 HEL /.* RAJ1 . .. ,,..,,... '.*:s$r . -200 -97 -69 97 69 -46 ,46 -30 .30 __ R NR R NR Fig 4. Immunoprecipitation of the 789 antigen from HE1and Raji cells under reducing (R) and nonreducing (NR) conditions. Antibodies 789. p67-6 (anti-CD33). 1F5 (anti-CDPO), and 31.A (an irrelevant control antibody) were tested. Molecular weight was determined with ['4ClLeucine-labeled markers (New England Nuclear, Boston, MA). On Raji cells, 789 precipitates a broad band of 63 to 73 Mr under reducing conditions, and a band of 60 to 70 Mr under nonreducing conditions. On HEL cells, 789 precipitates a band of 56 to 90 Mr under reducing conditions, and a band of 56 to 82 Mr under nonreducing conditions. On the Raji cells, the positive control, precipitated with antibody 1F5 (anti-CDPO),yielded a band at 37 Mr, just above a nonspecific band in all lanes. The negative control lysate, precipitated with antibody 31 .A, showed a nonspecific band at 75 Mr in the reducing lanes and severalfaint bands in the nonreducinglanes. On the HELcells, the positive control p67-6 (antLCD33 antibody) precipitated a broad band just below the 69 Mr marker on reduction and slightly lower in the nonreducinglane. Both 789 and p67-6 precipitated a nonspecific band at 35 Mr, and antibody 31 .A precipitated one nonspecific protein at 58 Mr under reducing conditions. Leukemia cells in IO of 14 acute lymphocytic leukemia (ALL) specimens, including 3 of 3 T-cell leukemias, bound 7B9 with between 35% and 85% ofthe cells showing reactivity. Four of five bone marrows from patients with acute myeloid leukemia (AML) were also found to express 7B9 antigen (data not shown). Characterization of the cell &ace molecule recognized by 7B9. Radioiodinated Raji cell lysates were precipitated with antibody 7B9 and electrophoresed. Under reducing conditions, a broad band of 63 to 73 Mr was consistently observed, whereas nonreducing conditions showed a band of slightly lower molecular weight, 60 to 70 Mr. (Fig 4). HEL cell lysates precipitated with 7B9 showed a broader band in the reducing lane of 56 to 90 Mr. The nonreducing band is slightly lower at 56 to 82 Mr. The broad bands ofthe 7B9 antigen precipitated from both cell lines are consistent with profiles seen for heavily glycosylated proteins. DISCUSSION The identification of cell surface antigens expressed by hematopoietic precursors at different stages of maturation has led to the development of methods for purifying distinct subpopulations of these cells. Thus, it has been possible to distinguish CFCs committed to express a single lineage from less mature cells capable of establishing hematopoiesis in long-term culture or giving rise to multipotential blast colony-forming The former cells have been shown to express the CD34 antigen as well as a variety of determinants associated with differentiative expression along the various hematopoietic lineages, including CD33 and HLA/DR, whereas the less mature cells appear to lack these differentiation antigens. In the present study, we have identified a novel epitope, identified by 7B9, that within the population of CD34+ marrow cells is, like CD33, expressed by CFCs and not their precursors detectable in long-term culture. In these experiments, virtually all of the BFU-E were 7B9+,whereas a variable percentage of CFU-GM were 7B9+. Of particular interest is the observed differential expression of the 7B9 and CD33 antigens on subsets of CFCs, with sole detection of 7B9 antigen on a portion of BFU-E and CFU-E, but only rarely on CFU-GM; and sole detection of CD33 on a portion of CFU-GM, but only rarely on BFU-E or CFU-E. The remaining progenitors display a 7B9+CD33+ phenotype From www.bloodjournal.org by guest on February 2, 2015. For personal use only. 798 and consist of a mixture of erythroid and myeloid progenitors. Thus, the populations expressing only one of the two antigens displayed greater enrichment for either erythroid or myeloid differentiation than the cells expressing both antigens, which displayed both differentiative potentials. These observations are consistent with the idea that CFCs selectively lose o r decrease their expression of 7B9 antigen as they differentiate along the granulocyte/monocyte pathway, or lose or decrease their expression of CD33 as they differentiate along the erythroid lineage. Furthermore, we do not know if the cells completely cease to express one of the antigens, or if they continue to express one antigen in a n amount that falls below our threshold ofdetection. Nonetheless, it is possible for antibodies detecting these antigens to be used to enrich for myeloid or erythroid subsets of CD34' progenitors. More intriguing is a potential role of these surface antigens in regulating the differentiative and proliferative potential of these cells. To date, we have been unable to influence the proliferation of CD34+ marrow cells in the presence ofhematopoieticgrowth factors by adding antibodies against these antigens either in solution or by adherence to plastic (data not shown). Although the CD33 gene has been cloned,"' sequence data has not provided sufficient information to determine function. However, sequence homology between CD22 and myelin-associated glycoprotein has been observed."' The former has been found to have a role in signaling B cells to proliferate, whereas the latter is thought to play a role in cell adhesion. Nevertheless, it is tempting to speculate that the common expression of 7B9 antigen and CD33 followed by exclusive decrease o r loss of one of these antigens by hematopoietic progenitors of different types suggests these molecules may play a role in influencing the differentiative expression of these cells. This could result, for example, if binding of these molecules to the appropriate ligand provided a synergistic signal in the response to growth factors, or if this binding interaction would cause these cells to bind in a n appropriate niche in the marrow space. The chemical nature of the 7B9 determinant is uncertain. Although it has been possible to precipitate a protein or glycoprotein from cells, the broad band o n polyacrylamide gel electrophoresis and differences in the molecular weight observed using different cell lines is consistent with the possibility that the epitope detected represents a carbohydrate determinant. This would also explain the lack of success in efforts to clone the gene encoding for this antigen (datanot shown). However, the antigen that we detected does appear novel because a similar one has not been previously described. Antibody 7B9 shows some similarity in its patterns of reactivity to the previously described antigen RFB- 1,'5,41 but differences in cell line reactivity suggest that this may not be the case, and biochemical data on the nature of the RFB- I antigen is not available. The development of antibody 7B9 has practical implications. Subsets of maturing progenitors could be isolated based o n the common o r selective expression of 7B9 antigen and CD33. 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For personal use only. 1993 82: 792-799 Ontogeny of hematopoietic stem cell development: reciprocal expression of CD33 and a novel molecule by maturing myeloid and erythroid progenitors C Brashem-Stein, DA Flowers, FO Smith, SJ Staats, RG Andrews and ID Bernstein Updated information and services can be found at: http://www.bloodjournal.org/content/82/3/792.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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