Journal of Clinical Immunology, Vot. 3, No. 3, 1983

Construction of an Antigenic Map for Human B-Cell Precursors G E O R G I A N N B. M E L I N K l and T U C K E R W. L e B I E N ~, 2

Accepted: February 3, 1983

cells and their differentiated progeny (plasma cells), studies of less mature ( s I g ) B cells are notably lacking (1). The first detectable human B-cell precursor, the pre-B cell, is characterized by small amounts of cytoplasmic tx heavy chain (clgM) in the absence of detectable cytoplasmic light chain and slg (2, 3). Based on the immunofluorescent detection of cIgM, pre-B cells have been reported to exist in 14- to 17week fetal bone marrow (I0.3%), liver (4.5%), spleen (0.9%), lymph node (2.9%), and blood (0.2%) (2). Subsequent to birth pre-B cells are not found in any of the above-mentioned organs, but they are found in bone marrow (1-2% of Ficoll-Hypaque interface nucleated cells) throughout life (3, 4). To date, little is known about the cell surface molecules expressed on normal pre-B cells. While lacking some of the characteristic markers of sIg ÷ B cells, such as the third c o m p o n e n t of complement and the Fc receptor for IgG (5), pre-B cells do (similarly to sIg ÷ B cells) express H L A - D R (6). Less than 5% of pre-B cells express the c o m m o n acute lymphoblastic leukemia antigen ( C A L L A ; 6) and the e n z y m e terminal deoxynucteotidyl transferase (7). The advent of hybridoma technology (8) has resulted in the production of numerous monoclonal antibodies to human lymphohematopoietic cell surface molecules. This report describes the results obtained when double fluorochrome analysis was used to analyze the binding of monoclonal antibodies to pre-B cells from fetal, pediatric, and adult bone marrow.

The purpose of this study was to evaluate the binding of a panel of monoclonal antibodies to human pre-B cells present in fetal, pediatric, and adult bone marrow. The antibodies included BA-1, BA-2, BA-3 (anti-CALLA), anti-Bl, L243 (anti-HLA-DR), and T101. Binding of the monoctonal antibodies to pre-B cells was evaluated at the single-cell level by double fluorochrome analysis. Percentages of BA-1 + and anti-B1 + pre-B cells were independent of age group. BA-1 bound to approximately 80% of fetal, pediatric, and adult bone marrow pre-B cells, whereas anti-B1 bound to approximately 50%. BA-2 bound to 55% of fetal pre-B cells, but this percentage decreased to 32% in pediatric and 16% in adult bone marrow. CALLA was expressed on less than 10% of fetal, pediatric, and adult bone marrow pre-B cells, and HLA-DR was expressed on greater than 95% of fetal, pediatric, and adult pre-B cells. Although T101 (an anti-Tcell monoclonal antibody) did not bind to pre-B cells, it did bind to approximately 25% of the sIgM + cells in fetal bone marrow. These results suggest a predominant phenotype of L243 (anti-HLA-DR) +, BA-I +, BA-3 (antiCALLA)-, T101- for the human pre-B cell while phenotypic heterogeneity exists for anti-B 1 and BA-2, KEY WORDS: Pre-B cells; monoclonal antibodies; bone marrOW,

INTRODUCTION The differentiative options and distinct functional stages of human B lymphocytes have been actively investigated during the last several years. While a considerable volume of information has accumulated on surface immunoglobulin-positive (sIg +) B

~Department of Laboratory Medicine and Pathology, University of Minnesota. Minneapolis, Minnesota 55455. 2To whom correspondence should be addressed at Box 609 Mayo, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455.

