Ficoll-Hypaque (Accurate Chemical & Scientific Corp., Westbury, NY) (600 g, ... (1:4) or a human IgG1 myeloma protein (30/~g/ml, provided by Dr. Gerald ...
Brief Definitive Report
POLYMORPHONUCLEAR COMMON
ACUTE
NEUTROPHILS
LYMPHOBLASTIC
EXPRESS
LEUKEMIA
THE
ANTIGEN
BY JEFFREY COSSMAN, LEONARD M. NECKERS, W A R R E N J. LEONARD, AND W A R N E R C. GREENE
From the Hematopathology Section, Laboratory of Pathology, and the Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 T h e a n t i g e n associated with most cases of non-T, non-B acute l y m p h o b l a s t i c leukemia, as first described by Greaves a n d referred to as the c o m m o n a c u t e l y m p h o b l a s t i c l e u k e m i a a n t i g e n ( C A L L A ) , is a 95,000-100,000-mol wt surface m e m b r a n e g l y c o p r o t e i n identified b y b o t h h e t e r o a n t i s e r a a n d m o n o c l o n a l antibodies, J5, V I L - A 1, a n d BA-3 (1-5). Because of the occurrence of C A L L A on the cells from most cases of non-T, non-B acute l y m p h o b l a s t i c l e u k e m i a ( c o m m o n A L L ) , it is a useful m a r k e r for the diagnosis o f this disorder. In a d d i t i o n , J 5 a n t i b o d y has been administered as passive s e r o t h e r a p y in c o m m o n A L L (6). Recently, this antigen has been d e t e c t e d on a variety of o t h e r cell types i n c l u d i n g some n o r m a l b o n e m a r r o w cells, as m a n y as 25% of cases o f T cell acute l y m p h o b l a s t i c l e u k e m i a ( T - A L L ) , Burkitt's l y m p h o m a , follicular l y m p h o m a , n o r m a l renal t u b u l a r a n d g l o m e r u l a r e p i t h e l i u m , fetal small intestine, a n d breast m y o e p i t h e l i u m (7, 8). D u r i n g the course of investigations on the expression of C A L L A in a variety of h u m a n h e m a t o p o i e t i c m a l i g n a n cies, we observed reactivity of m o n o c l o n a l a n t i - C A L L A a n t i b o d i e s with n o r m a l p o l y m o r p h o n u c l e a r n e u t r o p h i l s (PMN). This reactivity a p p e a r s to be specific a n d not m e d i a t e d by Fc receptors. Since the presence of C A L L A positive cells can be considered indicative of involvement b y l y m p h o m a or leukemia, the expression of C A L L A b y P M N c o u l d p r o d u c e falsely positive results when C A L L A is e x a m i n e d for in bone m a r r o w aspirates, p e r i p h e r a l b l o o d or other b o d y fluids. Materials and Methods Cells. PMN were isolated from the peripheral blood of 13 healthy normal adult volunteers. The granulocyte- and erythrocyte-rich fraction was separated by gradient centrifugation with Ficoll-Hypaque (Accurate Chemical & Scientific Corp., Westbury, NY) (600 g, 30 min) then mixed 1:1 with Plasma-Gel (HTI Corporation, Buffalo, NY) for 30 min at 37°C to allow the erythrocytes to settle out. The granulocyte-rich supernatant was washed three times in medium RPMI 1640 (Gibco Laboratories, Grand Island Biological Co., Grand Island, NY) and resuspended at 3 × 10*/ml. Purity of the preparations was documented morphologically on cytocentrifuge preparations. Cell lines studied included Raji (Burkitt's lymphoma), NALM-1 (lymphoblast crisis of chronic myelogenous leukemia), MOLT-4 (T ALL), and HL60 (promyelocytic leukemia). The induction of HL60 cells to more mature