MATERIALS AND METHODS A n t i b o d i e s . The preparation of affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat

260 0271-9142/83/0709-0260503.00/0 ~ 1983 Plenum Publishing Corporation

26t

ANTIGENS ON H U M A N B-CELL PRECURSORS

anti-human IgM has been described in detail (9), and it was used at a final concentration of 0.4 mg/ml for staining of both sIgM and cIgM. Tetramethylrhodamine isothiocyanate (TRtTC)-conjugated goat anti-mouse Ig polyvalent (Tago Inc., Burlingame, CA), was used at a final dilution of 1:40. Purified human IgM (9) was used for blocking of FITC goat anti-human IgM (see below). Monoclonal antibodies BA-1, BA-2, and BA-3 (anti-CALLA) were produced in our laboratory and have been described in detail (10-12). Briefly, BA-l identifies a cell surface molecule strongly expressed on all normal and malignant slg + cells that is lost as these cells differentiate into plasma cells (10). BA-1 does not bind to normal or malignant T cells but does weakly bind to granulocytes. BA-2 identifies a 24,000 molecular weight cell surface molecule primarily expressed on immature lymphoid cells and acute lymphoblastic leukemias (11). BA-3 identifies the 100,000 molecular weight CALLA (12). Anti-B 1 was purchased from Coulter Electronics (Hialeah, FL) and identifies an antigen expressed on normal and malignant sIg + cells (13). L243, an anti-HLA-DR antibody (14), was purchased from Becton Dickinson (Mountain View, CA). TI01, an antibody identifying normal and malignant T cells and slg + chronic lymphocytic leukemias (15), was kindly provided by Dr. Ivor Royston, UCSD, San Diego, CA. All antibodies were used at concentrations previously shown to give saturated binding conditions. Control ascitic fluid (CAF), obtained by injecting mice with non-antibody-secreting hybridomas, was used as a negative control. Preparation of Bone Marrow Cells. Fetal bone marrow specimens (18-24 weeks) were provided by Dr. R. Vessetla, Tumor Immunology Laboratory, Urologic Surgery, University of Minnesota and VA Medical Center, Minneapolis. Fetal bone marrow cells were obtained by cutting off the ends of long bones and flushing them with 3-10 mt of RPMI 1640-5% fetal bovine serum (FBS) using a 3-ml syringe fitted with a 27-gauge needle. Normal pediatric and adult bone marrow specimens were obtained through the University of Minnesota Bone Marrow Transplant Clinic. All bone marrow samples were initially washed in RPMI 1640-5% FBS by centrifugation at 1200 rpm for 10 rain to remove fatty material normally associated with bone marrow specimens. The cells were then separated on Ficoll-Hypaque gradients using the method of B6yum (16). Interface cells were collected and washed 3 × with RPMI 1640-5% FBS

Journal o f Clinical Immunology, Vol. 3, No, 3, 1983

and kept at 4°C thereafter. Viability of all specimens was greater than 95% when analyzed by trypan blue exclusion. Fluorescent Staining Protocol. Indirect immunofluorescence was used to evaluate the binding of monoclonal antibodies. The Ficoll-Hypaque-isolated bone marrow cells were adjusted to a final concentration of 1 x 107/ml in phosphate-buffered saline (PBS; 0.01 M, pH 7.2) containing 1% FBS. The appropriate dilutions of each antibody or CAF were incubated with equal volumes of bone marrow cells for 30 rain at 4°C. After two washes with cold (4°C) P B S - I % FBS, the celL,~ were stained with TRITC goat anti-mouse Ig for 5 rain at 37°C to enhance patching and capping. They were then washed 3x with cold P B S - I % FBS and stained with FITC goat anti-human IgM for 5 rain at 37°C to enhance capping of sIgM. FolMwing three washes with cold P B S - I % FBS the cells were adjusted to 1 × 106/ml, cytocentrifuged onlLo glass slides, and stained for cIgM as previously described (17). A specificity control for cIgM detection was accomplished by blocking the binding of FITC goat antihuman IgM with 10 ~g of purified human lgM. Detection of cytoplasmic fluorescence in pre-B cells was considerably enhanced by incorporating p-phenylenediamine into the Tris-glycerol mounting fluid (18). A range of 400-1100 cells per slide was examined using a Zeiss fluorescent microscope, with the requirement that at least 10 pre-B cells be included in the count. Ektachrome daylight ASA 400 film was used for photography. Exposure times for FITC, TRITC, and phase contrast were 2.5 rain, 3.25 rain, and 2-5 sec, respectively. Statistics. Statistical analysis was performed using the Kruskat-Wallis one-way ANOVA by ranks test (19) comparing values of fetal bone marrow samples to pediatric bone marrow samples, pediatric bone marrow samples to adult bone marrow samples, and fetal bone marrow samples to adult bone marrow samples. Results are expressed as P values, with 0.05 used as a cutoff for significance. RESULTS

Controls. The problem of distinguishing bone marrow B cells from small pre-B cells was considerably alleviated by capping slgM. A typical protocol for fluorescent staining of sIg + I3 cells in suspension calls for incubation with anti-Ig for 30 rain at 4°C (9), Thus, two controls were included to monitor

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Fig. 1. Immunofluorescent staining of normal pediatric bone marrow cells with FITC goat anti-human IgM. (A) Phase contrast. (B) Same field as in A photographed with a selective filter for FITC. Three capped B cells are shown. (C) Phase contrast of a different field than shown in A. (D) Same field as in C photographed with a selective filter for FITC. Two pre-B cells are shown with typical perinuclear staining. 1000x.

the staining of sIg. The first consisted of several preliminary experiments which indicated that staining with FITC goat anti-human lgM for 5 rain at 37°C gave the same results as staining for 30 rain at 4°C. The second consisted of directly staining an aliquot of cells with FITC goat anti-human IgM for 5 rain at 37°C and reading under wet mount. The

percentage of sIgM + B cells determined by this procedure was eventually compared to the number of sIgM + B cells on slides that had gone through the entire procedure. Examination of 29 bone marrow samples after surface staining gave 7.96 +-- 1.77% sIgM + cells, whereas examination of the same bone marrow samples after surface and cytoplasmic

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Table I. Pre-B Cells, B Cells, and Monoclonal Antibody-Positive Cells in Bone Marrow Specimens Monoclonal antibody reactivity b Source Fetal

Pediatric

Adult

Median age (range)

Pre-B a

B"

BA- 1

BA-2

BA-3

Anti-B 1

L243

TIO1

20 weeks (17-32)

1.8 ± 0.4 ~ (n = 14)d

8.9 ± 1,6 (n = 14)

30.2 +- 5.8 (n = 6)

39.7 -+ 4.0 (n = 10)

5.1 ± 0.7 (n = 3)

9.1 ± 2.4 (n = 7)

30.t ± 4.9 (n = 2)

4.4 -+- t,7

9 years (3-17)

1.95 -+ 0.4 ( n = 15)

8.2 -+ 1,7 (n = 15)

15.0 - 4.6 (n = 7 )

10.2 -+ 2.7 (n = 5)

2.0 ± 1.8 (n = 5)

7.3 +- 2.0 (n = 5 )

26.0 +-- 5.6 (n = 3)

6.8 -+ 3.0

26 years (19-34)

1.95 -+ 0.3 ( n = 7)

8.1 -+ 1.5 (n = 7)

7.2 --+ 3.8 (n=6)

3.6 - 1.2 ( n = 5)

1.6 --- 1.1 (n=4)

8.1 -+ 1.0 (n = 6 )

29.0 - 5.0 (n = 2 )

9.4 -+ 2.2

(n = 5) (n = 4) (n = 2)

~Percentage o f pre-B or B cells in Ficoll-Hypaque interface cells. bPercentage of monoctonal antibody positive cells in Ficoll-Hypaque interface cells. cMean percentage -+ SD. aNumber of individual specimens studied.