myeloid cells was performed with dimethyl sulfoxide (DMSO) (1.25%; Microbiological Associates, Walkersville, MD) and 12-tetradencanoyl-phorbol-13-acetate (TPA) (10 -s M; Sigma Chemical Co., St. Louis, MO) as previously described (9, 10). Cell suspensions were prepared from lymph nodes involved by follicular lymphoma (12 cases) by pressing teased-out cells through a stainless steel mesh and washing in RPMI 1640. Irnmunostaining. Indirect immunofluorescence was performed by incubating 2 × 105 ceils 1(164
Journal of Experimental Medicine • Volume 157, March 1983 1064-1069
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with 100 /.tl of primary antibody or control immunoglobulin in phosphate-buffered saline containing bovine serum albumin (2%) and sodium azide (0.2%) for 30 min on ice. After three washes, 100/tl of the fluorescein-conjugated, affinity-purified IgG fraction of goat anti-mouse IgG (1:40, Kirkegaard and Perry Co., Kensington, MD) was added for another 30 min on ice. Murine monoclonal anti-CALLA antibodies included (a) J5 in three forms (1/xg/ml): whole ascites and IgG-F(ab')2 (kindly provided by Dr. Jerome Ritz, Harvard University) and IgG fraction (Coulter Corp., Hialeah, FL); (b) BA-3 antibody (ascites, 1: 100) (4, 5) (courtesy of Dr. Tucker Le Bien, University of Minnesota); and (c) VIL-A1 antibody (3) (courtesy of Dr. Walter Knapp, University of Vienna). Other monoclonal antibodies included O K M 1, OKT6 (Ortho Pharmaceuticals, Raritan, NJ), and Leu-1 (Becton, Dickinson & Co., Sunnyvale, CA); all were used at 1 /~g/ml. Additional controls included a series of nonreactive, purified murine monoclonal (hybridoma) IgG proteins that were substituted for primary antibody and included one each of the IgG classes IgG1, IgG2a, IgG2b, and IgG3 (all at 30/xg/ml) (provided by Dr. Kenneth Schroer, National Institutes of Health). Nonreactive ascites from the IgGl-producing murine myeloma P3 × 63 (Bethesda Research Laboratories, Rockville, MD), was also used as a negative control. In some instances heat-inactivated (56°C, 30 minutes) normal human serum (1:4) or a human IgG1 myeloma protein (30/~g/ml, provided by Dr. Gerald Crabtree, National Institutes of Health) were added to PMN (37°C, 30 min) before immunostaining. Cells were subsequently washed in ice-cold buffer and stained as described above. Fluorescence Detection. Fluorescence was assayed on a fluorescence-activated cell sorter (FACSII, B-D FACS Systems, Becton, Dickinson & Co), at 500 mW of laser power (argon laser), 488 nm wavelength, 630 V photomultiplier tube sensitivity, and a fluorescence gain setting of 4 on a scale of 16. The data was recorded on a PDP-11/34 computer (Digital Electronic Corp., Marlboro, MA), 1023-channel histograms were generated and the percentage of fluorescent cells as well as the mean fluorescence intensity (mean channel number) were calculated. Fluorescence microscopy was performed on a Leitz Orthoplan equipped with epifluorescence at 450-490 nm excitation and 515 nm suppression filters. Immunoprecipitation and Gel Electrophoresis. Cell surface membrane proteins or intact cells were labeled with 125I using laetoperoxidase and hydrogen peroxide (11). The cells were then solubilized in 10 mM TRIS buffer (pH 7.4) containing 0.14 M NaCI, 1% Triton-x-100, and 100 /lg/ml phenylmethylsulfonyl fluoride. After centrifugation, these cellular extracts were then immunoprecipitated with J5 (2/~g/5-10 × 106 cells) and analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) as described previously (I 1). Parallel control immunoprecipitations were also performed with UPC10, a nonreactive murine IgG2a (Litton Bionetics, Bethesda, MD). Results T h e g r a n u l o c y t e fraction from all n o r m a l i n d i v i d u a l s tested stained w i t h J5, V I L A1, a n d BA-3 (mean 89%, range 55-99%). All three p r e p a r a t i o n s o f J5, n a m e l y , ascites, i n t a c t IgG, a n d F(ab')2, r e a c t e d with granulocytes (Fig. 1 A, B). G r e a t e r t h a n 90% o f cells s t a i n e d with O K M 1 , however, no fluorescence was observed with the n o n r e a c t i v e mouse ascites, purified m u r i n e IgG1, IgG2a, IgG2b, IgG3, a n t i b o d i e s O K T 6 a n d Leu-1, or after omission o f p r i m a r y a n t i b o d y . F u r t h e r , p r e i n c u b a t i o n o f cells w i t h n o r m a l h u m a n serum or h u m a n IgG1 d i d not affect either the p e r c e n t a g e o f cells s t a i n e d b y J 5 or the intensity o f fluorescence (Fig. 1 C). T o d e t e r m i n e w h e t h e r a n t i - C A L L A a n t i b o d y could distinguish between n e u t r o p h i l s a n d eosinophils, the leukocytes from n o r m a l i n d i v i d u a l s a n d from a p a t i e n t w i t h h y p e r e o s i n o p h i l i a were s t a i n e d w i t h the F(ab')2 fraction o f J 5 a n d sorted into fluorescent-positive a n d - n e g a t i v e fractions on the F A C S . T h e cells were collected, cytocentrifuged, s t a i n e d w i t h W r i g h t ' s stain, a n d differential counts were performed. T h e positive fractions were g r e a t l y e n r i c h e d for P M N (95 a n d 100% for n o r m a l s a n d 86% for the hypereos i n o p h i l i a p a t i e n t ) , whereas the negative fractions were p r e d o m i n a n t l y eosinophils, l y m p h o c y t e s , a n d monocytes. T h e fluorescence intensity of neutrophils stained with
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A /~ Control / I~I ~(LGranulocytes
=~ranulocytes
/',,/\
0~
uJ
J
LOG SCALE
Z -~ .J uJ
¢J
D
C
Follicular lyrnphoma anullcytes
Control , ARaji
FLUORESCENCEINTENSITY FIG. 1. (A) J5 staining of granulocytes as compared with control (murine IgG2a) shows 98% of cells stained. Linear scale. (B) Log transformation of histogram from A amplifies the positive staining reaction of granulocytes with J5. Log scale (two decades). (C) Granulocytes and follicular lymphoma had comparable fluorescence intensity when stained with J5. These granulocytes were pretreated with normal human serum and showed no diminution of fluorescence intensity when stained with J5. Preincubation with human IgG1 myeloma protein or staining with the F(ah')2 fraction of j5 showed similar fluorescence (not shown). The small negative peak in the granulocyte fraction was sorted and proved to be largely eosinophils.Linear scale. (D) The Burkitt's lymphoma ceil line, Raji, and a common ALL cell line, NALM-1, reacted with J5 and the relative intensity of the fluorescencewas greater than that of granulocytes. Linear scale, The control in all cases was a murine IgG~ monoctonal protein. All other negative controls produced similar results (see text). Each curve includes I0,000 cells counted.