staining gave 8. t0 --- 1.87% slgM + cells. Thus, the percentages of slgM + cells determined by the two methods did not differ. Examples of capped bone marrow B cells are shown in Fig. 1B and are readily distinguished from pre-B ceils with their perinuclear staining (Fig. 1D). Similar to the staining for slgM, preliminary experiments indicated the monoclonal antibodypositive pre-B cells could be more easily visualized if the monoclonal antibody was capped with TRITC goat anti-mouse Ig. Thus, several preliminary experiments were conducted which indicated that staining with TRITC goat anti-mouse Ig for 5 min at 37°C gave the same results as staining for 30 rain at 4°C. In addition, the number of monoclonal antibody positive cells detected by staining with TRITC goat anti-mouse Ig was compared to the number obtained from the corresponding slide that had gone through the entire procedure, and the percentage difference between the two sets of cells was never significant (data not shown). Analysis of Bone Marrow Cells with Monoclonal

Antibodies. Results in Table I show the percentages of pre-B, B, BA-1 +, BA-2 +, BA-3 +, anti-Bl +, L243 +, and T101 + cells in human bone marrow specimens. The percentages of pre-B and B cells did not vary significantly among fetal, pediatric, and adult bone marrow. Analysis with BA-1 and BA-2 showed a significant increase in positive ceils progressing from adult to fetal bone marrow (P < 0.001). No significant variation existed between bone marrow sources relative to the percentage of BA-3 +, anti-B1 +, L243 +, and T101 + cells. Data in Table II were obtained by double fluorochrome staining of Ficolt-Hypaque interface cells. The percentage of pre-B cells binding a given monoclonal antibody was relatively stable and independent of age group (with the exception of BA-2). The vast majority of pre-B cells from all three sources reacted with BA-1 and L243 (anti-HLA-DR). Monoclonal antibodies BA-2 and anti-Bl stained variable numbers of pre-B cells, while BA-3 (antiCALLA) and T10! were essentially unreactive. An example of a BA-2 + pre-B cell is shown in Fig. 2.

Table II. Characterization of" Pre-B Cells in Bone Marrow Specimens with Monoctonal Antibodies

Median age Source Fetal Pediatric Adult

Monoctonal antibody reactivity"

(range)

BA- 1

Anti-B 1

BA-2

BA-3

L243

T 101

20 weeks (17-32) 9 years (3-17) 26 years (19-34)

87,0 + 16b ( n = 9) c 74~0 -+ 12 (n = 14) 77.0 -+ 5 (n = 7)

62.0 --+ 5 (n = 7 ) 48.0 --+ 6 (n = 5) 50.0 ± t l (n = 6)

55,0 --+ 7 ( n = 10) 32.0 -+ 1t (n = t t ) 16.0 -+ 13 (n = 5)

4.5 --+ 7 (n=4) 5.8 + 6 (n = 5) 11.0 -_+ 7 (n = 4 )

96.0 +-- 4 (n=2) I00.0 + 0 (n = 3) 100.0 _+ 0 (n = 2)

2.0 --+ 3 ( n = 5) 0.0 -+ 0 (n = 3) 0.0 ± 0 (n = 2)

aPercentage of pre-B cells positive for a given monoclonal antibody determined by double fluorochrome analysis of Ficoll-Hypaque interface cells. bMean percentage -+ SD. ~Number of individual specimens studied.

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fetal bone marrow. In contrast, no T101 +, sIgM + cells were detected in eight pediatric and adult bone marrow specimens using the same staining procedure. Examples of the various T101, sIgM phenotypes are shown in Fig. 3. The vast majority (>90%) of sIgM + cells from fetal, pediatric, and adult bone marrow reacted with BA-1, anti-B1, and L243. The data obtained from the double staining experiments with BA-2 and BA3 gave somewhat variable results from donor to donor, with BA-2 binding to - 3 5 % of sIgM + cells and BA-3 binding to - 1 0 % of slgM + cells. DISCUSSION

Fig. 2. Double fluorochrome analysis of normal pediatric bone marrow with FITC goat anti-human IgM and BA-2, Cells were stained as described in Materials and Methods. (A) Phase contrast. (B) Same field as in A photographed with a selective filter for FITC showing a pre-B cell. (C) Same field as in A and B photographed with a selective filter for TRITC showing staining with BA-2.