j5
was c o m p a r a b l e to that observed with follicular l y m p h o m a cells b u t less t h a n that found with Raji cells (mean c h a n n e l 160) a n d NALM-1 cells (mean c h a n n e l 180) (Fig. 1 D). T h e average m e a n fluorescence c h a n n e l was 83 for P M N (10 samples) a n d 102 for follicular l y m p h o m a s (12 samples). IgG-J5 also reacted with HL60; however, this interaction a p p e a r e d to be m e d i a t e d t h r o u g h Fc receptors since (a) s t a i n i n g was i n h i b i t e d by p r e i n c u b a t i o n with h u m a n serum or h u m a n IgG1 m y e l o m a protein, a n d (b) F(ab')2 J5 did not react with HL60. A d d i t i o n of either T P A or D M S O to HL60 cells did not result in i n d u c t i o n of specific J5 reactivity d u r i n g a 5-d course. T h e m e m b r a n e d e t e r m i n a n t detected on P M N by a n t i - C A L L A antibodies was biochemically characterized by cell surface labeling, i m m u n o p r e c i p i t a t i o n , a n d SDSP A G E gel electrophoresis. A u t o r a d i o g r a p h s of these gels d e m o n s t r a t e d that J5 antibody identified a protein on the cell surface of P M N of ~95,000-110,000 mol wt, a n d was similar in size to that found on Raji cells (95,000-100,000 mol wt) (Fig. 2). These proteins recognized by J5 were not present in i m m u n o p r e c i p i t a t e s performed with control U P C 1 0 IgG2a m o n o c l o n a l antibodies or on the T cell leukemia cell line, MOLT-4. Discussion These studies d e m o n s t r a t e that the a n t i - C A L L A m o n o c l o n a l antibodies, J5, VILA1, a n d BA-3 consistently react with h u m a n p o l y m o r p h o n u c l e a r neutrophils b u t not
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FIG. 2. SDS-PAGE analysis of lzSI-surface-labeled cells after immunoprecipitation. Molecular weight standards are shown in the left lane. PMN (a, b), Raji (c, d), and Molt-4 (e, f) were immunoprecipltated with a nonreactive murine IgG2a 0JPC10) (a, c, e) or j5 (b, d, f). From PMN, J5 immunopreeipitated a single band that has a molecular weight similar to that of the band obtained with Raji cells. These proteins were not immunoprecipitated with Molt-4 cells or with UPC 10 monoclonal antibodies. with other leukocytes. This reactivity does not appear to be mediated by cell surface Fc receptors since staining persisted after saturation o f these receptors with either h u m a n IgG1 m y e l o m a protein or normal h u m a n serum. Furthermore, the F(ab')2 fraction a n d the intact IgG form of J5 showed equivalent reactivity. O t h e r routine IgG2a antibodies, including Leu-1, did not react with granulocytes. Preparative cell sorting of J5-positive and -negative fractions indicated that the J5-positive cells observed a m o n g granulocytes and unseparated peripheral blood leukocytes were, indeed, P M N . T h e antigen detected on the cell m e m b r a n e of P M N was similar in size to C A L L A present on Raji cells (95,000-110,000 tool wt for P M N a n d 95,000-100,000 mol wt for Raji) a n d was i m m u n o s t a i n e d by three different a n t i - C A L L A antibodies. Thus, the antigen detected on P M N not only shares an epitope with C A L L A , but these antigens m a y be identical. A l t h o u g h blasts from the lymphoblast crisis of chronic myelogenous leukemia (CML) are frequently CALLA-positive (2, 4, 7), C A L L A m a y not be expressed on myeloid precursor cells since; (a) the cells of the stable phase of C M L are reported to lack C A L L A (7); (b) a n t i - C A L L A (J5) treatment does not inhibit proliferation in myeloid clonal assays (12); a n d (c) as observed in the present study, the promyelocytic leukemia cell line, HL60, did not specifically stain with J5. Thus, C A L L A appears to be acquired at a late stage of P M N differentiation. Although a n t i - C A L L A antibodies have a role in the diagnosis of l y m p h o m a and leukemia, a d m i x e d P M N in patient samples could masquerade as CALLA-positive malignant cells. As an example, the pleural effusion from a patient with follicular l y m p h o m a
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Ill
Z LU
'
L) O0 LIJ
nr"
0 ::3 l._I U_
CELL SIZE Fro. 3. Dot plot display of FACS-II analysis of a pleural fluid from a patient with follicular lymphoma stained with J5. A cluster of large, stained cells is seen in the upper right. This stained cell population was sorted and was found to consist of polymorphonuclear neutrophils. c o n t a i n e d 6% J 5 - s t a i n e d cells (Fig. 3). This result was initially i n t e r p r e t e d as i n d i c a t i n g a low level o f i n v o l v e m e n t b y the neoplastic process. However, when these stained cells were sorted on the FA(3S, they p r o v e d to be entirely c o m p o s e d o f P M N . Therefore, c a u t i o n should be exercised when s t u d y i n g effusions, bone m a r r o w aspirates, a n d especially u n s e p a r a t e d leukocyte buffy coats as has been suggested for i m m u n o f l u o r e s c e n c e analysis b y flow c y t o m e t r y (13). Summary M o n o c l o n a l a n t i b o d i e s J5, V I L - A 1 , a n d BA-3, k n o w n to react with the c o m m o n acute l y m p h o b l a s t i c l e u k e m i a antigen ( C A L L A ) were found to specifically stain n o r m a l h u m a n p o l y m o r p h o n u c l e a r n e u t r o p h i l s ( P M N ) . T h e antigen detected on P M N h a d a m o l e c u l a r weight (95,000-110,000 mol wt) close to t h a t o f C A L L A (95,000-100,000 mol wt) a n d thus these surface m e m b r a n e antigens are likely related, if not identical. T h e fluorescent staining intensity of P M N is c o m p a r a b l e to that of C A L L A - p o s i t i v e leukemic cells a n d the presence o f P M N in p a t i e n t samples could p o t e n t i a l l y p r o d u c e false-positive results in diagnosis. Receivedfor publication 13 December 1983.
References 1. Greaves, M. F., G. Brown, N. T. Rapson, and T. A. Lasiter. 1975. Antisera to acute lymphoblastic leukemia cells. Chn. ImmunoL ImmunopathoL 4:67.
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2. Ritz, J., J. M. Pesando, J. Notis-McConarty, H. Lazarus, and S. F. Schlossman. 1980. A monoclonal antibody to human acute lymphoblastic leukemia antigen. Nature (Lond.). 283:583. 3. Knapp, W., O. Majdic, P. Bettelheim, and K. Liszka. 1982. VIL-A1, a monoclonal antibody reactive with common acute lymphoblastic leukemia cells. Leuk. Res. 6:137. 4. LeBien, T., J. Kersey, S. Nakazawa, K. Minato, and J. Minowada. 1982. Analysis of human leukemia/lymphoma cell lines with monoelonal antibodies BA-1, BA-2 and BA-3. Leuk. Res. 6:299. 5. LeBien, T. W., D. R. Boue, J. G. Bradley, and J. H. Kersey. 1982. Antibody affinity may influence antigenic modulation of the common acute lymphoblastic leukemia antigen m vitro. J. Irnmunol. 29:2287. 6. Ritz, J., J. M. Pesando, S. E. Sullan, L. A. Clavell, J. Notis-McConarty, P. Rosenthal, and S. F. Schlossman. 1981. Serotherapy of acute lymphoblastic leukemia with monoclonal antibody. Blood. 58:141. 7. Ritz, J., L. M. Nadler, A. K. Bahn, J. Notis-MeConarty, J. M. Pesando, and S. F. Sehlossman. 1981. Expression of common acute lymphoblastic leukemia antigen (CALLA) by lymphomas of B-cell and T-cell lineage. Blood. 58:648. 8. Metzgar, R. S., M. J. Borowitz, N. H. Jones, and B. L. Dowell. 1981. Distribution of common acute lymphoblastic leukemia antigen in non-hematopoietic tissues. J. Exp. Med. 154:1249. 9. Collins, S. J., F. W. Ruscetti, R. E. Gallagher, and R. C. Gallo. I978. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc. Natl. Acad. Sci. USA. 75:2458. 10. Huberman, E., and M. F. Callahan. 1979. Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents. Proc. Natl. Acad. ScL USA. 76:1293. 11. Leonard, W. J., J. M. Depper, T. Uchiyama, K. A. Smith, T. A. Waldmann, and W. C. Greene. 1982. A monoclonal antibody that appears to recognize the membrane receptor for human T-cell growth factor receptor; partial characterization of the receptor. Nature (Lond. ) . 300:267. 12. Clavell, L. A.,J. M. Lipton, R. C. Bast, M. Kudisch, J. Pesando, S. F. Schlossman, a n d J . Ritz. 1981. Absence of common ALL antigen on normal bipotent myeloid, erythroid, and granulocyte precursors. Blood. 58:333. 13. Hoffman, R. A., P. C. Kung, W. P. Hansen, and G. Goldstein. 1980. Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood. Proc. Natl. Acad. Sci. USA. 77:4914.