Reactivity of B Cells with the Anti-T-Cell Monoctonal Antibody TIOt. During the course of studying the binding of T101 to pre-B cells, we noted that T101 bound to approximately 25% of sIgM + cells in

The hallmark characteristic of the human pre-B cell is the expression of cytoplasmic ~ heavy chains in the absence of detectable cytoplasmic light chains and sIg (2, 3). Since the pre-B cell is a rare constituent of normal bone marrow (3; Table I), studies of its surface characteristics have been limited. The purpose of this study was to construct a surface antigenic map of the human pre-B cell using a panel of well-characterized monoclonal antibodies. Several reports from Cooper and his colleagues (2, 20, 21) have indicated that the percentage of preB cells is increased during the early stages of fetal bone marrow development (14-17 weeks of gestation), pre-B cells constituting 5-10% of the total nucleated cells. We did not detect such a high frequency of pre-B cells in fetal bone marrow although our specimens were obtained from 17- to 32-week fetuses. This possible discrepancy could be resolved only by analyzing multiple fetal bone marrow specimens from different gestational time points. We have previously shown that BA-I identifies a cell surface molecule strongly expressed by 100% of normal slg + cells, 100% of pre-B ALLs, and the vast majority of sIg + non-Hodgkin's lymphomas (10, 22-24). A monoclonal antibody designated antiB1 has also been shown to bind to normal and malignant sIg + cells (13, 25). Because the cell surface molecules identified by BA-1 and anti-B1 show similar (but not identical) patterns of expression on normal and malignant sIg + cells, we were interested in analyzing normal pre-B cells with the two antibodies. Data shown in Table II indicate that B A d binds to a greater percentage of normal pre-B cells than anti-B1. Thus, BA-I appears to be more broadly reactive with pre-B cells than anti-B1. This

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Fig. 3. Double fluorochrome analysis of fetal bone marrow with FITC goat anti-human IgM and T101. Cells were stained as described in Materials and Methods. (A) Phase contrast. (B, C) Same field as in A photographed with selective filters for FITC and TRITC, respectively. Arl'owheads point to a T101 +, slgM + cell in A, B, and C. Also visible are two T101-, slgM + cells in B. (D) Phase contrast. (E, F) Same field as in D photographed with selective filters for FITC and TRITC, respectively. Arrows point to a T101 +, slgM- cell in D, E, and F. Also visible are three T101-, slgM + cells in E.

observation is consistent with data obtained on preB ALL, the majority of which is BA-1 +, anti-B1(22, 26). Whether the BA-I + and anti-B1 + pre-B cells represent a different lineage or differ functionally from the B A - I - and anti-Bl- pre-B cells is not known. The reactivity of pre-B cells with BA-2 revealed an interesting pattern. The total pre-B cell population showed a significant decrease in BA-2 reactivity as the age of the donor bone marrow group increased (Table II). We have reported a similar phenomenon while conducting double fluorochrome experiments with BA-2 and an antibody to terminal deoxynucleotidyl transferase (27). The implication of this finding is not clear but may relate to cell proliferation. The cell surface molecule recognized by BA-2 (designated p24; Ref. 11) is known to be strongly expressed on hematopoietic and nonhematopoietic tumor cells as well as activated T cells (28, 29; T. W. LeBien and J. H. Kersey, unpublished observations). It is conceivable that the higher percentage of BA-2 + pre-B cells in fetal bone marrow reflects a higher proliferative rate of these cells early in ontogeny. In this regard, studies in both mouse (30) and human (5) have demonstrated the existence of two types of pre-B cells, a large, rapidly proliferating population and a small, slowly proliferating population.

Journal of Clinical Immunology, Vol. 3, No. 3, 1983

The results obtained with L243 (anti-HLA-DR) and BA-3 (anti-CALLA) can be compared to those of previous studies. As shown in Table II, L243 binds to virtually all pre-B cells, in agreement with data from two other laboratories (6, 20). BA-3 binds to very few pre-B cells, consistent with data reported by Greaves et al. (6). Royston et al. (15) have shown that both T- and B-cell markers are expressed on peripheral blood cells from patients with chronic lymphocytic leukemia, hypothesizing that a common stem-cell origin for T and B cells explains this phenotype. Recently, Caligaris-Cappio et al. (31) have identified what may be a Leu l(T101) +, sIg + nonmalignant counterpart of the chronic lymphocytic leukemia cell in normal lymph node and tonsil. Results from this study provide additional evidence for the existence of a normal B cell with the T101(Leu 1)+, sIgM + phenotype. T101(Leu I) +, sIgM + B cells were detected in ° normal fetal bone marrow but were discriminatingly not found in pediatric or adult bone marrow. Perhaps these Tl01(Leu I) +, sIgM + cells originate from the fetal bone marrow and migrate to the lymph node or tonsil, remaining there throughout life. It is worth noting that a Leu I(Lyt-1) +, sIgM + population of normal mouse B cells has recently been reported (32, 33), although this population was found principally in the spleen.

266

As is quite evident in Tables I and II, the vast majority of bone marrow cells reacting with BA-1, BA-2, anti-B 1, and L243 are n o t pre-B cells. These reagents are known to bind to a variety of other cell populations (10, 11, 13, 14) and are, therefore, not specifically reactive with pre-B cells. However, they are useful in defining the cellular phenotype of a rare cell in normal bone marrow and we can now begin to construct an antigenic map of the human pre-B cell. A recent report by Landreth et al. (34) described a rat anti-mouse monoclonal antibody (177.17) which binds to >95% of human pre-B cells. Coupled with the data reported herein, we can now assign a predominant phenotype of L243 +, BA-1 ÷, 177.17 ÷, BA-3-, and T101- for the human pre-B cell, while phenotypic heterogeneity exists for antiB1 and BA-2. Further studies defining the maturation requirements of these cells in vitro will probably be facilitated by the development of clonogenic assays for human lymphocyte precursors.

MELINK AND LeBIEN

7.

8.

9.

10.

11.

12.

13. ACKNOWLEDGMENTS This w o r k w a s s u p p o r t e d b y G r a n t CA-31685 f r o m the N a t i o n a l C a n c e r I n s t i t u t e , N a t i o n a l Instit u t e s o f H e a l t h , a n d w a s s u b m i t t e d in partial fulfillm e n t (by G B M ) for the d e g r e e o f M a s t e r of S c i e n c e in L a b o r a t o r y M e d i c i n e , U n i v e r s i t y of M i n n e s o t a . T h e a u t h o r s w i s h to t h a n k Dr. A n n e G o l d m a n for a s s i s t a n c e w i t h the statistical a n a l y s i s , Drs. J o h n K e r s e y a n d N e i l K a y for critically r e v i e w i n g the m a n u s c r i p t , a n d M a u r e e n R e a g a n for p r e p a r i n g the manuscript.

14. 15.

16.

17.

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29, Kemshead JT, Fritschy J, Asser U, Sutherland R, Greaves MF: Monoclonal antibodies defining markers with apparent selectivity for particular hematopoietic cell types may also detect antigens on cells of neural crest origin. Hybridoma 1:109-115, 1982 30. Landreth KS, Rosse C, Claggett J: Myelogenous production and maturation of B lymphocytes in the mouse. J Immunot t27:2027-2034, 1981 31. Caligaris-Cappio F, Gobbi M. Bofilt M, Janossy G: Infrequent normal B lymphocytes express features of B-chronic lymphocytic leukemia. J Exp Med 155:623-628, 1982 32. Manohar V, Brown E, Leiserson WM, Chused TM: Expression of LYT-1 by a subset of B lymphocytes. J Immunol 129:532-538, t982 33. Hyakawa K, Hardy RR, Parks DR, Herzenberg LA: The "LY-1 B" celt subpopulation in normal, imm~anodefective, and autoimmune mice. J Exp Med t57:202-218, 1983 34. Landreth KD, Kincade PW, Lee G, Gathings WE, Fu SM: Enrichment of human marrow lymphocytes with monoclonat antibodies to murine antigens. Proc Natl Acad Sci USA 79:2370-2374, 1982